# Patent

## Title
Polynucleotides, polypeptides and methods for increasing oil content, growth rate and biomass of plants

## Abstract

Provided are method of increasing oil content, growth rate, biomass, yield and/or vigor of a plant. The methods are effected by upregulating in the plant an expression level of a polypeptide comprising an amino acid sequence at least 90% homologous to the amino acid sequence selected from the group consisting of SEQ ID NOs: 199, 166-198, 200-221, 229-307, 311-330, 351-353, 355-361, 363-364, 366-368, 218, 222-228, 308-310, 350, 354, 362, 365, 523-649, 786-920, 1047 and 1048. Also provided are polynucleotides, nucleic acid constructs, polypeptides and transgenic plants expressing same which can be used to increase oil content, growth rate, biomass, yield and/or vigor of a plant and produce oil.

## Background
RELATED APPLICATIONS
This Application is a National Phase of PCT Patent Application No. PCT/IL2008/000489 having International filing date of Apr. 9, 2008, which claims the benefit of U.S. Provisional Patent Application No. 60/907,568 filed on Apr. 9, 2007. The contents of the above Applications are all incorporated herein by reference.


FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments thereof, relates to polypeptides, polynucleotides encoding same, transgenic plants expressing same and methods of producing and using same, and, more particularly, but not exclusively, to methods of increasing oil content, seed yield, growth rate, biomass and/or yield of a plant.
Vegetable or seed oils are the major source of energy and nutrition in human and animal diet. They are also used for the production of industrial products, such as paints, inks and lubricants. In addition, plant oils represent renewable sources of long-chain hydrocarbons which can be used as fuel. Since the currently used fossil fuels are finite resources and are gradually being depleted, fast growing biomass crops may be used as alternative fuels or for energy feedstocks and may reduce the dependence on fossil energy supplies. However, the major bottleneck for increasing the consumption of plant oils as bio-fuel is the oil price, which is still higher than fossil fuel [Hypertext Transfer Protocol://World Wide Web (dot) eia (dot) doe (dot) gov/oiaf/analysispaper/biodiesel/; Hypertext Transfer Protocol://World Wide Web (dot) njbiz (dot)com/weekly_article.asp?aID=19755147 (dot) 6122555 (dot) 957931 (dot) 7393254 (dot) 4337383 (dot) 561&aID2=73678]. In addition, the production rate of plant oil is limited by the availability of agricultural land and water. Thus, increasing plant oil yields from the same growing area can effectively overcome the shortage in production space and can decrease vegetable oil prices at the same time.
Studies aiming at increasing plant oil yields focus on the identification of genes involved in oil metabolism as well as in genes capable of increasing plant and seed yields in transgenic plants.
Genes known to be involved in increasing plant oil yields include those participating in fatty acid synthesis or sequestering such as desaturase [e.g., DELTA6, DELTA12 or acyl-ACP (Ssi2; Arabidopsis Information Resource (TAIR; Hypertext Transfer Protocol://World Wide Web (dot) arabidopsis (dot) org/), TAIR No. AT2G43710)], OleosinA (TAIR No. AT3G01570) or FAD3 (TAIR No. AT2G29980), and various transcription factors and activators such as Lec1 [TAIR No. AT1G21970, Lotan et al. 1998. Cell. 26; 93(7):1195-205], Lec2 [TAIR No. AT1G28300, Santos Mendoza et al. 2005, FEBS Lett. 579(21):4666-70], Fus3 (TAIR No. AT3G26790), ABI3 [TAIR No. AT3G24650, Lara et al. 2003. J Biol Chem. 278(23): 21003-11] and Wri1 [TAIR No. AT3G54320, Cernac and Benning, 2004. Plant J. 40(4): 575-85].
Zabrouskov V., et al., 2002 (Physiol Plant. 116:172-185) demonstrated that upregulation of endoplasmic reticulum (FAD3) and plastidal (FAD7) fatty acid desaturases in potato increases the total lipid fraction in transgenic clones.
Wang H W et al., 2007 (Plant J. 52:716-29. Epub 2007 Sep. 18) found that transgenic plant seeds over-expressing the GmDof4 and GmDof11 transcription factors exhibit increased content of total fatty acids and lipids.
Vigeolas H, et al. [Plant Biotechnol J. 2007, 5(3):431-41] and U.S. Pat. Appl. No. 20060168684 disclose increased seed oil content in oil-seed rape (Brassica napus L.) by over-expression of a yeast glycerol-3-phosphate dehydrogenase under the control of a seed-specific promoter.
Katavic V, et al., 2000 (Biochem Soc Trans. 28:935-7) describe the use of the Arabidopsis FAE1 and yeast SLC1-1 genes for improvements in erucic acid and oil content in rapeseed.
U.S. Pat. Appl. No. 20080076179 discloses an isolated moss nucleic acid encoding a lipid metabolism protein (LMP) and transgenic plants expressing same with increased lipid levels.
U.S. Pat. Appl. No. 20060206961 discloses a method of increasing oil content in plants (e.g., in plant seeds), by expressing in the plant the Ypr140w polypeptide.
U.S. Pat. Appl. No. 20060174373 discloses a method of increasing oil content in plants by expressing a nucleic acid encoding a triacylglycerols (TAG) synthesis enhancing protein (TEP) in the plant.
U.S. Pat. Appl. Nos. 20070169219, 20070006345, 20070006346 and 20060195943, disclose transgenic plants with improved nitrogen use efficiency which can be used for the conversion into fuel or chemical feedstocks.
SUMMARY OF THE INVENTION
According to an aspect of some embodiments of the present invention there is provided a method of increasing oil content, growth rate, biomass, yield and/or vigor of a plant, comprising introducing into the plant an exogenous polynucleotide encoding a polypeptide comprising an amino acid sequence at least 90% homologous to the amino acid sequence selected from the group consisting of SEQ ID NOs: 199, 166-198, 200-221, 229-307, 311-330, 351-353, 355-361, 363-364, 366-368, 218, 222-228, 308-310, 350, 354, 362, 365, 523-649, 786-920, 1047 and 1048, thereby increasing the oil content, growth rate, biomass, yield and/or vigor of the plant.
According to an aspect of some embodiments of the present invention there is provided a method of producing oil, comprising: (a) providing the plant according to the method of the invention, and (b) extracting the oil from the plant; thereby producing the oil.
According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence at least 90% identical to SEQ ID NOs: 34, 1-33, 35-52, 54-56, 64-165, 332-334, 336-342, 344-345, 347-349, 53, 57-63, 143-145, 331, 335, 343, 346, 369-522, 650-785, 1016-1046.
According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct, comprising the isolated polynucleotide of the invention and a promoter for directing transcription of the nucleic acid sequence.
According to an aspect of some embodiments of the present invention there is provided an isolated polypeptide, comprising an amino acid sequence at least 90% homologous to SEQ ID NO: 199, 166-198, 200-221, 229-307, 311-330, 351-353, 355-361, 363-364, 366-368, 218, 222-228, 308-310, 350, 354, 362, 365, 523-649, 786-920, 1047 and 1048.
According to an aspect of some embodiments of the present invention there is provided a plant cell exogenously expressing the polypeptide of the invention.
According to an aspect of some embodiments of the present invention there is provided a plant cell exogenously expressing the polynucleotide of the invention.
According to some embodiments of the invention, the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 34, 1-33, 35-52, 54-56, 64-165, 332-334, 336-342, 344-345, 347-349, 53, 57-63, 143-145, 331, 335, 343, 346, 369-522, 650-785, 1016-1046.
According to some embodiments of the invention, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 199, 166-198, 200-221, 229-307, 311-330, 351-353, 355-361, 363-364, 366-368, 218, 222-228, 308-310, 350, 354, 362, 365, 523-649, 786-920, 1047 and 1048.
According to some embodiments of the invention, the polynucleotide is selected from the group consisting of SEQ ID NOs: 34, 1-33, 35-52, 54-56, 64-165, 332-334, 336-342, 344-345, 347-349, 53, 57-63, 143-145, 331, 335, 343, 346, 369-522, 650-785, 1016-1046.
According to some embodiments of the invention, the polypeptide is selected from the group consisting of SEQ ID NOs: 199, 166-198, 200-221, 229-307, 311-330, 351-353, 355-361, 363-364, 366-368, 218, 222-228, 308-310, 350, 354, 362, 365, 523-649, 786-920, 1047 and 1048.
According to some embodiments of the invention, the oil comprises a seed oil.
According to some embodiments of the invention, the oil comprises a vegetative portion oil.
According to some embodiments of the invention, the plant cell forms a part of a plant.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.



BRIEF DESCRIPTION OF THE DRAWINGS
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
FIGS. 1a-d are digital images of leaves depicting leaf length (FIG. 1a, the leaf length is represented by the arrow), laminar length (FIG. 1b, the laminar length is represented by the arrow), laminar area (FIG. 1c, the laminar area is represented by the white ellipse) and laminar width (FIG. 1d, the laminar width is represented by the arrow). Blade circularity was calculated as laminar width divided by laminar length.
FIGS. 2a-b are images depicting root development of plants grown in transparent agar plates. The different ecotypes were grown in transparent agar plates for 17 days and the plates were photographed every 2 days starting at day 7. An exemplary image is shown in FIG. 2a (taken following 12 days on agar plates). The length of the root measured is represented by the red arrow (FIG. 2b).
FIG. 3 is an image depicting iodine vapor staining of lipids isolated from the transgenic plants expressing the genes listed in Table 56, Example 7 of the Examples section which follows. The arrow points at the tri acyl glycerol bands.



DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention, in some embodiments thereof, relates to isolated polypeptides and polynucleotides encoding same, and more particularly, but not exclusively, to methods of using same for increasing oil content, growth rate, yield, biomass and/or vigor of a plant.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
While reducing the present invention to practice, the present inventors have identified novel polypeptides and polynucleotides which can be used to increase oil content, seed yield, growth rate, biomass, yield and/or vigor of a plant.
Thus, as shown in the Examples section which follows, the present inventors have employed a bioinformatics approach which compares the expression pattern of Arabidopsis-derived genes in 79 tissues or developmental stages to that of the oil hook genes (OHGs) known to play a role in embryogenesis, seed development and oil synthesis and accumulation, and genes exhibiting a significant correlation were identified (Table 1, Example 1). In addition, using an oligonucleotide micro-array, the present inventors determined the expression profile of identified genes in tissues and developmental stages of various Arabidopsis ecotypes (Table 3; Example 2) and correlated the expression profile to selected yield or vigor related parameters (Tables 4, 5 and 6; Example 2). Genes exhibiting a significant correlation between the expression profile and the yield or vigor parameters of the ecotypes were identified (Table 7; Example 2). Of them, several genes were found to modulate seed yield (Table 8), oil yield (Table 9), growth rate (Table 10), organ shape/size/length (Table 11), harvest index (Table 12), oil content per seed (Table 13), plant dry matter (Table 14) and seed number per silique (Table 15). Additional genes which are predicted to increase oil content, seed yield, growth rate, yield and/or biomass of a plant were identified using bioinformatics tools (Table 2, Example 1). In addition, polypeptides and polynucleotides encoding same which are homologous to the predicted polypeptides of Tables 1 and 2 were also identified (Table 18, Example 5). Furthermore, as described in Examples 3, 4 and 6 of the Examples section which follows, transgenic plants expressing the identified polynucleotides exhibit increased seed yield, oil yield, dry matter, harvest index, growth rate, rosette area, oil percentage in seed and weight of 1000 seeds (Tables 19-55; Example 6). In addition, transgenic plants expressing the polynucleotides of the invention exhibited increased oil content as compared to control plants (FIG. 3, Example 7). Altogether, these results suggest the use of the novel polynucleotides and polypeptides of the invention for increasing oil content, yield (including seed yield), growth rate, biomass, and/or vigor of a plant.
It should be noted that since oil content is affected by intrinsic oil production, or mass/size of oil producing tissue per plant/per growth period, any gene which affects these aforementioned processes is contemplated in accordance with the teachings of the present invention.
Thus, according to one aspect of the invention there is provided a method of increasing oil content, yield, growth rate, biomass and/or vigor of a plant. The method is effected by introducing into the plant an exogenous polynucleotide encoding a polypeptide comprising an amino acid sequence at least 90% homologous to the amino acid sequence selected from the group consisting of SEQ ID NOs:166-221, 229-307, 311-330, 351-353, 355-361, 363-364, 366-368, 218, 222-228, 308-310, 350, 354, 362, 365, 523-649, 786-920, 1047 and 1048.
The phrase “oil content” as used herein refers to the amount of lipids in a given plant organ, either the seeds (seed oil content) or the vegetative portion of the plant (vegetative oil content) and typically expressed as percentage of dry weight (10% humidity of seeds) or wet weight (for vegetative portion).
As mentioned, in one embodiment, increase in oil content of the plant can be achieved by increasing the size/mass of a plant's tissue(s) which comprise oil per growth period. Thus, increased oil content of a plant can be achieved by increasing the yield, growth rate, biomass and vigor of the plant.
As used herein the phrase “plant yield” refers to the amount (as determined by weight/size) or quantity (numbers) of tissue (e.g., seed, referred to “seed yield” and vegetative portion) produced per plant or per growing season. Hence increased yield could affect the economic benefit one can obtain from the plant in a certain growing area and/or growing time.
As used herein the phrase “plant biomass” refers to the amount (measured in grams of air-dry tissue) of a tissue produced from the plant in a growing season, which could also determine or affect the plant yield or the yield per growing area.
As used herein the phrase “plant vigor” refers to the amount (measured by weight) of tissue produced by the plant in a given time. Hence increase vigor could determine or affect the plant yield or the yield per growing time or growing area.
As used herein the term “increasing” refers to at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, increase in plant oil content, seed yield (seed yield per plant and/or seed yield per growing area), plant yield, growth rate, biomass, and/or vigor as compared to a native plant [i.e., a plant not modified with the biomolecules (polynucleotide or polypeptides) of the invention, e.g., a non-transformed plant of the same species which is grown under the same growth conditions).
As used herein, the phrase “exogenous polynucleotide” refers to a heterologous nucleic acid sequence which may not be naturally expressed within the plant or which overexpression in the plant is desired. The exogenous polynucleotide may be introduced into the plant in a stable or transient manner, so as to produce a ribonucleic acid (RNA) molecule and/or a polypeptide molecule. It should be noted that the exogenous polynucleotide may comprise a nucleic acid sequence which is identical or partially homologous to an endogenous nucleic acid sequence of the plant.
According to some embodiments of the invention, the exogenous polynucleotide encodes a polypeptide comprising an amino acid sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more say 100% homologous to the amino acid sequence selected from the group consisting of SEQ ID NOs:166-221, 229-307, 311-330, 351-353, 355-361, 363-364, 366-368, 218, 222-228, 308-310, 350, 354, 362, 365, 523-649, 786-920, 1047 and 1048.
Homology (e.g., percent homology) can be determined using any homology comparison software, including for example, the BlastP or TBLASTN softwares of the National Center of Biotechnology Information (NCBI) such as by using default parameters, when starting from a polypeptide sequence; or the tBLASTX algorithm (available via the NCBI) such as by using default parameters, which compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database.
Homologous sequences include both orthologous and paralogous sequences. The term “paralogous” relates to gene-duplications within the genome of a species leading to paralogous genes. The term “orthologous” relates to homologous genes in different organisms due to ancestral relationship.
One option to identify orthologues in monocot plant species is by performing a reciprocal blast search. This may be done by a first blast involving blasting the sequence-of-interest against any sequence database, such as the publicly available NCBI database which may be found at: Hypertext Transfer Protocol://World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov. If orthologues in rice were sought, the sequence-of-interest would be blasted against, for example, the 28,469 full-length cDNA clones from Oryza sativa Nipponbare available at NCBI. The blast results may be filtered. The full-length sequences of either the filtered results or the non-filtered results are then blasted back (second blast) against the sequences of the organism from which the sequence-of-interest is derived. The results of the first and second blasts are then compared. An orthologue is identified when the sequence resulting in the highest score (best hit) in the first blast identifies in the second blast the query sequence (the original sequence-of-interest) as the best hit. Using the same rational a paralogue (homolog to a gene in the same organism) is found. In case of large sequence families, the ClustalW program may be used [Hypertext Transfer Protocol://World Wide Web (dot) ebi (dot) ac (dot) uk/Tools/clustalw2/index (dot) html], followed by a neighbor joining tree (Hypertext Transfer Protocol://en (dot) wikipedia (dot) org/wiki/Neighbor-joining) which helps visualizing the clustering.
According to some embodiments of the invention, the exogenous polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOs:166-221, 229-307, 311-330, 351-353, 355-361, 363-364, 366-368, 218, 222-228, 308-310, 350, 354, 362, 365, 523-649, 786-920, 1047 and 1048.
According to some embodiments of the invention the exogenous polynucleotide comprises a nucleic acid sequence which is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g., 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1-52, 54-56, 64-165, 332-334, 336-342, 344-345, 347-349, 53, 57-63, 143-145, 331, 335, 343, 346, 369-522, 650-785, 1016-1046.
Identity (e.g., percent homology) can be determined using any homology comparison software, including for example, the BlastN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters.
According to some embodiments of the invention the exogenous polynucleotide is set forth by SEQ ID NOs:1-52, 54-56, 64-165, 332-334, 336-342, 344-345, 347-349, 53, 57-63, 143-145, 331, 335, 343, 346, 369-522, 650-785, 1016-1046.
As used herein the term “polynucleotide” refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
As used herein the phrase “complementary polynucleotide sequence” refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent DNA polymerase.
As used herein the phrase “genomic polynucleotide sequence” refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.
As used herein the phrase “composite polynucleotide sequence” refers to a sequence, which is at least partially complementary and at least partially genomic. A composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing therebetween. The intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.
Nucleic acid sequences encoding the polypeptides of the present invention may be optimized for expression. Non-limiting examples of optimized nucleic acid sequences are provided in SEQ ID NOs:1040, 1041, 1042, 1043, 1044, 1045, and 1046 which encodes polypeptides comprising the amino acid sequences set forth by SEQ ID NOs: 167, 169, 1047, 181, 185, 189 and 196, respectively. Examples of such sequence modifications include, but are not limited to, an altered G/C content to more closely approach that typically found in the plant species of interest, and the removal of codons atypically found in the plant species commonly referred to as codon optimization.
The phrase “codon optimization” refers to the selection of appropriate DNA nucleotides for use within a structural gene or fragment thereof that approaches codon usage within the plant of interest. Therefore, an optimized gene or nucleic acid sequence refers to a gene in which the nucleotide sequence of a native or naturally occurring gene has been modified in order to utilize statistically-preferred or statistically-favored codons within the plant. The nucleotide sequence typically is examined at the DNA level and the coding region optimized for expression in the plant species determined using any suitable procedure, for example as described in Sardana et al. (1996, Plant Cell Reports 15:677-681). In this method, the standard deviation of codon usage, a measure of codon usage bias, may be calculated by first finding the squared proportional deviation of usage of each codon of the native gene relative to that of highly expressed plant genes, followed by a calculation of the average squared deviation. The formula used is: 1 SDCU=n=1N[(Xn−Yn)/Yn]2/N, where Xn refers to the frequency of usage of codon n in highly expressed plant genes, where Yn to the frequency of usage of codon n in the gene of interest and N refers to the total number of codons in the gene of interest. A Table of codon usage from highly expressed genes of dicotyledonous plants is compiled using the data of Murray et al. (1989, Nuc Acids Res. 17:477-498).
One method of optimizing the nucleic acid sequence in accordance with the preferred codon usage for a particular plant cell type is based on the direct use, without performing any extra statistical calculations, of codon optimization tables such as those provided on-line at the Codon Usage Database through the NIAS (National Institute of Agrobiological Sciences) DNA bank in Japan (http://www.kazusa.or.jp/codon/). The Codon Usage Database contains codon usage tables for a number of different species, with each codon usage table having been statistically determined based on the data present in Genbank.
By using the above Tables to determine the most preferred or most favored codons for each amino acid in a particular species (for example, rice), a naturally-occurring nucleotide sequence encoding a protein of interest can be codon optimized for that particular plant species. This is effected by replacing codons that may have a low statistical incidence in the particular species genome with corresponding codons, in regard to an amino acid, that are statistically more favored. However, one or more less-favored codons may be selected to delete existing restriction sites, to create new ones at potentially useful junctions (5′ and 3′ ends to add signal peptide or termination cassettes, internal sites that might be used to cut and splice segments together to produce a correct full-length sequence), or to eliminate nucleotide sequences that may negatively effect mRNA stability or expression.
The naturally-occurring encoding nucleotide sequence may already, in advance of any modification, contain a number of codons that correspond to a statistically-favored codon in a particular plant species. Therefore, codon optimization of the native nucleotide sequence may comprise determining which codons, within the native nucleotide sequence, are not statistically-favored with regards to a particular plant, and modifying these codons in accordance with a codon usage table of the particular plant to produce a codon optimized derivative. A modified nucleotide sequence may be fully or partially optimized for plant codon usage provided that the protein encoded by the modified nucleotide sequence is produced at a level higher than the protein encoded by the corresponding naturally occurring or native gene. Construction of synthetic genes by altering the codon usage is described in for example PCT Patent Application 93/07278.
According to some embodiments of the invention, expression of the polynucleotide of the invention results in downregulation of the expression level or activity of the corresponding endogenous polypeptide (e.g., homologue).
According to some embodiments of the invention, the exogenous polynucleotide is used for co-suppression or sense suppression of an endogenous polypeptide. Thus, introducing the exogenous polynucleotide to the plant cells results in transcription of an RNA molecule (in a sense direction with respect to the corresponding endogenous gene) which suppresses translation of the corresponding endogenous RNA molecule, such as described in U.S. Pat. No. 5,231,020 to Jorgensen, which is fully incorporated herein by reference. For co-suppression, the exogenous polynucleotide does not require the entire nucleic acid sequence of the corresponding endogenous gene, nor does it require that the introduced sequence be exactly identical to the endogenous gene. However, as with antisense suppression, the suppressive efficiency is enhanced as specificity of hybridization is increased, e.g., as the introduced sequence is lengthened, and/or as the sequence similarity between the introduced sequence and the endogenous gene is increased. For further details see U.S. Pat. Appl. No. 20050172364 which is fully incorporated herein by reference.
According to some embodiments of the invention, the exogenous polynucleotide comprises an untranslatable nucleic acid sequence, e.g., a sequence comprising one or more pre-mature stop codons, or nonsense mutations, such as described in U.S. Pat. No. 5,583,021.
Thus, the invention encompasses isolated polynucleotides described hereinabove; fragments thereof, sequences hybridizable therewith, sequences homologous thereto, sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or man induced, either randomly or in a targeted fashion.
As mentioned, the present inventors have uncovered previously uncharacterized polypeptides.
Thus, the invention provides an isolated polypeptide having an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more say 100% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs:166-221, 229-307, 311-330, 351-353, 355-361, 363-364, 366-368, 218, 222-228, 308-310, 350, 354, 362, 365, 523-649, 786-920, 1047 and 1048.
According to some embodiments of the invention, there is provided an exogenous polypeptide selected from the group consisting of SEQ ID NOs:166-221, 229-307, 311-330, 351-353, 355-361, 363-364, 366-368, 218, 222-228, 308-310, 350, 354, 362, 365, 523-649, 786-920, 1047 and 1048.
The invention also encompasses fragments of the above described polypeptides and polypeptides having mutations, such as deletions, insertions or substitutions of one or more amino acids, either naturally occurring or man induced, either randomly or in a targeted fashion.
The term “plant” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), and plant cells, tissues and organs. The plant may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores. Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalypfus spp., Euclea schimperi, Eulalia vi/losa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane, sunflower, tomato, squash tea, maize, wheat, barely, rye, oat, peanut, pea, lentil and alfalfa, cotton, rapeseed, canola, pepper, sunflower, tobacco, eggplant, eucalyptus, a tree, an ornamental plant, a perennial grass and a forage crop. Alternatively algae and other non-Viridiplantae can be used for the methods of the present invention.
According to some embodiments of the invention, the oil producing plant can be oilseed crops, soybeans, sunflower, Brassica napus, Brassica Juncea, zea maize, cotton, olive (Olea europaea), flax, Brassica nigra, Jatropha curcas, and Castorbean (Ricinus communis).
Introducing the exogenous polynucleotide of the invention into the plant can be effected by transforming one or more cells of the plant with the exogenous polynucleotide, followed by generating a mature plant from the transformed cells and cultivating the mature plant under conditions suitable for expressing the exogenous polynucleotide within the mature plant.
According to some embodiments of the invention, the transformation is effected by introducing to the plant cell a nucleic acid construct which includes the exogenous polynucleotide of some embodiments of the invention and at least one promoter capable of directing transcription of the exogenous polynucleotide in the plant cell. Further details of suitable transformation approaches are provided hereinbelow.
As used herein, the term “promoter” refers to a region of DNA which lies upstream of the transcriptional initiation site of a gene to which RNA polymerase binds to initiate transcription of RNA. The promoter controls where (e.g., which portion of a plant) and/or when (e.g., at which stage or condition in the lifetime of an organism) the gene is expressed.
Any suitable promoter sequence can be used by the nucleic acid construct of the present invention. According to some embodiments of the invention, the promoter is a constitutive promoter, a tissue-specific, or a developmental or embryonic-specific promoter.
Suitable constitutive promoters include, for example, CaMV 35S promoter (SEQ ID NO:921; Odell et al., Nature 313:810-812, 1985); Arabidopsis At6669 promoter (SEQ ID NO:1015; see PCT Publication No. WO2004/104162); maize Ubi 1 (Christensen et al., Plant Sol. Biol. 18:675-689, 1992); rice actin (McElroy et al., Plant Cell 2:163-171, 1990); pEMU (Last et al., Theor. Appl. Genet. 81:581-588, 1991); CaMV 19S (Nilsson et al., Physiol. Plant 100:456-462, 1997); GOS2 (de Pater et al, Plant J November; 2(6):837-44, 1992); Rice cyclophilin (Bucholz et al, Plant Mol. Biol. 25(5):837-43, 1994); Maize H3 histone (Lepetit et al, Mol. Gen. Genet. 231: 276-285, 1992); Actin 2 (An et al, Plant J. 10(1); 107-121, 1996) and Synthetic Super MAS (Ni et al., The Plant Journal 7: 661-76, 1995). Other constitutive promoters include those in U.S. Pat. Nos. 5,659,026, 5,608,149; 5,608,144; 5,604,121; 5,569,597: 5,466,785; 5,399,680; 5,268,463; and 5,608,142.
Suitable tissue-specific promoters include, but not limited to, seed-preferred promoters [e.g., from seed specific genes (Simon, et al., Plant Mol. Biol. 5. 191, 1985; Scofield, et al., J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990), Brazil Nut albumin (Pearson' et al., Plant Mol. Biol. 18: 235-245, 1992), legumin (Ellis, et al. Plant Mol. Biol. 10: 203-214, 1988), Glutelin (rice) (Takaiwa, et al., Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa, et al., FEBS Letts. 221: 43-47, 1987), Zein (Matzke et al Plant Mol Biol, 143: 323-32 1990), napA (Stalberg, et al, Planta 199: 515-519, 1996), Wheat SPA (Albanietal, Plant Cell, 9: 171-184, 1997), sunflower oleosin (Cummins, et al., Plant Mol. Biol. 19: 873-876, 1992)], leaf-specific promoters [such as described, for example, by Yamamoto et al., Plant J. 12:255-265, 1997; Kwon et al., Plant Physiol. 105:357-67, 1994; Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994; Gotor et al., Plant J. 3:509-18, 1993; Orozco et al., Plant Mol. Biol. 23:1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA 90:9586-9590, 1993], endosperm specific promoters [e.g., wheat LMW and HMW, glutenin-1 (Mol Gen Genet. 216:81-90, 1989; NAR 17:461-2), wheat a, b and g gliadins (EMBO3:1409-15, 1984), Barley ltrl promoter, barley B1, C, D hordein (Theor Appl Gen 98:1253-62, 1999; Plant J 4:343-55, 1993; Mol Gen Genet. 250:750-60, 1996), Barley DOF (Mena et al, The Plant Journal, 116(1): 53-62, 1998), Biz2 (EP99106056.7), Synthetic promoter (Vicente-Carbajosa et al., Plant J. 13: 629-640, 1998), rice prolamin NRP33, rice-globulin Glb-1 (Wu et al, Plant Cell Physiology 39(8) 885-889, 1998), rice alpha-globulin REB/OHP-1 (Nakase et al. Plant Mol. Biol. 33: 513-S22, 1997), rice ADP-glucose PP (Trans Res 6:157-68, 1997), maize ESR gene family (Plant J 12:235-46, 1997), sorgum gamma-kafirin (PMB 32:1029-35, 1996)], embryo specific promoters [e.g., rice OSH1 (Sato et al, Proc. Natl. Acad. Sci. USA, 93: 8117-8122), KNOX (Postma-Haarsma et al, Plant Mol. Biol. 39:257-71, 1999), rice oleosin (Wu et at, J. Biochem., 123:386, 1998)], and flower-specific promoters [e.g., AtPRP4, chalene synthase (chsA) (Van der Meer, et al., Plant Mol. Biol. 15, 95-109, 1990), LAT52 (Twell et al Mol. Gen. Genet. 217:240-245; 1989), apetala-3].
The nucleic acid construct of some embodiments of the invention can further include an appropriate selectable marker and/or an origin of replication. According to some embodiments of the invention, the nucleic acid construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible with propagation in cells. The construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
The nucleic acid construct of some embodiments of the invention can be utilized to stably or transiently transform plant cells. In stable transformation, the exogenous polynucleotide is integrated into the plant genome and as such it represents a stable and inherited trait. In transient transformation, the exogenous polynucleotide is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait.
There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (Potrykus, I., Annu Rev. Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al., Nature (1989) 338:274-276).
The principle methods of causing stable integration of exogenous DNA into plant genomic DNA include two main approaches:
(i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S, and Arntzen, C. J., Butterworth Publishers, Boston, Mass. (1989) p. 93-112.
(ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 52-68; including methods for direct uptake of DNA into protoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. DNA uptake induced by brief electric shock of plant cells: Zhang et al. Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986) 319:791-793. DNA injection into plant cells or tissues by particle bombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al. Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990) 79:206-209; by the use of micropipette systems: Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant. (1990) 79:213-217; glass fibers or silicon carbide whisker transformation of cell cultures, embryos or callus tissue, U.S. Pat. No. 5,464,765 or by the direct incubation of DNA with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.
The Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. See, e.g., Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.
There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field. In microinjection, the DNA is mechanically injected directly into the cells using very small micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.
Following stable transformation plant propagation is exercised. The most common method of plant propagation is by seed. Regeneration by seed propagation, however, has the deficiency that due to heterozygosity there is a lack of uniformity in the crop, since seeds are produced by plants according to the genetic variances governed by Mendelian rules. Basically, each seed is genetically different and each will grow with its own specific traits. Therefore, it is preferred that the transformed plant be produced such that the regenerated plant has the identical traits and characteristics of the parent transgenic plant. Therefore, it is preferred that the transformed plant be regenerated by micropropagation which provides a rapid, consistent reproduction of the transformed plants.
Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue expressing the fusion protein. The new generation plants which are produced are genetically identical to, and have all of the characteristics of, the original plant. Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant. The advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.
Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages. Thus, the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening. During stage one, initial tissue culturing, the tissue culture is established and certified contaminant-free. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production goals. During stage three, the tissue samples grown in stage two are divided and grown into individual plantlets. At stage four, the transformed plantlets are transferred to a greenhouse for hardening where the plants' tolerance to light is gradually increased so that it can be grown in the natural environment.
According to some embodiments of the invention, the transgenic plants are generated by transient transformation of leaf cells, meristematic cells or the whole plant.
Transient transformation can be effected by any of the direct DNA transfer methods described above or by viral infection using modified plant viruses.
Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, TMV and BV. Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants are described in WO 87/06261.
According to some embodiments of the invention, the virus used for transient transformations is avirulent and thus is incapable of causing severe symptoms such as reduced growth rate, mosaic, ring spots, leaf roll, yellowing, streaking, pox formation, tumor formation and pitting. A suitable avirulent virus may be a naturally occurring avirulent virus or an artificially attenuated virus. Virus attenuation may be effected by using methods well known in the art including, but not limited to, sub-lethal heating, chemical treatment or by directed mutagenesis techniques such as described, for example, by Kurihara and Watanabe (Molecular Plant Pathology 4:259-269, 2003), Gal-on et al. (1992), Atreya et al. (1992) and Huet et al. (1994).
Suitable virus strains can be obtained from available sources such as, for example, the American Type culture Collection (ATCC) or by isolation from infected plants. Isolation of viruses from infected plant tissues can be effected by techniques well known in the art such as described, for example by Foster and Tatlor, Eds. “Plant Virology Protocols From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)”, Humana Press, 1998. Briefly, tissues of an infected plant believed to contain a high concentration of a suitable virus, preferably young leaves and flower petals, are ground in a buffer solution (e.g., phosphate buffer solution) to produce a virus infected sap which can be used in subsequent inoculations.
Construction of plant RNA viruses for the introduction and expression of non-viral exogenous polynucleotide sequences in plants is demonstrated by the above references as well as by Dawson, W. O. et al., Virology (1989) 172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French et al. Science (1986) 231:1294-1297; and Takamatsu et al. FEBS Letters (1990) 269:73-76.
When the virus is a DNA virus, suitable modifications can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat protein which will encapsidate the viral DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all of the constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral RNA.
Construction of plant RNA viruses for the introduction and expression in plants of non-viral exogenous polynucleotide sequences such as those included in the construct of the present invention is demonstrated by the above references as well as in U.S. Pat. No. 5,316,931.
In one embodiment, a plant viral polynucleotide is provided in which the native coat protein coding sequence has been deleted from a viral polynucleotide, a non-native plant viral coat protein coding sequence and a non-native promoter, preferably the subgenomic promoter of the non-native coat protein coding sequence, capable of expression in the plant host, packaging of the recombinant plant viral polynucleotide, and ensuring a systemic infection of the host by the recombinant plant viral polynucleotide, has been inserted. Alternatively, the coat protein gene may be inactivated by insertion of the non-native polynucleotide sequence within it, such that a protein is produced. The recombinant plant viral polynucleotide may contain one or more additional non-native subgenomic promoters. Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or polynucleotide sequences in the plant host and incapable of recombination with each other and with native subgenomic promoters. Non-native (foreign) polynucleotide sequences may be inserted adjacent the native plant viral subgenomic promoter or the native and a non-native plant viral subgenomic promoters if more than one polynucleotide sequence is included. The non-native polynucleotide sequences are transcribed or expressed in the host plant under control of the subgenomic promoter to produce the desired products.
In a second embodiment, a recombinant plant viral polynucleotide is provided as in the first embodiment except that the native coat protein coding sequence is placed adjacent one of the non-native coat protein subgenomic promoters instead of a non-native coat protein coding sequence.
In a third embodiment, a recombinant plant viral polynucleotide is provided in which the native coat protein gene is adjacent its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the viral polynucleotide. The inserted non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in a plant host and are incapable of recombination with each other and with native subgenomic promoters. Non-native polynucleotide sequences may be inserted adjacent the non-native subgenomic plant viral promoters such that the sequences are transcribed or expressed in the host plant under control of the subgenomic promoters to produce the desired product.
In a fourth embodiment, a recombinant plant viral polynucleotide is provided as in the third embodiment except that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.
The viral vectors are encapsidated by the coat proteins encoded by the recombinant plant viral polynucleotide to produce a recombinant plant virus. The recombinant plant viral polynucleotide or recombinant plant virus is used to infect appropriate host plants. The recombinant plant viral polynucleotide is capable of replication in the host, systemic spread in the host, and transcription or expression of foreign gene(s) (exogenous polynucleotide) in the host to produce the desired protein.
Techniques for inoculation of viruses to plants may be found in Foster and Taylor, eds. “Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)”, Humana Press, 1998; Maramorosh and Koprowski, eds. “Methods in Virology” 7 vols, Academic Press, New York 1967-1984; Hill, S. A. “Methods in Plant Virology”, Blackwell, Oxford, 1984; Walkey, D. G. A. “Applied Plant Virology”, Wiley, New York, 1985; and Kado and Agrawa, eds. “Principles and Techniques in Plant Virology”, Van Nostrand-Reinhold, New York.
In addition to the above, the polynucleotide of the present invention can also be introduced into a chloroplast genome thereby enabling chloroplast expression.
A technique for introducing exogenous polynucleotide sequences to the genome of the chloroplasts is known. This technique involves the following procedures. First, plant cells are chemically treated so as to reduce the number of chloroplasts per cell to about one. Then, the exogenous polynucleotide is introduced via particle bombardment into the cells with the aim of introducing at least one exogenous polynucleotide molecule into the chloroplasts. The exogenous polynucleotides selected such that it is integratable into the chloroplast's genome via homologous recombination which is readily effected by enzymes inherent to the chloroplast. To this end, the exogenous polynucleotide includes, in addition to a gene of interest, at least one polynucleotide stretch which is derived from the chloroplast's genome. In addition, the exogenous polynucleotide includes a selectable marker, which serves by sequential selection procedures to ascertain that all or substantially all of the copies of the chloroplast genomes following such selection will include the exogenous polynucleotide. Further details relating to this technique are found in U.S. Pat. Nos. 4,945,050; and 5,693,507 which are incorporated herein by reference. A polypeptide can thus be produced by the protein expression system of the chloroplast and become integrated into the chloroplast's inner membrane.
Since increasing of the oil content, yield, biomass, growth rate and/or vigor in plants can involve multiple genes acting additively or in synergy (see, for example, in Quesda et al., Plant Physiol. 130:951-063, 2002), the invention also envisages expressing a plurality of exogenous polynucleotides in a single host plant to thereby achieve superior increase of oil content, yield, biomass, growth rate and/or vigor in plants.
Expressing a plurality of exogenous polynucleotides in a single host plant can be effected by co-introducing multiple nucleic acid constructs, each including a different exogenous polynucleotide, into a single plant cell. The transformed cell can then be regenerated into a mature plant using the methods described hereinabove.
Alternatively, expressing a plurality of exogenous polynucleotides in a single host plant can be effected by co-introducing into a single plant-cell a single nucleic-acid construct including a plurality of different exogenous polynucleotides. Such a construct can be designed with a single promoter sequence which can transcribe a polycistronic messager RNA including all the different exogenous polynucleotide sequences. To enable co-translation of the different polypeptides encoded by the polycistronic messager RNA, the polynucleotide sequences can be inter-linked via an internal ribosome entry site (IRES) sequence which facilitates translation of polynucleotide sequences positioned downstream of the IRES sequence. In this case, a transcribed polycistronic RNA molecule encoding the different polypeptides described above will be translated from both the capped 5′ end and the two internal IRES sequences of the polycistronic RNA molecule to thereby produce in the cell all different polypeptides. Alternatively, the construct can include several promoter sequences each linked to a different exogenous polynucleotide sequence.
The plant cell transformed with the construct including a plurality of different exogenous polynucleotides, can be regenerated into a mature plant, using the methods described hereinabove.
Alternatively, expressing a plurality of exogenous polynucleotides in a single host plant can be effected by introducing different nucleic acid constructs, including different exogenous polynucleotides into a plurality of plants. The regenerated transformed plants can then be cross-bred and resultant progeny selected for superior oil content, growth rate, biomass, yield and/or vigor, using conventional plant breeding techniques.
Thus, the invention encompasses plants exogenously expressing (as described above) the polynucleotide(s) and/or polypeptide(s) of the invention. Once expressed within the plant cell or the entire plant, the level of the polypeptide encoded by the exogenous polynucleotide can be determined by methods well known in the art such as, activity assays, Western blots using antibodies capable of specifically binding the polypeptide, Enzyme-Linked ImmunoSorbent Assay (ELISA), radio-immuno-assays (RIA), immunohistochemistry, immunofluorescence and the like.
Methods of determining the level in the plant of the RNA transcribed from the exogenous polynucleotide are well known in the art and include, for example, Northern blot analysis, reverse transcription polymerase chain reaction (RT-PCR) analysis (including quantitative, semi-quantitative or real-time RT-PCR) and RNA-in situ hybridization.
The polynucleotides and polypeptides described hereinabove can be used in a wide range of economical plants, in a safe and cost effective manner.
The effect of the transgene (the exogenous polynucleotide encoding the polypeptide) on oil content, plant yield, seed yield, biomass, growth rate and/or vigor can be determined using known methods.
The oil content of a plant can be determined by extraction of the oil from the seed or the vegetative portion of the plant. Briefly, lipids (oil) can be removed from the plant (e.g., seed) by grinding the plant tissue in the presence of specific solvents (e.g., hexane or petroleum ether) and extracting the oil in a continuous extractor. Indirect oil content analysis can be carried out using various known methods such as Nuclear Magnetic Resonance (NMR) Spectroscopy, which measures the resonance energy absorbed by hydrogen atoms in the liquid state of the sample [See for example, Conway T F. and Earle F R., 1963, Journal of the American Oil Chemists' Society; Springer Berlin/Heidelberg, ISSN: 0003-021X (Print) 1558-9331 (Online)]; the Near Infrared (NI) Spectroscopy, which utilizes the absorption of near infrared energy (1100-2500 nm) by the sample; and a method described in WO/2001/023884, which is based on extracting oil a solvent, evaporating the solvent in a gas stream which forms oil particles, and directing a light into the gas stream and oil particles which forms a detectable reflected light. Another method of determining oil content is described in Example 7 of the Examples section which follows.
The plant vigor can be calculated by the increase in growth parameters such as leaf area, rosette diameter, plant fresh weight and the like per time.
The growth rate can be measured using digital analysis of growing plants. For example, images of plants growing in greenhouse on plot basis can be captured every 3 days and the rosette area can be calculated by digital analysis. Rosette area growth is calculated using the difference of rosette area between days of sampling divided by the difference in days between samples.
Measurements of seed yield can be done by collecting the total seeds from 8-16 plants together, weighting them using analytical balance and dividing the total weight by the number of plants. Seed per growing area can be calculated in the same manner while taking into account the growing area given to a single plant. Increase seed yield per growing area could be achieved by increasing seed yield per plant, and/or by increasing number of plants capable of growing in a given area.
Evaluation of the seed yield per plant can be done by measuring the amount (weight or size) or quantity (i.e., number) of dry seeds produced and harvested from 8-16 plants and divided by the number of plants.
Evaluation of growth rate can be done by measuring plant biomass produced, rosette area, leaf size or root length per time (can be measured in cm2 per day of leaf area).
Thus, the present invention is of high agricultural value for promoting the yield of commercially desired crops (e.g., seeds).
Any of the transgenic plants described hereinabove or parts thereof may be processed to produce a feed, meal, protein or oil preparation, such as for ruminant animals.
The transgenic plants described hereinabove, which exhibit an increased oil content can be used to produce plant oil (by extracting the oil from the plant).
The plant oil (including the seed oil and/or the vegetative portion oil) produced according to the method of the invention may be combined with a variety of other ingredients. The specific ingredients included in a product are determined according to the intended use. Exemplary products include animal feed, raw material for chemical modification, biodegradable plastic, blended food product, edible oil, biofuel, cooking oil, lubricant, biodiesel, snack food, cosmetics, and fermentation process raw material. Exemplary products to be incorporated to the plant oil include animal feeds, human food products such as extruded snack foods, breads, as a food binding agent, aquaculture feeds, fermentable mixtures, food supplements, sport drinks, nutritional food bars, multi-vitamin supplements, diet drinks, and cereal foods.
As used herein the term “about” refers to ±10%.
The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
The term “consisting of means “including and limited to”.
The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
EXAMPLES
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., Eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
Example 1
Gene Identification and Gene Role Prediction Using Bioinformatics Tools
Genes encoding polypeptides, suitable for increasing seed oil and seed yield were identified by in-depth analysis of RNA expression profiles, sequence similarities, gene annotations, biochemical pathways, DNA, ESTs, protein and expression databases deposited in the internet.
Bioinformatics Tools
In-Silico Gene Identification
To identify novel genes which could greatly affect seed oil yield, Arabidopsis genes, already found to play key role in embryogenesis, seed development and oil synthesis and accumulation were identified in the literature (‘oil hook genes’—OHGs). OHGs number is according to TAIR website [Hypertext Transfer Protocol://World Wide Web (dot) arabidopsis (dot) org/] and includes all information on the OHGs. OHGs include wild-type alleles of Ssi2 (AT2G43710), OleosinA (AT3G01570), Lec1 (AT1G21970), Lec2 (AT1G28300), Fus3 (AT3G26790), FAD3 (AT2G29980), ABI3 (AT3G24650) and Wri1 (AT3G54320). Comparison of gene expression profile in 79 different developmental stages of Arabidopsis was done on the OHGs genes and all other genes printed on the Nottingham Arabidopsis Stock Centre [(NASC), Hypertext Transfer Protocol://affymetrix (dot) arabidopsis (dot) info/)] micro-arrays describing anatomy, development and various stress experiments. Correlation was determined using the Pearson correlation statistic analysis [Hypertext Transfer Protocol://davidmlane (dot) com/hyperstat/A34739 (dot) html].
The criteria used for each of the genes are described in detail in Table 1 below and cover a variety of biological rationales that use various bioinformatics approaches. The genes were selected to cause changes in seed size and/or seed oil yield based on their highest expression correlation (given as Pearson R values between 0.7<R<1) to one or more of the OHGs. The list of genes identified and their correlation (R value) to each of the OHGs are provided in Table 1, hereinbelow.







TABLE 1







Table 1




















Nucl.
Prot.













SEQ
SEQ


Serial
ID
ID
BDL
TAIR-gene

R

R



R


No
NO:
NO:
No
name
R wri1
abi3
R fus3
oleosin A
R ssi2
R fad3
R lec1
lec2






















1
1
166
  3
AT5G50770

0.891
0.986
0.897
0.791
0.882




2
2
167
  1
AT1G65090

0.995
0.921
0.997
0.715
0.902


3
3
168
  2
AT1G34580






0.955
0.915


4
4
169
  4
AT2G45420
0.933

0.893
0.713
0.74
0.716
0.759
0.76


5
5
170
  5
AT3G14360

0.969
0.96
0.97
0.731
0.914


6
6
171
  6
AT4G10490
0.912

0.88
0.725
0.76
0.71
0.757
0.755


7
7
172
  7
AT5G51490
0.901
0.722
0.92
0.745
0.79
0.797


8
8
173
  8
AT3G03240

0.947
0.982
0.956
0.775
0.912


9
9
174
  9
AT5G24130

0.988
0.917
0.987

0.91


10
10
175
 10
AT5G09640
0.719
0.905
0.98
0.91
0.8
0.908


11
11
176
 11
AT5G12460
0.815





0.969
0.911


12
12
177
 12
AT4G08530






0.931
0.919


13
13
178
 14
AT1G53690
0.931

0.792

0.74


14
14
179
 15
AT1G68510
0.905





0.938
0.913


15
15
180
 16
AT5G03800
0.8
0.878
0.966
0.894
0.797
0.882


16
16
181
 17
AT5G36770






0.922
0.921


17
17
182
 18
AT5G40420

0.997
0.894
0.9996

0.886


18
18
183
 19
AT2G02080

0.702
0.741
0.72
0.748


19
19
184
 20a
AT1G47540.1

0.993
0.915
0.995
0.71
0.892


20
20
185
 20b
AT1G47540.2

0.993
0.915
0.995
0.71
0.892


21
21
186
 21
AT3G62730

0.995
0.92
0.993
0.711
0.903


22
22
187
 22
AT2G27380

0.995
0.873
0.997

0.875


23
23
188
 23
AT3G27785
0.939





0.867
0.81


24
24
189
2991
AT5G15000

0.955
0.959
0.957
0.739
0.902


25
25
190
 25
AT3G20910

0.963
0.943
0.962

0.883


26
26
191
 26a
AT1G11170.1

0.926
0.981
0.929
0.765
0.894


27
27
192
 26b
AT1G11170.2

0.926
0.981
0.929
0.765
0.894


28
28
193
 27
AT1G68380

0.97
0.965
0.977
0.77
0.92


29
29
194
 28
AT1G09380
0.705
0.899
0.95
0.91
0.756
0.897


30
30
195
 29
AT1G60970
0.92
0.709
0.908
0.746
0.78
0.747
0.742
0.745


31
31
196
 30
AT1G72580






0.935
0.917


32
32
197
 31
AT2G28490

0.998
0.871
0.995

0.882


33
33
198
 32a
AT2G46960.1
0.89





0.937
0.9


34
34
199
 32b
AT2G46960.2
0.89





0.937
0.9


35
35
200
 166
AT1G71691
0.938

0.71



0.723
0.713


36
36
201
 330
AT1G73220

0.761
0.755
0.759

0.768


37
37
202
3004
AT5G01790
0.792





0.899
0.85


38
38
203
 333
AT1G71120
0.866





0.925
0.856


39
39
204
 334
AT5G38170
0.937

0.869

0.744

0.81
0.793


40
40
205
 335
AT3G25160
0.88

0.874

0.747
0.761


41
41
206
 336
AT1G18100
0.917

0.851

0.751
0.711


42
42
207
 337
AT2G22620
0.906





0.927
0.888


43
43
208
 339
AT3G26480

0.785
0.717
0.784


44
44
209
 340
AT1G64660

0.872
0.854
0.882

0.808


45
45
210
 341
AT5G52330
0.811





0.796
0.774


46
46
211
 341
AT5G52330
0.811





0.796
0.774


47
47
212
 342
AT1G52670
0.802


48
48
213
 343
AT5G64080

0.923
0.876
0.923

0.92


49
49
214
 343
AT5G64080

0.923
0.876
0.923

0.92


50
50
215
 344
AT2G43060
0.726





0.857
0.794


51
51
216
 345
AT1G27330

0.839
0.856
0.837

0.814


52
52
217
2999
AT2G41340
0.816

0.745

0.744


53
54
219
2810
AT2G13290

0.878
0.76
0.876

0.74


54
55
220
 349
AT4G33670

0.861

0.855

0.734


55
56
221
 350
AT5G04500

0.899
0.702
0.894

0.756


56
64
229
 358
AT3G01570

0.996
0.904
1

0.891


57
65
230
 359
AT2G15010

0.944
0.955
0.942
0.763
0.924


58
66
231
 362
AT2G25940
0.791
0.873
0.977
0.885
0.777
0.873


59
67
232
 364
AT1G04660
0.94

0.882

0.763
0.715
0.777
0.768


60
68
233
 365
AT1G05160
0.945





0.857
0.814


61
69
234
2992
AT1G05280
0.939

0.805



0.859
0.84


62
70
235
2993
AT1G19900

0.975
0.909
0.962

0.898


63
71
236
 368
AT1G23200
0.852





0.957
0.906


64
72
237
 369
AT1G26680
0.93





0.738
0.717


65
73
238
 370
AT1G28590
0.937





0.855
0.813


66
74
239
 371
AT1G48910
0.877
0.753
0.912
0.77
0.808
0.807


67
75
240
2995
AT1G51000
0.906





0.785
0.77


68
76
241
 373
AT1G62340
0.712





0.978
0.903


69
77
242
 374
AT1G62610

0.946
0.909
0.938

0.891


70
78
243
 374
AT1G62610

0.946
0.909
0.938

0.891


71
79
244
 374
AT1G62610

0.946
0.909
0.938

0.891


72
80
245
 375
AT1G76290
0.735
0.91
0.967
0.923
0.803
0.904


73
81
246
 376
AT1G68470
0.917

0.814


74
82
247
 377
AT1G71250
0.922





0.93
0.881


75
83
248
 379
AT3G58200
0.719
0.897
0.973
0.907
0.771
0.914


76
84
249
 380
AT1G78500
0.731
0.844
0.964
0.843
0.788
0.879


77
85
250
 381
AT2G14690






0.972
0.916


78
86
251
 382
AT3G63040

0.949
0.979
0.962
0.783
0.907


79
87
252
 383
AT2G15325






0.978
0.929


80
88
253
 384
AT2G23510
0.804
0.767
0.943
0.777
0.789
0.85


81
89
254
 385
AT2G26070
0.927





0.827
0.762


82
90
255
2997
AT2G28650
0.811

0.711



0.953
0.939


83
91
256
2998
AT2G41290
0.827

0.869

0.779
0.786


84
92
257
 389
AT2G42860
0.903

0.829

0.727

0.825
0.813


85
93
258
 390
AT2G47750
0.906

0.744



0.784
0.754


86
94
259
 391
AT3G03230
0.828
0.844
0.954
0.854
0.783
0.833


87
95
260
 392
AT3G04200
0.912

0.827

0.733


88
96
261
 393
AT3G21840
0.702





0.968
0.928


89
97
262
3000
AT3G22640

0.995
0.855
0.991

0.873


90
98
263
 395
AT3G49380
0.919

0.724



0.843
0.784


91
99
264
3001
AT4G03050
0.93

0.847

0.749


92
100
265
3001
AT4G03050
0.93

0.847

0.749


93
101
266
3003
AT4G19380
0.783
0.792
0.913
0.803
0.826
0.839


94
102
267
 398
AT4G27460

0.992
0.896
0.985

0.897


95
103
268
 399
AT4G33280
0.885
0.715
0.912
0.732
0.811
0.8


96
104
269
 400
AT4G33600
0.917





0.908
0.851


97
105
270
 401
AT5G07260
0.956

0.82

0.73


98
106
271
3007
AT5G08460
0.955

0.768

0.702

0.757
0.747


99
107
272
 403
AT2G34700
0.932

0.903

0.783
0.741


100
108
273
 404
AT5G15740
0.911

0.712



0.883
0.818


101
109
274
 405
AT5G16230
0.812
0.82
0.961
0.834
0.773
0.858


102
110
275
 406
AT5G18290
0.905

0.722



0.821
0.803


103
111
276
2814
AT5G25470
0.901





0.748
0.711


104
112
277
 408
AT5G39130
0.951

0.726



0.769
0.75


105
113
278
 409
AT5G39160
0.94

0.729



0.829
0.789


106
114
279
 409
AT5G39160
0.94

0.729



0.829
0.789


107
115
280
 410
AT5G39190
0.951

0.795

0.706

0.754
0.737


108
116
281
 411
AT5G44360
0.828
0.833
0.975
0.855
0.804
0.849


109
117
282
 412
AT5G47670
0.957





0.797
0.759


110
118
283
3008
AT5G49820
0.905



0.715


111
119
284
 414
AT5G56300
0.936

0.823

0.717

0.712


112
120
285
 416
AT5G59170

0.995
0.852
0.991

0.87


113
121
286
 418
AT1G28640

0.967
0.949
0.975
0.752
0.92


114
122
287
 419
AT1G22990

0.789
0.889
0.794

0.738


115
123
288
2816a
AT1G64110.1

0.883

0.869

0.701


116
124
289
2816b
AT1G64110.2

0.883

0.869

0.701


117
125
290
 421
AT1G04380
0.971

0.798

0.717

0.772
0.749


118
126
291
2817
AT1G08810

0.888
0.948
0.885
0.831
0.862


119
127
292
2817
AT1G08810

0.888
0.948
0.885
0.831
0.862


120
128
293
 423
AT1G28170






0.962
0.903


121
129
294
 424
AT1G28650
0.821
0.843
0.974
0.853
0.801
0.844


122
130
295
 425
AT3G10590






0.969
0.944


123
131
296
 426
AT3G58740
0.948

0.842

0.745


124
132
297
 427
AT4G02360

0.941
0.941
0.937
0.731
0.915


125
133
298
 428
AT4G36700

0.965
0.967
0.976
0.768
0.899


126
134
299
 429
AT5G07200
0.957

0.851

0.753

0.725
0.71


127
135
300
 430
AT5G22810
0.958

0.702



0.86
0.834


128
136
301
 431
AT5G43860

0.866
0.916
0.868
0.776
0.817


129
137
302
 432
AT5G57390

0.989
0.914
0.987
0.713
0.916


130
138
303
 433
AT5G62800

0.961
0.967
0.962
0.769
0.913


131
139
304
 435
AT5G52500






0.956
0.876


132
140
305
 436
AT5G24600

0.956
0.902
0.954

0.863


133
141
306
2818
AT2G23550
0.829





0.928
0.839


134
142
307
2818
AT2G23550
0.829





0.928
0.839


135
146
311
 441
AT5G48100
0.737
0.923
0.95
0.944
0.761
0.864


136
147
312
 442
AT1G14760
0.708
0.874
0.93
0.876
0.831
0.877


137
148
313
 443
AT1G15150
0.871





0.971
0.92


138
149
314
 444
AT1G20500
0.92

0.783



0.904
0.874


139
150
315
 445
AT1G56170
0.966

0.751



0.782
0.751


140
151
316
2996
AT1G62070
0.956





0.847
0.797


141
152
317
 447
AT1G67100

0.967
0.969
0.973
0.761
0.914


142
153
318
 448
AT3G21090
0.902

0.724


143
154
319
 449
AT3G24250
0.826





0.986
0.931


144
155
320
 450
AT3G50990
0.715





0.982
0.914


145
156
321
 451
AT4G00220
0.905
0.741
0.923
0.773
0.782
0.779
0.7
0.703


146
157
322
 452
AT4G10150
0.706
0.875
0.95
0.883
0.821
0.886


147
158
323
3006
AT5G07190

0.998
0.903
0.997

0.901


148
159
324
3006
AT5G07190

0.998
0.903
0.997

0.901


149
160
325
 455
AT5G10220
0.722





0.984
0.917


150
161
326
 456
AT5G20940

0.969
0.901
0.961

0.901


151
162
327
 457
AT5G51210
0.907
0.7
0.925
0.734
0.788
0.752
0.702
0.7


152
163
328
 458
AT5G55620
0.704
0.769
0.898
0.776
0.871
0.8


153
164
329
 459
AT5G60460

0.987
0.931
0.988

0.902


154
165
330
 460
AT5G65590
0.793
0.725
0.882
0.754
0.783
0.77


155
332
351
2991
AT5G15000

0.955
0.959
0.957
0.739
0.902


156
333
352
2992
AT1G05280
0.939

0.805



0.859
0.84


157
334
353
2993
AT1G19900

0.975
0.909
0.962

0.898


158
336
355
2995
AT1G51000
0.906





0.785
0.77


159
337
356
2996
AT1G62070
0.956





0.847
0.797


160
338
357
2997
AT2G28650
0.811

0.711



0.953
0.939


161
339
358
2998
AT2G41290
0.827

0.869

0.779
0.786


162
340
359
2999
AT2G41340
0.816

0.745

0.744


163
341
360
3000
AT3G22640

0.995
0.855
0.991

0.873


164
342
361
3001
AT4G03050
0.93

0.847

0.749


165
344
363
3003
AT4G19380
0.783
0.792
0.913
0.803
0.826
0.839


166
345
364
3004
AT5G01790
0.792





0.899
0.85


167
347
366
3006
AT5G07190

0.998
0.903
0.997

0.901


168
348
367
3007
AT5G08460
0.955

0.768

0.702

0.757
0.747


169
349
368
3008
AT5G49820
0.905



0.715









Additional genes which are predicted to affect seed oil synthesis and which were identified using bioinformatics tools are provided in Table 2, below.







TABLE 2







Table 2












Polynucleotide
Polypeptide




Serial No
SEQ ID NO:
SEQ ID NO:
BDL No.
TAIR-gene name














1
53
218
3005
AT5G03450.1


2
57
222
351
AT1G27120.1


3
58
223
352
AT5G01820.1


4
59
224
353
AT2G32780.1


5
60
225
354
AT3G16490.1


6
61
226
355
AT5G23050.1


7
62
227
3002
AT4G16050.1


8
63
228
2994
AT1G44760.1


9
143
308
438
AT1G72040


10
144
309
439
AT1G53070


11
145
310
440
AT1G50510


12
331
350
2990
AT5G14995


13
335
354
2994
AT1G44760


14
343
362
3002
AT4G16050


15
346
365
3005
AT5G03450









Example 2
Production of Arabidopsis Transcriptom and High Throughput Correlation Analysis Using 44K Arabidopsis Full Genome Oligonucleotide Micro-Array
In order to produce a high throughput correlation analysis, the present inventors utilized an Arabidopsis thaliana oligonucleotide micro-array, produced by Agilent Technologies [Hypertext Transfer Protocol://World Wide Web (dot) chem. (dot) agilent (dot) com/Scripts/PDS (dot) asp?1 Page=50879]. The array oligonucleotide represents about 40,000 A. thaliana genes and transcripts designed based on data from the TIGR ATH1 v.5 database and Arabidopsis MPSS (University of Delaware) databases. In order to define correlations between the levels of RNA expression and yield components or vigor related parameters, various plant characteristics of 15 different Arabidopsis ecotypes were analyzed. Among them, nine ecotypes encompassing the observed variance were selected for RNA expression analysis. The correlation between the RNA levels and the characterized parameters was analyzed using Pearson correlation test [Hypertext Transfer Protocol://World Wide Web (dot) davidmlane (dot) com/hyperstat/A34739 (dot) html].
Experimental Procedures
RNA Extraction
Five tissues at different developmental stages [root, leaf, flower at anthesis, seed at 5 days after flowering (DAF) and seed at 12 DAF], representing different plant characteristics, were sampled and RNA was extracted using TRIzol Reagent from Invitrogen [Hypertext Transfer Protocol://World Wide Web (dot) invitrogen (dot) com/content (dot)cfm?pageid=469]. For convenience, each micro-array expression information tissue type has received a Set ID as summarized in Table 3 below.







TABLE 3







Table 3


Arabidopsis transcriptom experimental sets










Expression Set
Set ID







Root
A



Leaf
B



Flower
C



Seed 5 DAF
D



Seed 12 DAF
E










Approximately 30-50 mg of tissue was taken from samples. The weighed tissues were ground using pestle and mortar in liquid nitrogen and resuspended in 500 μl of TRIzol Reagent. To the homogenized lysate, 100 μl of chloroform was added followed by precipitation using isopropanol and two washes with 75% ethanol. The RNA was eluted in 30 μl of RNase-free water. RNA samples were cleaned up using Qiagen's RNeasy minikit clean-up protocol as per the manufacturer's protocol.
Yield Component and Vigor Related Parameters Assessment
8 Arabidopsis ecotypes in 5 repetitive blocks (named A, B, C, D and E), each containing 20 plants per plot were grown at control conditions greenhouse 22° C., 20:20:20 (weight ratios) N:P:K [nitrogen (N), phosphorus (P) and potassium (K)] fertilizer was added. During this time data was collected documented and analyzed. Additional data was collected through the seedling stage of plants grown at tissue culture in vertical grown transparent agar plates. Data parameters collected are summarized in Table 4, below.







TABLE 4







Table 4


Arabidopsis correlated parameters (vectors)








Correlated parameter with
Correlation Id











Root length day 13 (cm)
1


Root length day 7 (cm)
2


Relative root growth (cm/day) day 13
3


Fresh weight per plant (gr) at bolting stage
4


Dry matter per plant (gr)
5


Vegetative growth rate (cm2/day) till 8 true leaves
6


Blade circularity
7


Lamina width (cm)
8


Lamina length (cm)
9


Total leaf area per plant (cm)
10


1000 Seed weight (gr)
11


Oil % per seed
12


Seeds per silique
13


Silique length (cm)
14


Seed yield per plant (gr)
15


Oil yield per plant (mg)
16


Harvest Index
17


Leaf width/length
18









Most of chosen parameters were analyzed by digital imaging.
Digital Imaging
A laboratory image acquisition system, which consists of a digital reflex camera (Canon EOS 300D) attached with a 55 mm focal length lens (Canon EF-S series), mounted on a reproduction device (Kaiser R S), which included 4 light units (4×150 Watts light bulb) and located in a darkroom, was used for capturing images of plantlets sawn in square agar plates.
The image capturing process was repeated every 2 days starting at day 7 till day 14. The same camera attached with a 24 mm focal length lens (Canon EF series), placed in a custom made iron mount, was used for capturing images of larger plants sawn in white tubs in an environmental controlled greenhouse (as seen on FIG. 2b). The white tubs were square shape with measurements of 36×26.2 cm and 7.5 cm deep. During the capture process, the tubs were placed beneath the iron mount, while avoiding direct sun light and casting of shadows. This process was repeated every 3-4 days for up to 30 days.
An image analysis system was used, which consists of a personal desktop computer (Intel P4 3.0 GHz processor) and a public domain program—ImageJ 1.37 (Java based image processing program which was developed at the U.S National Institutes of Health and freely available on the internet at Hypertext Transfer Protocol://rsbweb (dot) nih (dot) gov/). Images were captured in resolution of 6 Mega Pixels (3072×2048 pixels) and stored in a low compression JPEG (Joint Photographic Experts Group standard) format. Next, analyzed data was saved to text files and processed using the JMP statistical analysis software (SAS institute).
Leaf Analysis
Using the digital analysis leaves data was calculated, including leaf number, area, perimeter, length and width. On day 30, 3-4 representative plants were chosen from each plot of blocks A, B and C. The plants were dissected, each leaf was separated and was introduced between two glass trays, a photo of each plant was taken and the various parameters (such as leaf total area, laminar length etc.) were calculated from the images (FIGS. 1a-d). The blade circularity was calculated as laminar width divided by laminar length.
Root Analysis
During 17 days, the different ecotypes were grown in transparent agar plates. The plates were photographed every 2 days starting at day 7 in the photography room and the roots development was documented (FIGS. 2a-b).
The growth rate was calculated according to the following formula I.

Relative growth area rate=(ΔArea/Δt)*(1/Area t0)  Formula I

Δt is the current analyzed image day subtracted from the initial day (t−t0).

Thus, the relative growth area rate is in units of 1/day and length growth rate is in units of 1/day.

Vegetative Growth Rate Analysis
The growth rate was calculated by dividing the area added (ΔArea) by the number of days for each interval (Δt). The analysis was ended with the appearance of overlapping plants.
The growth rate was calculated according to formula II.

Growth rate=ΔArea/Δt.  Formula II

For comparison between ecotypes the calculated rate was normalized using plant developmental stage as represented by the number of true leaves. In cases where plants with 8 leaves had been sampled twice (for example at day 10 and day 13), only the largest sample was chosen and added to the Anova comparison.
Seeds in Siliques Analysis
On day 70, 15-17 siliques were collected from each plot in blocks D and E. The chosen siliques were light brown color but still intact. The siliques were opened in the photography room and the seeds were scatter on a glass tray, a high resolution digital picture was taken for each plot. Using the images the number of seeds per silique was determined.
Seeds Average Weight
At the end of the experiment all seeds from plots A-C were collected. An average weight of 0.02 grams was measured from each sample, the seeds were scattered on a glass tray and a picture was taken. Using the digital analysis, the number of seeds in each sample was calculated.
Oil Percentage in Seeds
At the end of the experiment all seeds from plots A-C were collected. Columbia seeds from 3 plots were mixed grounded and then mounted onto the extraction chamber. 210 ml of n-Hexane (Cat No. 080951 Biolab Ltd.) were used as the solvent. The extraction was performed for 30 hours at medium heat 50° C. Once the extraction has ended the n-Hexane was evaporated using the evaporator at 35° C. and vacuum conditions. The process was repeated twice. The information gained from the Soxhlet extractor (Soxhlet, F. Die gewichtsanalytische Bestimmung des Milchfettes, Polytechnisches J. (Dingler's) 1879, 232, 461) was used to create a calibration curve for the Low Resonance NMR. The content of oil of all seed samples was determined using the Low Resonance NMR (MARAN Ultra-Oxford Instrument) and its MultiQuant software package.
Silique Length Analysis
On day 50 from sowing, 30 siliques from different plants in each plot were sampled in block A. The chosen siliques were green-yellow in color and were collected from the bottom parts of a grown plant's stem. A digital photograph was taken to determine silique's length.
Dry weight and seed yield—On day 80 from sowing, the plants from blocks A-C were harvested and left to dry at 30° C. in a drying chamber. The biomass and seed weight of each plot was separated, measured and divided by the number of plants. Dry weight=total weight of the vegetetative portion above ground (excluding roots) after drying at 30° C. in a drying chamber; Seed yield per plant=total seed weight per plant (gr).
Oil Yield
The oil yield was calculated using Formula III.

Seed Oil yield=Seed yield per plant (gr)*Oil % in seed  Formula III

Harvest Index
The harvest index was calculated using Formula IV.

Harvest Index=Average seed yield per plant/Average dry weight  Formula IV

Experimental Results
Nine different Arabidopsis ecotypes were grown and characterized for 18 parameters (named as vectors). The characterized values are summarized in Tables 5 and 6 below.







TABLE 5







Table 5


Arabidopsis ecotypes, measured parameters

















Seed
Oil


Dry

Total





yield
yield

1000
matter

leaf area



per
per

Seed
per

per
Seeds
Silique



plant
plant
Oil %
weight
plant
Harvest
plant
per
length


Ecotype
(gr)
(mg)
per seed
(gr)
(gr)
Index
(cm)
silique
(cm)



















An-1
0.34
118.63
34.42
0.0203
0.64
0.53
46.86
45.44
1.06


Col-0
0.44
138.73
31.19
0.0230
1.27
0.35
109.89
53.47
1.26


Ct-1
0.59
224.06
38.05
0.0252
1.05
0.56
58.36
58.47
1.31


Cvi
0.42
116.26
27.76
0.0344
1.28
0.33
56.80
35.27
1.47


(N8580)


Gr-6
0.61
218.27
35.49
0.0202
1.69
0.37
114.66
48.56
1.24


Kondara
0.43
142.11
32.91
0.0263
1.34
0.32
110.82
37.00
1.09


Ler-1
0.36
114.15
31.56
0.0205
0.81
0.45
88.49
39.38
1.18


Mt-0
0.62
190.06
30.79
0.0226
1.21
0.51
121.79
40.53
1.18


Shakdara
0.55
187.62
34.02
0.0235
1.35
0.41
93.04
25.53
1.00
















TABLE 6







Table 6


Arabidopsis ecotypes, additional measured parameters





















Fresh







Vegetative
Relative


weight



growth
root


per



rate
growth
Root
Root
plant



(cm2/day)
(cm/
length
length
(gr) at
Lamina
Lamina
Leaf



till 8 true
day)
day 7
day 13
bolting
length
width
width/
Blade


Ecotype
leaves
day 13
(cm)
(cm)
stage
(cm)
(cm)
length
circularity





An-1
0.313
0.631
0.937
4.419
1.510
2.767
1.385
0.353
0.509


Col-0
0.378
0.664
1.759
8.530
3.607
3.544
1.697
0.288
0.481


Ct-1
0.484
1.176
0.701
5.621
1.935
3.274
1.460
0.316
0.450


Cvi
0.474
1.089
0.728
4.834
2.082
3.785
1.374
0.258
0.370


(N8580)


Gr-6
0.425
0.907
0.991
5.957
3.556
3.690
1.828
0.356
0.501


Kondara
0.645
0.774
1.163
6.372
4.338
4.597
1.650
0.273
0.376


Ler-1
0.430
0.606
1.284
5.649
3.467
3.877
1.510
0.305
0.394


Mt-0
0.384
0.701
1.414
7.060
3.479
3.717
1.817
0.335
0.491


Shakdara
0.471
0.782
1.251
7.041
3.710
4.149
1.668
0.307
0.409









The selected genes, their R (calculated using Pearson correlation), the characterized parameters used as x axis for correlation and the tissue transcriptom correlated with are summarized in Table 7, below.







TABLE 7







Table 7


Arabidopsis selected genes and their correlation with yield components among


different transcriptom sets















Nucleotide
Polypeptide








SEQ ID
SEQ ID


Exp.
Correl.



NO:
NO:
Gene Name
Cluster Name
Set
Vector
R


















1
3
168
BDL2
arabidopsis|6|AT1G34580
B
8
0.77


2
3
168
BDL2
arabidopsis|6|AT1G34580
D
15
0.75


3
3
168
BDL2
arabidopsis|6|AT1G34580
D
16
0.71


4
6
171
BDL6
arabidopsis|6|AT4G10490
E
12
−0.7


5
7
172
BDL7
arabidopsis|6|AT5G51490
A
15
0.76


6
7
172
BDL7
arabidopsis|6|AT5G51490
A
16
0.74


7
7
172
BDL7
arabidopsis|6|AT5G51490
B
4
−0.78


8
7
172
BDL7
arabidopsis|6|AT5G51490
B
9
−0.77


9
7
172
BDL7
arabidopsis|6|AT5G51490
B
10
−0.73


10
7
172
BDL7
arabidopsis|6|AT5G51490
B
17
0.88


11
8
173
BDL8
arabidopsis|6|AT3G03240
D
15
0.87


12
8
173
BDL8
arabidopsis|6|AT3G03240
D
16
0.89


13
9
174
BDL9
arabidopsis|6|AT5G24130
D
15
0.75


14
9
174
BDL9
arabidopsis|6|AT5G24130
D
16
0.75


15
9
174
BDL9
arabidopsis|6|AT5G24130
E
13
0.75


16
10
175
BDL10
arabidopsis|6|AT5G09640
E
11
0.72


17
13
178
BDL14
arabidopsis|6|AT1G53690
B
11
0.87


18
13
178
BDL14
arabidopsis|6|AT1G53690
B
12
−0.71


19
13
178
BDL14
arabidopsis|6|AT1G53690
B
14
0.71


20
13
178
BDL14
arabidopsis|6|AT1G53690
E
11
0.72


21
14
179
BDL15
arabidopsis|6|AT1G68510
E
15
0.72


22
16
181
BDL17
arabidopsis|6|AT5G36770
D
15
0.75


23
18
183
BDL19
arabidopsis|6|AT2G02080
C
16
0.7


24
18
183
BDL19
arabidopsis|6|AT2G02080
D
17
0.72


25
19
184
BDL20a
arabidopsis|6|AT1G47540
A
11
0.85


26
20
185
BDL20b
arabidopsis|6|AT1G47540
A
11
0.85


27
21
186
BDL21
arabidopsis|6|AT3G62730
D
17
0.8


28
21
186
BDL21
arabidopsis|6|AT3G62730
E
11
0.79


29
21
186
BDL21
arabidopsis|6|AT3G62730
E
14
0.79


30
22
187
BDL22
arabidopsis|6|AT2G27380
A
11
0.81


31
22
187
BDL22
arabidopsis|6|AT2G27380
A
12
−0.75


32
23
188
BDL23
arabidopsis|6|AT3G27785
E
11
0.7


33
23
188
BDL23
arabidopsis|6|AT3G27785
E
12
−0.86


34
23
188
BDL23
arabidopsis|6|AT3G27785
E
14
0.71


35
25
190
BDL25
arabidopsis|6|AT3G20910
A
5
0.77


36
25
190
BDL25
arabidopsis|6|AT3G20910
A
8
0.7


37
25
190
BDL25
arabidopsis|6|AT3G20910
B
12
0.72


38
25
190
BDL25
arabidopsis|6|AT3G20910
B
16
0.75


39
25
190
BDL25
arabidopsis|6|AT3G20910
C
15
0.77


40
25
190
BDL25
arabidopsis|6|AT3G20910
C
16
0.81


41
25
190
BDL25
arabidopsis|6|AT3G20910
D
12
0.77


42
25
190
BDL25
arabidopsis|6|AT3G20910
D
15
0.73


43
25
190
BDL25
arabidopsis|6|AT3G20910
D
16
0.8


44
26
191
BDL26a
arabidopsis|6|AT1G11170
C
15
−0.77


45
27
192
BDL26b
arabidopsis|6|AT1G11170
C
15
−0.77


46
28
193
BDL27
arabidopsis|6|AT1G68380
A
13
−0.71


47
28
193
BDL27
arabidopsis|6|AT1G68380
C
13
−0.75


48
28
193
BDL27
arabidopsis|6|AT1G68380
E
11
0.71


49
28
193
BDL27
arabidopsis|6|AT1G68380
E
14
0.74


50
29
194
BDL28
arabidopsis|6|AT1G09380
C
11
0.87


51
29
194
BDL28
arabidopsis|6|AT1G09380
C
12
−0.79


52
29
194
BDL28
arabidopsis|6|AT1G09380
C
14
0.73


53
29
194
BDL28
arabidopsis|6|AT1G09380
E
15
0.83


54
29
194
BDL28
arabidopsis|6|AT1G09380
E
16
0.8


55
30
195
BDL29
arabidopsis|6|AT1G60970
B
9
−0.74


56
30
195
BDL29
arabidopsis|6|AT1G60970
C
11
0.76


57
30
195
BDL29
arabidopsis|6|AT1G60970
D
12
0.87


58
30
195
BDL29
arabidopsis|6|AT1G60970
D
15
0.88


59
30
195
BDL29
arabidopsis|6|AT1G60970
D
16
0.93


60
30
195
BDL29
arabidopsis|6|AT1G60970
E
11
0.8


61
32
197
BDL31
arabidopsis|6|AT2G28490
A
11
0.85


62
32
197
BDL31
arabidopsis|6|AT2G28490
A
12
−0.74


63
32
197
BDL31
arabidopsis|6|AT2G28490
A
14
0.71


64
35
200
BDL166
arabidopsis|6|AT1G71691
D
12
0.78


65
35
200
BDL166
arabidopsis|6|AT1G71691
D
17
0.72


66
36
201
BDL_unnamed_330
arabidopsis|6|AT1G73220
B
6
0.8


67
36
201
BDL_unnamed_330
arabidopsis|6|AT1G73220
C
12
−0.78


68
36
201
BDL_unnamed_330
arabidopsis|6|AT1G73220
C
17
−0.77


69
36
201
BDL_unnamed_330
arabidopsis|6|AT1G73220
D
17
−0.76


70
37
202
BDL_unnamed_331
arabidopsis|6|AT5G01790
B
5
0.85


71
37
202
BDL_unnamed_331
arabidopsis|6|AT5G01790
E
14
0.72


72
38
203
BDL_unnamed_333
arabidopsis|6|AT1G71120
B
12
−0.77


73
38
203
BDL_unnamed_333
arabidopsis|6|AT1G71120
B
14
0.77


74
38
203
BDL_unnamed_333
arabidopsis|6|AT1G71120
E
11
0.82


75
38
203
BDL_unnamed_333
arabidopsis|6|AT1G71120
E
14
0.88


76
39
204
BDL_unnamed_334
arabidopsis|6|AT5G38170
D
15
0.82


77
39
204
BDL_unnamed_334
arabidopsis|6|AT5G38170
D
16
0.81


78
39
204
BDL_unnamed_334
arabidopsis|6|AT5G38170
E
11
0.87


79
39
204
BDL_unnamed_334
arabidopsis|6|AT5G38170
E
12
−0.75


80
39
204
BDL_unnamed_334
arabidopsis|6|AT5G38170
E
14
0.79


81
40
205
BDL_unnamed_335
arabidopsis|6|AT3G25160
A
1
−0.89


82
40
205
BDL_unnamed_335
arabidopsis|6|AT3G25160
A
2
−0.76


83
40
205
BDL_unnamed_335
arabidopsis|6|AT3G25160
E
11
0.71


84
42
207
BDL_unnamed_337
arabidopsis|6|AT2G22620
A
13
−0.76


85
42
207
BDL_unnamed_337
arabidopsis|6|AT2G22620
E
15
0.86


86
42
207
BDL_unnamed_337
arabidopsis|6|AT2G22620
E
16
0.79


87
43
208
BDL_unnamed_339
arabidopsis|6|AT3G26480
A
11
0.84


88
43
208
BDL_unnamed_339
arabidopsis|6|AT3G26480
A
14
0.73


89
43
208
BDL_unnamed_339
arabidopsis|6|AT3G26480
C
11
0.76


90
43
208
BDL_unnamed_339
arabidopsis|6|AT3G26480
C
14
0.88


91
44
209
BDL_unnamed_340
arabidopsis|6|AT1G64660
A
1
0.83


92
44
209
BDL_unnamed_340
arabidopsis|6|AT1G64660
A
2
0.7


93
46
211
BDL_unnamed_341
arabidopsis|6|AT5G52330
E
17
0.85


94
49
214
BDL_unnamed_343
arabidopsis|6|AT5G64080
C
12
0.74


95
49
214
BDL_unnamed_343
arabidopsis|6|AT5G64080
C
16
0.77


96
50
215
BDL_unnamed_344
arabidopsis|6|AT2G43060
B
11
0.89


97
50
215
BDL_unnamed_344
arabidopsis|6|AT2G43060
B
12
−0.73


98
50
215
BDL_unnamed_344
arabidopsis|6|AT2G43060
B
18
−0.81


99
50
215
BDL_unnamed_344
arabidopsis|6|AT2G43060
E
15
0.8


100
52
217
BDL_unnamed_346
arabidopsis|6|AT2G41340
A
13
−0.72


101
52
217
BDL_unnamed_346
arabidopsis|6|AT2G41340
B
5
0.72


102
52
217
BDL_unnamed_346
arabidopsis|6|AT2G41340
B
8
0.81


103
53
218
BDL_unnamed_347
arabidopsis|6|AT5G03450
A
3
0.76


104
53
218
BDL_unnamed_347
arabidopsis|6|AT5G03450
A
5
0.74


105
53
218
BDL_unnamed_347
arabidopsis|6|AT5G03450
A
15
0.74


106
53
218
BDL_unnamed_347
arabidopsis|6|AT5G03450
D
15
0.78


107
53
218
BDL_unnamed_347
arabidopsis|6|AT5G03450
D
16
0.82


108
55
220
BDL_unnamed_349
arabidopsis|6|AT4G33670
A
5
0.74


109
55
220
BDL_unnamed_349
arabidopsis|6|AT4G33670
A
15
0.78


110
55
220
BDL_unnamed_349
arabidopsis|6|AT4G33670
A
16
0.73


111
55
220
BDL_unnamed_349
arabidopsis|6|AT4G33670
B
5
0.86


112
56
221
BDL_unnamed_350
arabidopsis|6|AT5G04500
A
13
−0.72


113
56
221
BDL_unnamed_350
arabidopsis|6|AT5G04500
C
15
0.85


114
56
221
BDL_unnamed_350
arabidopsis|6|AT5G04500
C
16
0.83


115
56
221
BDL_unnamed_350
arabidopsis|6|AT5G04500
E
11
−0.72


116
56
221
BDL_unnamed_350
arabidopsis|6|AT5G04500
E
12
0.73


117
56
221
BDL_unnamed_350
arabidopsis|6|AT5G04500
E
17
0.74


118
57
222
BDL_unnamed_351
arabidopsis|6|AT1G27120
B
7
0.78


119
57
222
BDL_unnamed_351
arabidopsis|6|AT1G27120
B
13
0.74


120
57
222
BDL_unnamed_351
arabidopsis|6|AT1G27120
C
15
0.79


121
57
222
BDL_unnamed_351
arabidopsis|6|AT1G27120
C
16
0.82


122
57
222
BDL_unnamed_351
arabidopsis|6|AT1G27120
D
17
0.74


123
58
223
BDL_unnamed_352
arabidopsis|6|AT5G01820
B
4
−0.71


124
58
223
BDL_unnamed_352
arabidopsis|6|AT5G01820
B
8
−0.7


125
58
223
BDL_unnamed_352
arabidopsis|6|AT5G01820
C
15
−0.74


126
58
223
BDL_unnamed_352
arabidopsis|6|AT5G01820
E
16
0.71


127
60
225
BDL_unnamed_354
arabidopsis|6|AT3G16490
C
16
0.73


128
61
226
BDL_unnamed_355
arabidopsis|6|AT5G23050
D
12
0.72


129
62
227
BDL_unnamed_356
arabidopsis|6|AT4G16050
E
11
0.95


130
62
227
BDL_unnamed_356
arabidopsis|6|AT4G16050
E
14
0.77


131
63
228
BDL_unnamed_357
arabidopsis|6|AT1G44760
B
15
0.73


132
63
228
BDL_unnamed_357
arabidopsis|6|AT1G44760
B
16
0.7


133
64
229
BDL_unnamed_358
arabidopsis|6|AT3G01570
C
16
0.71


134
66
231
BDL_unnamed_362
arabidopsis|6|AT2G25940
B
15
0.83


135
66
231
BDL_unnamed_362
arabidopsis|6|AT2G25940
B
16
0.84


136
67
232
BDL_unnamed_364
arabidopsis|6|AT1G04660
D
12
0.88


137
67
232
BDL_unnamed_364
arabidopsis|6|AT1G04660
D
15
0.84


138
67
232
BDL_unnamed_364
arabidopsis|6|AT1G04660
D
16
0.91


139
68
233
BDL_unnamed_365
arabidopsis|6|AT1G05160
C
16
0.71


140
68
233
BDL_unnamed_365
arabidopsis|6|AT1G05160
D
15
0.72


141
68
233
BDL_unnamed_365
arabidopsis|6|AT1G05160
D
16
0.72


142
70
235
BDL_unnamed_367
arabidopsis|6|AT1G19900
B
6
0.8


143
70
235
BDL_unnamed_367
arabidopsis|6|AT1G19900
C
12
−0.86


144
70
235
BDL_unnamed_367
arabidopsis|6|AT1G19900
C
14
0.73


145
70
235
BDL_unnamed_367
arabidopsis|6|AT1G19900
E
15
0.71


146
71
236
BDL_unnamed_368
arabidopsis|6|AT1G23200
D
13
−0.78


147
71
236
BDL_unnamed_368
arabidopsis|6|AT1G23200
E
17
−0.73


148
72
237
BDL_unnamed_369
arabidopsis|6|AT1G26680
A
1
0.84


149
72
237
BDL_unnamed_369
arabidopsis|6|AT1G26680
A
2
0.75


150
73
238
BDL_unnamed_370
arabidopsis|6|AT1G28590
E
11
0.9


151
73
238
BDL_unnamed_370
arabidopsis|6|AT1G28590
E
12
−0.72


152
74
239
BDL_unnamed_371
arabidopsis|6|AT1G48910
B
12
0.72


153
74
239
BDL_unnamed_371
arabidopsis|6|AT1G48910
B
15
0.79


154
74
239
BDL_unnamed_371
arabidopsis|6|AT1G48910
B
16
0.86


155
74
239
BDL_unnamed_371
arabidopsis|6|AT1G48910
C
17
0.79


156
79
244
BDL_unnamed_374
arabidopsis|6|AT1G62610
D
15
−0.74


157
80
245
BDL_unnamed_375
arabidopsis|6|AT1G76290
B
16
0.72


158
80
245
BDL_unnamed_375
arabidopsis|6|AT1G76290
C
17
0.77


159
81
246
BDL_unnamed_376
arabidopsis|6|AT1G68470
B
4
0.76


160
81
246
BDL_unnamed_376
arabidopsis|6|AT1G68470
B
5
0.77


161
81
246
BDL_unnamed_376
arabidopsis|6|AT1G68470
B
8
0.96


162
81
246
BDL_unnamed_376
arabidopsis|6|AT1G68470
B
10
0.89


163
81
246
BDL_unnamed_376
arabidopsis|6|AT1G68470
C
15
0.83


164
81
246
BDL_unnamed_376
arabidopsis|6|AT1G68470
C
16
0.74


165
81
246
BDL_unnamed_376
arabidopsis|6|AT1G68470
D
13
−0.81


166
81
246
BDL_unnamed_376
arabidopsis|6|AT1G68470
D
14
−0.82


167
82
247
BDL_unnamed_377
arabidopsis|6|AT1G71250
E
11
0.72


168
82
247
BDL_unnamed_377
arabidopsis|6|AT1G71250
E
14
0.8


169
82
247
BDL_unnamed_377
arabidopsis|6|AT1G71250
E
17
−0.7


170
83
248
BDL_unnamed_379
arabidopsis|6|AT3G58200
B
6
0.75


171
84
249
BDL_unnamed_380
arabidopsis|6|AT1G78500
A
1
−0.74


172
84
249
BDL_unnamed_380
arabidopsis|6|AT1G78500
B
7
0.75


173
84
249
BDL_unnamed_380
arabidopsis|6|AT1G78500
B
18
0.84


174
85
250
BDL_unnamed_381
arabidopsis|6|AT2G14690
E
15
−0.72


175
88
253
BDL_unnamed_384
arabidopsis|6|AT2G23510
B
12
0.74


176
88
253
BDL_unnamed_384
arabidopsis|6|AT2G23510
B
15
0.71


177
88
253
BDL_unnamed_384
arabidopsis|6|AT2G23510
B
16
0.8


178
89
254
BDL_unnamed_385
arabidopsis|6|AT2G26070
B
15
0.91


179
89
254
BDL_unnamed_385
arabidopsis|6|AT2G26070
B
16
0.88


180
90
255
BDL_unnamed_386
arabidopsis|6|AT2G28650
D
13
−0.93


181
90
255
BDL_unnamed_386
arabidopsis|6|AT2G28650
D
14
−0.87


182
90
255
BDL_unnamed_386
arabidopsis|6|AT2G28650
E
15
0.7


183
91
256
BDL_unnamed_388
arabidopsis|6|AT2G41290
E
11
0.78


184
93
258
BDL_unnamed_390
arabidopsis|6|AT2G47750
B
8
0.79


185
93
258
BDL_unnamed_390
arabidopsis|6|AT2G47750
D
14
0.84


186
93
258
BDL_unnamed_390
arabidopsis|6|AT2G47750
E
14
0.71


187
93
258
BDL_unnamed_390
arabidopsis|6|AT2G47750
E
17
−0.79


188
94
259
BDL_unnamed_391
arabidopsis|6|AT3G03230
D
15
0.96


189
94
259
BDL_unnamed_391
arabidopsis|6|AT3G03230
D
16
0.95


190
94
259
BDL_unnamed_391
arabidopsis|6|AT3G03230
E
14
−0.73


191
95
260
BDL_unnamed_392
arabidopsis|6|AT3G04200
B
7
0.85


192
95
260
BDL_unnamed_392
arabidopsis|6|AT3G04200
B
9
−0.94


193
95
260
BDL_unnamed_392
arabidopsis|6|AT3G04200
B
13
0.78


194
98
263
BDL_unnamed_395
arabidopsis|6|AT3G49380
B
4
−0.78


195
98
263
BDL_unnamed_395
arabidopsis|6|AT3G49380
B
9
−0.77


196
98
263
BDL_unnamed_395
arabidopsis|6|AT3G49380
B
10
−0.73


197
98
263
BDL_unnamed_395
arabidopsis|6|AT3G49380
B
17
0.88


198
98
263
BDL_unnamed_395
arabidopsis|6|AT3G49380
C
12
0.71


199
98
263
BDL_unnamed_395
arabidopsis|6|AT3G49380
C
15
0.75


200
98
263
BDL_unnamed_395
arabidopsis|6|AT3G49380
C
16
0.82


201
98
263
BDL_unnamed_395
arabidopsis|6|AT3G49380
E
11
0.82


202
104
269
BDL_unnamed_400
arabidopsis|6|AT4G33600
B
12
0.8


203
104
269
BDL_unnamed_400
arabidopsis|6|AT4G33600
E
11
0.84


204
104
269
BDL_unnamed_400
arabidopsis|6|AT4G33600
E
14
0.8


205
106
271
BDL_unnamed_402
arabidopsis|6|AT5G08460
D
15
0.77


206
106
271
BDL_unnamed_402
arabidopsis|6|AT5G08460
D
16
0.78


207
107
272
BDL_unnamed_403
arabidopsis|6|AT2G34700
C
11
0.89


208
107
272
BDL_unnamed_403
arabidopsis|6|AT2G34700
C
12
−0.71


209
108
273
BDL_unnamed_404
arabidopsis|6|AT5G15740
B
5
0.74


210
108
273
BDL_unnamed_404
arabidopsis|6|AT5G15740
B
8
0.71


211
108
273
BDL_unnamed_404
arabidopsis|6|AT5G15740
E
15
0.8


212
109
274
BDL_unnamed_405
arabidopsis|6|AT5G16230
A
1
−0.75


213
109
274
BDL_unnamed_405
arabidopsis|6|AT5G16230
B
8
0.83


214
109
274
BDL_unnamed_405
arabidopsis|6|AT5G16230
C
12
−0.8


215
109
274
BDL_unnamed_405
arabidopsis|6|AT5G16230
D
12
0.73


216
109
274
BDL_unnamed_405
arabidopsis|6|AT5G16230
D
16
0.74


217
110
275
BDL_unnamed_406
arabidopsis|6|AT5G18290
E
11
−0.76


218
112
277
BDL_unnamed_408
arabidopsis|6|AT5G39130
B
12
0.79


219
112
277
BDL_unnamed_408
arabidopsis|6|AT5G39130
B
13
0.76


220
112
277
BDL_unnamed_408
arabidopsis|6|AT5G39130
B
16
0.79


221
112
277
BDL_unnamed_408
arabidopsis|6|AT5G39130
C
14
0.79


222
112
277
BDL_unnamed_408
arabidopsis|6|AT5G39130
D
14
0.79


223
112
277
BDL_unnamed_408
arabidopsis|6|AT5G39130
E
12
0.73


224
114
279
BDL_unnamed_409
arabidopsis|6|AT5G39160
B
12
0.79


225
114
279
BDL_unnamed_409
arabidopsis|6|AT5G39160
B
13
0.76


226
114
279
BDL_unnamed_409
arabidopsis|6|AT5G39160
B
16
0.79


227
114
279
BDL_unnamed_409
arabidopsis|6|AT5G39160
C
14
0.79


228
114
279
BDL_unnamed_409
arabidopsis|6|AT5G39160
D
14
0.79


229
114
279
BDL_unnamed_409
arabidopsis|6|AT5G39160
E
12
0.73


230
115
280
BDL_unnamed_410
arabidopsis|6|AT5G39190
B
12
0.79


231
115
280
BDL_unnamed_410
arabidopsis|6|AT5G39190
B
13
0.76


232
115
280
BDL_unnamed_410
arabidopsis|6|AT5G39190
B
16
0.79


233
115
280
BDL_unnamed_410
arabidopsis|6|AT5G39190
C
14
0.79


234
115
280
BDL_unnamed_410
arabidopsis|6|AT5G39190
D
14
0.79


235
115
280
BDL_unnamed_410
arabidopsis|6|AT5G39190
E
12
0.73


236
116
281
BDL_unnamed_411
arabidopsis|6|AT5G44360
B
10
−0.74


237
117
282
BDL_unnamed_412
arabidopsis|6|AT5G47670
E
11
0.86


238
117
282
BDL_unnamed_412
arabidopsis|6|AT5G47670
E
14
0.72


239
119
284
BDL_unnamed_414
arabidopsis|6|AT5G56300
C
15
0.77


240
119
284
BDL_unnamed_414
arabidopsis|6|AT5G56300
C
16
0.78


241
119
284
BDL_unnamed_414
arabidopsis|6|AT5G56300
D
15
0.78


242
119
284
BDL_unnamed_414
arabidopsis|6|AT5G56300
D
16
0.82


243
121
286
BDL_unnamed_418
arabidopsis|6|AT1G28640
B
18
0.81


244
122
287
BDL_unnamed_419
arabidopsis|6|AT1G22990
E
11
0.95


245
122
287
BDL_unnamed_419
arabidopsis|6|AT1G22990
E
14
0.8


246
123
288
BDL_unnamed_420
arabidopsis|6|AT1G64110
B
6
0.78


247
125
290
BDL_unnamed_421
arabidopsis|6|AT1G04380
D
15
0.73


248
126
291
BDL_unnamed_422
arabidopsis|6|AT1G08810
B
8
0.8


249
126
291
BDL_unnamed_422
arabidopsis|6|AT1G08810
D
14
−0.79


250
126
291
BDL_unnamed_422
arabidopsis|6|AT1G08810
D
15
−0.82


251
126
291
BDL_unnamed_422
arabidopsis|6|AT1G08810
D
16
−0.82


252
128
293
BDL_unnamed_423
arabidopsis|6|AT1G28170
B
16
−0.71


253
128
293
BDL_unnamed_423
arabidopsis|6|AT1G28170
C
11
0.78


254
128
293
BDL_unnamed_423
arabidopsis|6|AT1G28170
C
12
−0.79


255
128
293
BDL_unnamed_423
arabidopsis|6|AT1G28170
C
14
0.75


256
130
295
BDL_unnamed_425
arabidopsis|6|AT3G10590
E
13
0.72


257
131
296
BDL_unnamed_426
arabidopsis|6|AT3G58740
E
14
0.75


258
131
296
BDL_unnamed_426
arabidopsis|6|AT3G58740
E
17
−0.72


259
132
297
BDL_unnamed_427
arabidopsis|6|AT4G02360
A
1
0.85


260
132
297
BDL_unnamed_427
arabidopsis|6|AT4G02360
A
2
0.76


261
134
299
BDL_unnamed_429
arabidopsis|6|AT5G07200
C
13
−0.76


262
134
299
BDL_unnamed_429
arabidopsis|6|AT5G07200
D
15
0.73


263
134
299
BDL_unnamed_429
arabidopsis|6|AT5G07200
D
16
0.73


264
135
300
BDL_unnamed_430
arabidopsis|6|AT5G22810
D
12
0.86


265
135
300
BDL_unnamed_430
arabidopsis|6|AT5G22810
D
15
0.71


266
135
300
BDL_unnamed_430
arabidopsis|6|AT5G22810
D
16
0.8


267
136
301
BDL_unnamed_431
arabidopsis|6|AT5G43860
A
11
0.75


268
136
301
BDL_unnamed_431
arabidopsis|6|AT5G43860
A
13
−0.77


269
136
301
BDL_unnamed_431
arabidopsis|6|AT5G43860
C
11
0.72


270
136
301
BDL_unnamed_431
arabidopsis|6|AT5G43860
C
17
−0.7


271
136
301
BDL_unnamed_431
arabidopsis|6|AT5G43860
D
14
0.71


272
137
302
BDL_unnamed_432
arabidopsis|6|AT5G57390
C
15
0.72


273
137
302
BDL_unnamed_432
arabidopsis|6|AT5G57390
C
16
0.76


274
137
302
BDL_unnamed_432
arabidopsis|6|AT5G57390
D
17
0.71


275
138
303
BDL_unnamed_433
arabidopsis|6|AT5G62800
D
11
0.76


276
138
303
BDL_unnamed_433
arabidopsis|6|AT5G62800
E
17
−0.73


277
139
304
BDL_unnamed_435
arabidopsis|6|AT5G52500
B
5
−0.75


278
139
304
BDL_unnamed_435
arabidopsis|6|AT5G52500
B
8
−0.73


279
140
305
BDL_unnamed_436
arabidopsis|6|AT5G24600
A
3
−0.78


280
143
308
BDL_unnamed_438
arabidopsis|6|AT1G72040
D
13
0.71


281
145
310
BDL_unnamed_440
arabidopsis|6|AT1G50510
B
8
0.75


282
146
311
BDL_unnamed_441
arabidopsis|6|AT5G48100
E
17
−0.77


283
147
312
BDL_unnamed_442
arabidopsis|6|AT1G14760
B
6
0.83


284
147
312
BDL_unnamed_442
arabidopsis|6|AT1G14760
B
7
−0.76


285
147
312
BDL_unnamed_442
arabidopsis|6|AT1G14760
B
9
0.75


286
148
313
BDL_unnamed_443
arabidopsis|6|AT1G15150
B
11
0.9


287
148
313
BDL_unnamed_443
arabidopsis|6|AT1G15150
E
11
0.76


288
149
314
BDL_unnamed_444
arabidopsis|6|AT1G20500
D
13
−0.78


289
150
315
BDL_unnamed_445
arabidopsis|6|AT1G56170
B
6
0.73


290
150
315
BDL_unnamed_445
arabidopsis|6|AT1G56170
D
15
0.94


291
150
315
BDL_unnamed_445
arabidopsis|6|AT1G56170
D
16
0.93


292
151
316
BDL_unnamed_446
arabidopsis|6|AT1G62070
A
1
0.77


293
151
316
BDL_unnamed_446
arabidopsis|6|AT1G62070
A
2
0.77


294
153
318
BDL_unnamed_448
arabidopsis|6|AT3G21090
C
13
0.9


295
154
319
BDL_unnamed_449
arabidopsis|6|AT3G24250
B
6
0.8


296
154
319
BDL_unnamed_449
arabidopsis|6|AT3G24250
C
11
0.73


297
155
320
BDL_unnamed_450
arabidopsis|6|AT3G50990
D
13
−0.85


298
157
322
BDL_unnamed_452
arabidopsis|6|AT4G10150
B
17
−0.75


299
159
324
BDL_unnamed_454
arabidopsis|6|AT5G07190
B
17
0.77


300
159
324
BDL_unnamed_454
arabidopsis|6|AT5G07190
B
18
0.82


301
159
324
BDL_unnamed_454
arabidopsis|6|AT5G07190
D
15
−0.92


302
159
324
BDL_unnamed_454
arabidopsis|6|AT5G07190
D
16
−0.91


303
160
325
BDL_unnamed_455
arabidopsis|6|AT5G10220
A
10
−0.72


304
160
325
BDL_unnamed_455
arabidopsis|6|AT5G10220
E
16
−0.72


305
161
326
BDL_unnamed_456
arabidopsis|6|AT5G20940
D
15
0.76


306
161
326
BDL_unnamed_456
arabidopsis|6|AT5G20940
D
16
0.7


307
162
327
BDL_unnamed_457
arabidopsis|6|AT5G51210
C
17
0.81


308
163
328
BDL_unnamed_458
arabidopsis|6|AT5G55620
A
13
−0.76


309
163
328
BDL_unnamed_458
arabidopsis|6|AT5G55620
E
11
−0.81


310
163
328
BDL_unnamed_458
arabidopsis|6|AT5G55620
E
14
−0.71


311
164
329
BDL_unnamed_459
arabidopsis|6|AT5G60460
C
14
0.84


312
164
329
BDL_unnamed_459
arabidopsis|6|AT5G60460
E
17
−0.72


313
165
330
BDL_unnamed_460
arabidopsis|6|AT5G65590
D
16
0.72





Correlation vector (correl. Vector).






The following Tables 8-15 present polynucleotides which are predicted based on the microarray correlation analysis to increase in a plant the seed yield (Table 8), oil yield (Table 9), growth rate (Table 10), organ shape/size/length (Table 11), harvest index (Table 12), oil content per seed (Table 13), plant dry matter (Table 14) and seed number per silique (Table 15). It should be noted that additional polynucleotides described in the instant application can be used to change the above characteristics in plants.







TABLE 8







Table 8.


Polynucleotides which impact seed yield












SEQ ID NO:





of the




polypeptide



Polynucleotide
encoded by the



SEQ ID NO:
polynucleotide
Gene Name














1
3
168
BDL2


2
8
173
BDL8


3
9
174
BDL9


4
14
179
BDL15


5
16
181
BDL17


6
26
191
BDL26a


7
27
192
BDL26b


8
29
194
BDL28


9
30
195
BDL29


10
39
204
BDL_unnamed_334


11
42
207
BDL_unnamed_337


12
50
215
BDL_unnamed_344


13
53
218
BDL_unnamed_347


14
55
220
BDL_unnamed_349


15
56
221
BDL_unnamed_350


16
57
222
BDL_unnamed_351


17
63
228
BDL_unnamed_357


18
66
231
BDL_unnamed_362


19
68
233
BDL_unnamed_365


20
70
235
BDL_unnamed_367


21
74
239
BDL_unnamed_371


22
79
244
BDL_unnamed_374


23
81
246
BDL_unnamed_376


24
88
253
BDL_unnamed_384


25
89
254
BDL_unnamed_385


26
94
259
BDL_unnamed_391


27
98
263
BDL_unnamed_395


28
106
271
BDL_unnamed_402


29
108
273
BDL_unnamed_404


30
119
284
BDL_unnamed_414


31
125
290
BDL_unnamed_421


32
126
291
BDL_unnamed_422


33
134
299
BDL_unnamed_429


34
137
302
BDL_unnamed_432


35
150
315
BDL_unnamed_445


36
159
324
BDL_unnamed_454


37
161
326
BDL_unnamed_456
















TABLE 9







Table 9


Polynucleotides which impact oil yield












SEQ ID NO: of the





polypeptide



Polynucleotide SEQ
encoded by the



ID NO:
polynucleotide
Gene Name














1
18
183
BDL19


2
25
190
BDL25


3
49
214
BDL_unnamed_343


4
57
222
BDL_unnamed_351


5
60
225
BDL_unnamed_354


6
64
229
BDL_unnamed_358


7
67
232
BDL_unnamed_364


8
109
274
BDL_unnamed_405


9
135
300
BDL_unnamed_430


10
160
325
BDL_unnamed_455


11
165
330
BDL_unnamed_460
















TABLE 10







Table 10


Polynucleotides which impact growth rate












SEQ ID NO: of the




Polynucleotide
polypeptide encoded by



SEQ ID NO:
the polynucleotide
Gene Name














1
36
201
BDL_unnamed_330


2
70
235
BDL_unnamed_367


3
83
248
BDL_unnamed_379


4
123
288
BDL_unnamed_420


5
140
305
BDL_unnamed_436


6
147
312
BDL_unnamed_442


7
150
315
BDL_unnamed_445


8
154
319
BDL_unnamed_449
















TABLE 11







Table 11.


Polynucleotides which impact organ shape/size/length












SEQ ID NO: of the




Polynucleotide
polypeptide encoded by



SEQ ID NO:
the polynucleotide
Gene Name














1
10
175
BDL10


2
13
178
BDL14


3
19
184
BDL20a


4
20
185
BDL20b


5
21
186
BDL21


6
22
187
BDL22


7
28
193
BDL27


8
38
203
BDL_unnamed_333


9
40
205
BDL_unnamed_335


10
40
205
BDL_unnamed_335


11
43
208
BDL_unnamed_339


12
44
209
BDL_unnamed_340


13
62
227
BDL_unnamed_356


14
72
237
BDL_unnamed_369


15
73
238
BDL_unnamed_370


16
81
246
BDL_unnamed_376


17
82
247
BDL_unnamed_377


18
84
249
BDL_unnamed_380


19
91
256
BDL_unnamed_388


20
93
258
BDL_unnamed_390


21
95
260
BDL_unnamed_392


22
104
269
BDL_unnamed_400


23
109
274
BDL_unnamed_405


24
110
275
BDL_unnamed_406


25
116
281
BDL_unnamed_411


26
117
282
BDL_unnamed_412


27
121
286
BDL_unnamed_418


28
122
287
BDL_unnamed_419


29
126
291
BDL_unnamed_422


30
128
293
BDL_unnamed_423


31
132
297
BDL_unnamed_427


32
136
301
BDL_unnamed_431


33
138
303
BDL_unnamed_433


34
145
310
BDL_unnamed_440


35
148
313
BDL_unnamed_443


36
151
316
BDL_unnamed_446


37
154
319
BDL_unnamed_449


38
163
328
BDL_unnamed_458





Organ shape/size/length include for example, leaf length, leaf width, leaf circularity, seed size, or root length.













TABLE 12







Table 12


Polynucleotides which impact harvest index












SEQ ID NO: of the




Polynucleotide
polypeptide encoded




SEQ ID NO:
by the polynucleotide
Gene Name














1
7
172
BDL7


2
18
183
BDL19


3
36
201
BDL_unnamed_330


4
46
211
BDL_unnamed_341


5
56
221
BDL_unnamed_350


6
80
245
BDL_unnamed_375


7
93
258
BDL_unnamed_390


8
98
263
BDL_unnamed_395


9
131
296
BDL_unnamed_426


10
136
301
BDL_unnamed_431


11
138
303
BDL_unnamed_433


12
146
311
BDL_unnamed_441


13
157
322
BDL_unnamed_452


14
162
327
BDL_unnamed_457


15
164
329
BDL_unnamed_459
















TABLE 13







Table 13


Polynucleotides which impact oil content per seed












SEQ ID NO: of the




Polynucleotide
polypeptide encoded by



SEQ ID NO:
the polynucleotide
Gene Name














1
6
171
BDL6


2
23
188
BDL23


3
56
221
BDL_unnamed_350


4
61
226
BDL_unnamed_355


5
112
277
BDL_unnamed_408


6
114
279
BDL_unnamed_409


7
115
280
BDL_unnamed_410


8
128
293
BDL_unnamed_423


9
135
300
BDL_unnamed_430
















TABLE 14







Table 14


Polynucleotides which impact plant dry matter












SEQ ID NO: of the




Polynucleotide
polypeptide encoded by



SEQ ID NO:
the polynucleotide
Gene Name














1
37
202
BDL_unnamed_331


2
52
217
BDL_unnamed_346


3
55
220
BDL_unnamed_349


4
139
304
BDL_unnamed_435
















TABLE 15







Table 15


Polynucleotides which impact seed number per silique












SEQ ID NO: of the




Polynucleotide
polypeptide encoded by



SEQ ID NO:
the polynucleotide
Gene Name














1
57
222
BDL_unnamed_351


2
71
236
BDL_unnamed_368


3
81
246
BDL_unnamed_376


4
90
255
BDL_unnamed_386


5
112
277
BDL_unnamed_408


6
114
279
BDL_unnamed_409


7
115
280
BDL_unnamed_410


8
131
296
BDL_unnamed_426


9
143
308
BDL_unnamed_438


10
149
314
BDL_unnamed_444


11
153
318
BDL_unnamed_448


12
155
320
BDL_unnamed_450









Example 3
Gene Cloning and Creation of Binary Vectors for Plant Expression
Cloning Strategy
Selected genes from those listed in Examples 1 and 2 above were cloned into binary vectors for the generation of transgenic plants. For cloning, the full-length open reading frame (ORF) was first identified. In case of ORF-EST clusters and in some cases mRNA sequences were analyzed to identify the entire open reading frame by comparing the results of several translation algorithms to known proteins from other plant species. To clone the full-length cDNAs, Reverse Transcription followed by PCR(RT-PCR) was performed on total RNA extracted from Arabidopsis siliques collected 3 and 13 days after flowering (3 and 13 DAF). RNA was extracted using Hot Borate RNA Extraction protocol according to World Wide Web (dot) www (dot) eeob (dot) iastate (dot) edu/faculty/WendelJ/ultramicrorna (dot) html. Production of cDNA (using random hexamer and poly dT primers) and PCR amplification was performed using standard protocols described elsewhere (Sambrook J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning. A Laboratory Manual., 2nd Ed. Cold Spring Harbor Laboratory Press, New York.) and are routine for those skilled in the art.
To clone the full-length genomic region of a gene, genomic DNA was extracted from wild type (WT) Arabidopsis thaliana leaves (DNeasy plant mini kit, Qiagen, Germany). All genes were amplified by nested PCR. PCR products were purified using Mini Elute PCR purification kit (Qiagen) and sequencing of the amplified PCR products is performed, using ABI 377 sequencer (Applied Biosystems). To facilitate cloning of the cDNAs/genomic sequences, a 8-12 bp extension was added to the 5′ prime end of each primer. The primer extension includes an endonuclease restriction site. The restriction sites are selected using two parameters: (a). The site does not exist in the cDNA sequence; and (b). The restriction sites in the forward and reverse primers are designed so the digested cDNA is inserted in the sense formation into the binary vector utilized for transformation.
PCR products were purified (Mini Elute PCR Purification Kit, Qiagen, Germany) and digested with the restriction sites according to the primers used (Roche, Switzerland). The digested PCR products were first subcloned into a high copy vector [(originated from the pBlue-script KS plasmid vector http://www.stratagene.com/manuals/212205.pdf)] with the 35S promoter (SEQ ID NO:921), and the NOS terminator (SEQ ID NO:922) originated from pBI 101.3 binary vector (GenBank Accession No. U12640, bp 4417 to 4693)), followed by cloning the entire cassette into the binary vectors pGI or pMBArt (according to Table 16, hereinbelow). The digested PCR products and the linearized plasmid vector were ligated using T4 DNA ligase enzyme (Roche, Switzerland). The following polynucleotides were cloned from RNA extracted from the tissues described above or genomic DNA using the primers as provided in Table 17, below.







TABLE 16







Table 16:


Genes cloned into different binary vectors















Bioinf.
Bioinf.


Cloned





identified
identified


polynucleotide



Polynucleotide
Polypeptide
TAIR gene
Internal
SEQ
Cloned In
Cloned In



SEQ ID NO:
SEQ ID NO:
name
name
ID NO:
pGI
pMBart


















1
1
166
AT5G50770
BDL3
1017

V


2
4
169
AT2G45420
BDL4
1041

V


3
5
170
AT3G14360
BDL5
1018

V


4
6
171
AT4G10490
BDL6
1019
V


5
7
172
AT5G51490
BDL7
1020
V


6
8
173
AT3G03240
BDL8
1021

V


7
9
174
AT5G24130
BDL9
1022

V


8
3
168
AT1G34580
BDL2
1016

V


9
11
176
AT5G12460
BDL11
1042

V


10
12
177
AT4G08530
BDL12
1023
V


11
2
167
AT1G65090
BDL1
1040
V


12
13
178
AT1G53690
BDL14
1024
V


13
14
179
AT1G68510
BDL15
1025
V


14
15
180
AT5G03800
BDL16
1026

V


15
16
181
AT5G36770
BDL17
1043

V


16
17
182
AT5G40420
BDL18
1027
V


17
19
184
AT1G47540.1
BDL20a
1029
V


18
20
185
AT1G47540.2
BDL20b
1044
V


19
21
186
AT3G62730
BDL21
1030

V


20
23
188
AT3G27785
BDL23
1031

V


21
24
189
AT5G15000
BDL24
1045
V


22
25
190
AT3G20910
BDL25
1032

V


23
26
191
AT1G11170.1
BDL26a
1033
V


24
27
192
AT1G11170.2
BDL26b
1034
V


25
28
193
AT1G68380
BDL27
1035

V


26
29
194
AT1G09380
BDL28
1036

V


27
30
195
AT1G60970
BDL29
1037

V


28
31
196
AT1G72580
BDL30
1046
V


29
33
198
AT2G46960.1
BDL32a
1038

V


30
34
199
AT2G46960.2
BDL32b
1039

V


31
933
183
AT2G02080.1
BDL19gD
1028

V






NA


32
—
AY254038
WRINKLED1
WRI
1050
V





Provided are the sequence identifiers of the polynucleotides and polypeptides identified bioinformatically (bioinf.), as well as the sequence identifiers of the cloned polynucleotides. In two cases, the translated polypeptide sequences of the cloned genes were different from the predicted bioinformatically identified polypeptides (SEQ ID NOs: 176 and 178) and new sequence identifiers were provided (i.e., SEQ ID NO: 1047 for the translated polypeptide of cloned gene SEQ ID NO: 1042 and SEQ ID NO: 1048 for the translated polypeptide of cloned gene SEQ ID NO: 1024).













TABLE 17







Table 17


Polynucleotides cloned from cDNA libraries, genomic


DNA or synthetically produced and the primers used


for the cloning











Restriction





Enzymes




Gene
used for

SEQ ID


Name
cloning
Primers used for amplification (5′→3′)
NO:













BDL3
SalI, XbaI
Fwd Nested: BDL3_ORF_NF_SalI-
923




AATGTCGACGATGCATGGATTCAATCAACA



 










Fwd External: BDL3_ORF_EF_SalI-
924



TTTGTCGACCATTGTGAAGTATAGTCCTTGATG



 



Rev Nested: BDL3_ORF_NR_XbaI-
925



TATCTAGAACATAAACGGGGAGACTCAAG



 



Rev External: BDL3_ORF_ER_XbaI-
926



AATCTAGACTATGGTAACCCGAAGTTGTATAC



 










BDL4
SacI, XbaI
synthetic product
1041


 


BDL5
SalI, XbaI
Fwd Nested: BDL5_ORF_NF_Sal-
927




ACTGTCGACAGACATGCACAAAGACAACG



 










Fwd External: BDL5_ORF_EF_SalI-
928



ATAGTCGACCAAAACCCAGAGACATGCAC



 



Rev Nested: BDL5_ORF_NR_XbaI-
929



AATCTAGACACTTTTCAAAGAGAGGACATCT



 



Rev External: BDL5_ORF_ER_XbaI-
930



ACTCTAGACCGGTTCACTTAAGATTTATTC



 










BDL6
SalI, XbaI
Fwd: BDL6_ORF_F1_SalI-
931




AAAGTCGACCAATCATGGCAGCATCAAAAC



 










Rev Nested: BDL6_ORF_NR_XbaI-
932



AGTCTAGACGGATGATTGATTCGATAGTACAC




Phaseolus vulgaris



 



Rev External: BDL6_ORF_ER_SacI-
933



TGAGCTCCCAATCAAGAACTAAGGACCG



 










BDL7
SalI, XbaI
Fwd: BDL7_ORF_F1_Sal-
934




AATGTCGACAACAATGAATATGATGATGCAAAAACTC



 










Rev Nested: BDL7_ORF_NR_XbaI-
935



AATCTAGACGGTCTTTAGAGTCCAGAAGTG



 



Rev External: BDL7_ORF_ER_XbaI-
936



AATCTAGAATCATTGCAACTTAAACACGA



 










BDL8
XbaI, SalI
Fwd: BDL8_gDNA_F_Sal-
937




AATGTCGACCCTCTGTCTTGTCTTTTGGTTAGTA



 










Rev: BDL8_gDNA_R_Xb-
938



AATCTAGACCTTCAACTACAAGCGGCTT



 










BDL9
SalI, XbaI
Fwd Nested: BDL9_ORF_NF_SalI-
939




acggtcgacCTTACAATAAAATGGTGAAACTCG



 










Fwd External: BDL9_ORF_EF_SalI-
940



aatgtcgacCTCTCTAAACGCATAATCTTACA



 



Rev Nested: BDL9_ORF_NR_XbaI-
941



AATCTAGACAAAATATGTGGTCTCCGCAG



 



Rev External: BDL9_ORF_ER_XbaI-
942



AGTCTAGACAAAAAGGAAACGAATCACA



 










BDL2
SalI, XbaI
Fwd Nested: BDL2_ORF_NF_SalI-
943




CAAGTCGACCGTAAGACATAAGCAAAATGGC



 










Fwd External BDL2_ORF_EF_SalI-
944



TTAGTCGACCACTTCATGCGTAAGACATAAGC



 



Rev Nested: BDL2_ORF_NR_XbaI-
945



GCTCTAGAGCATCTTTTAAGTTGACGTCG



 



Rev External: BDL2_ORF_ER_XbaI-
946



AATCTAGATCCATTGAAAATGCGAACC



 










BDL11
SacI, XbaI
synthetic product



 


BDL12
SalI, SacI
Fwd Nested: BDL12_gDNA_NF_SalI-
947




AATGTCGACGTTCTATCCCCAACTCTAAATG



 










Fwd External: BDL12_gDNA_EF_XbaI-
948



ATTCTAGATTGTTGTTTGTATCACTTTATTGG



 



Rev Nested: BDL12_gDNA_NR_SacI-
949



AGAGCTCCTTAAAGTTCTATCGAGATAGTGC



 



Rev External: BDL12_gDNA_ER_SacI-
950



AGAGCTCTCAATGAAATTTTACATAACCATC



 










BDL1
XbaI, SacI
synthetic product



 


BDL14
SalI, XbaI
Fwd: BDL14_ORF_F1_SalI-
951




AATGTCGACAACAATGGATCTACAACAGTCCGAAAC



 










Rev Nested: BDL14_ORF_NR_XbaI-
952



AATCTAGACACTCAGACAGCTGGGTATTAAAC



 



Rev External: BDL14_ORF_ER_SacI-
953



AGAGCTCGTTGTGGCACTCAGACAGCTG



 










BDL15
SalI, XbaI
Fwd Nested: BDL15_ORF_NF_Sal-
954




TTCGTCGACAAAGGAATATGAGAATCAGCTG



 










Fwd External: BDL15_ORF_EF_Sal-
955



AACGTCGACCAAACACACATCATACGTATATTTG



 



Rev Nested: BDL15_ORF_NR_XbaI-
956



ATTCTAGAGAGTTTATGATAACCTAATGATTGAC



 



Rev External: BDL15_ORF_ER_XbaI-
957



GTTCTAGACAGAGTGAGTTTATGATAACCTAATG



 










BDL16
SalI, XbaI
Fwd: BDL16_ORF_F1_SalI-
958




AATGTCGACAACAATGTCCACCGTTAATCATCAC



 










Rev Nested: BDL16_ORF_NR_XbaI-
959



AATCTAGACAGAACCAAAACTCTCGTATTAAC



 



Rev External: BDL16_ORF_ER_XbaI-
960



AATCTAGAGAAACTTTGAATGGACTATGTAGC



 










BDL17
SacI, XbaI
synthetic product
1043


 


BDL18
XbaI, SacI
Fwd Nested: BDL18_ORF_NF_XbaI-
961




AATCTAGATACAATGGCGGATACACACC



 










Fwd External: BDL18_ORF_EF_XbaI-
962



ATTCTAGAGCTTACAATGGCGGATACACA



 



Rev Nested: BDL18_ORF_NR_SacI-
963



AGAGCTCGTGAAAACACATATCTACCGTTC



 



Rev External: BDL18_ORF_ER_SacI-
964



AGAGCTCCTTGCGATCTTTCATGCTTAC



 










BDL19
SacI
Fwd Nested: BDL19_gDNA_NF_SacI-
965




AGAGCTCAGAGAGAGATAGGGCTTTGAGG



 










Fwd External: BDL19_g_DNA_EF_SacI-
966



AGAGCTCGAAGAAGAACACAAAACAGTAGAG



 



Rev: BDL19_gDNA_R1_SacI-
967



AGAGCTCGTGATTATGAAAACAACAAGCG



 










BDL20a
SalI, XbaI
Fwd: BDL20a_ORF_F1_SalI-
968




AAAGTCGACAGAGACAAAGAAGTTGGCCA



 










Rev Nested: BDL20a_ORF_NR_XbaI-
969



TTTCTAGATGCAAGATTCAAATACGACTTAG



 



Rev External: BDL20a_ORF_ER_SacI-
970



AGAGCTCGGACCATTTACCTTGATTTGTTAC



 










BDL20b
SmaI + SacI
synthetic product
1044


 


BDL21
SalI, XbaI
Fwd Nested: BDL21-ORF-NF-Sal-
971




AATGTCGACAAGCATGTTTAAACTCTGTCTCG



 










Fwd External: BDL21-ORF-EF-Sal-
972



TTAGTCGACGAAAGGAAAAGCATGTTTAAAC



 



Rev Nested: BDL21-ORF-NR-XbaI-
973



CCGTCTAGAGGAAACTTTTAATTGTCATGTGA



 



Rev External: BDL21-ORF-ER-XbaI-
974



GGCTCTAGATTTTCTAGTGAATTGTATCAATGG



 










BDL23
XbaI, SacI
Fwd Nested: BDL23_ORF_NF_XbaI-
975




AATCTAGACATCATAATCATATGGAGTTCGA



 










Fwd External: BDL23_ORF_EF_XbaI-
976



AATCTAGAGATCTAGGGTTTCATGCTTCAC



 



Rev: BDL23_ORF_R1_SacI-
977



AGAGCTCGTTCGACTTGTTTATATTGCACG



 










BDL24
SmaI, SacI
synthetic product
1045


 


BDL25
XbaI
Fwd Nested: BDL25_ORF_NF_XbaI-
978




ATTCTAGACTCCGAGACTGTCTCCGATTG



 










Fwd External: BDL25_ORF_EF_XbaI-
979



ATTCTAGACAATCACCGTGGACACCTC



 



Rev: BDL25_ORF_R_XbaI-
980



ATTCTAGAGTGGCAACATCTGAAGTATTCC



 










BDL26a
SacI
Fwd Nested: BDL26a_ORF_NF_SacI-
981




AGAGCTCTCATTACAGTGACTCTGCATGC



 










Fwd External: BDL26a_ORF_EF_SacI-
982



AGAGCTCTCTTGTCTACTTTCATTACAGTGAC



 



Rev Nested: BDL26a + b_ORF_NR_SacI-
983



TAGAGCTCGAAAGTACATAATGGACATGAGC



 



Rev External: BDL26a + b_ORF_ER_SacI-
984



TAGAGCTCGATTTTTAAAGTAGTTATAGTGATGAA



 










BDL26b
SacI
Fwd Nested: BDL26b_ORF_NF_SacI-
985




AGAGCTCGTAATATTACCATAAGGTTCAGAAG



 










Fwd External: BDL26b_ORF_EF_SacI-
986



AGAGCTCCATAATTTTTTCGTATTTAACTCTT



 



Rev Nested: BDL26a + b_ORF_NR_SacI-
987



TAGAGCTCGAAAGTACATAATGGACATGAGC



 



Rev external: BDL26a + b_ORF_ER_SacI-
988



TAGAGCTCGATTTTTAAAGTAGTTATAGTGATGAA



 










BDL27
XbaI, SacI
Fwd Nested: BDL27_ORF_NF_XbaI-
989




AATCTAGACTCTTACACATGTATCGGTAGTTG



 










Fwd External: BDL27_ORF_EF_XbaI-
990



AATCTAGACTTAAAACATTGGAAACAAGAATTC



 



Rev Nested: BDL27_ORF_NR_SacI-
991



AGAGCTCGATCAGAAATACATGACGATAGATG



 



Rev External: BDL27_ORF_ER_SacI-
992



AGAGCTCGCATCTTTGTTTTTGGACGA



 










BDL28
SalI, xbaI
Fwd Nested: BDL28_ORF_NF_SalI-
993




AAAGTCGACGAGAGATGGCTAAATCAGATATG



 










Fwd External: BDL28_ORF_EF_SalI-
994



AATGTCGACGAGAGTGAGAGATGGCTAAATCAG



 



Rev Nested: BDL28_ORF_NR_XbaI-
995



ATTCTAGAAGAAGCAATCACCATTTTAAGG



 



Rev External: BDL28_ORF_ER_XbaI-
996



ATTCTAGACCGAAAATCCAATTTAGTTGC



 










BDL29
SalI, XbaI
Fwd Nested: BDL29_ORF_NF_SalI-
997




AATGTCGACGATTTCTTCTCCTTAAGCCATG



 










Fwd External: BDL29_ORF_EF_SalI-
998



AATGTCGACGGAGAGTTTTTCTTTATTACTAGGG



 



Rev Nested: BDL29_ORF_NR_XbaI-
999



AATCTAGACACACATCATTTCATAAGTGACC



 



Rev External: BDL29_ORF_ER_XbaI-
1000



AATCTAGACAACCATTATTACCGAAGAGC



 










BDL30
SmaI, SacI
synthetic product
1046


 


BDL32a
XbaI, SacI
Fwd Nested: BDL32a_ORF_NF_XbaI-
1001




AATCTAGAGAGGATAATGCGTAACACACAAG



 










Fwd External: BDL32a_ORF_EF_XbaI-
1002



AATCTAGAGATTTTATTCGAGGATAATGCG



 



Rev Nested: BDL32a + b_ORF_NR_SacI-
1003



AGAGCTCCATTAAGACATCCGATTTATTTG



 



Rev External: BDL32a + b_ORF_ER_SacI-
1004



AGAGCTCGAGACTTGTCACACACGTGAGG



 










BDL32b
XbaI, SacI
Fwd nested: BDL32b_ORF_NF_XbaI-
1005




AATCTAGACACACACACAAACATAAGGAAA



 










Fwd External: BDL32b_ORF_EF_XbaI-
1006



AATCTAGAAACAATACACACACACAAACATAAG



 



Rev Nested: BDL32a + b_ORF_NR_SacI-
1007



AGAGCTCCATTAAGACATCCGATTTATTTG



 



Rev External: BDL32a + b_ORF_ER_SacI-
1008



AGAGCTCGAGACTTGTCACACACGTGAGG



 










Wrinkle
SalI, XbaI
Fwd nested: WRI_NF_ORF_SalI-
1009


d1

AATGTCGACCAGAGTTTAATGAAGAAGCGCT



 










Fwd External: WRI_EF_Art_SalI-
1010



AATGTCGACAAATCTAAACTTTCTCAGAG



 



Rev Nested: WRI_NR_ORF_XbaI-
1011



AATCTAGACTCTCTCAGACCAAATAGTTACAAG



 



Rev External: WRI_ER_Art_XbaI-
1012



AATCTAGAGGCAAAGACATTGATTATTC



 










Napin
HindIII, SalI
Fwd: Napin F HindIII-
1013




ATAAGCTTATTGATTCCTTTAAAGACTTATGTT



 










Rev: Napin R SalI-
1014



TCGTCGACGGGTGTATGTTTTTAATCTTGTTT










To optimize the coding sequence (in silico design), codon-usage Tables calculated from plant transcriptoms were used (example of such Tables can be found in the Codon Usage Database available online at Hypertext Transfer Protocol://World Wide Web (dot) kazusa (dot) or (dot) jp/codon/). The optimized coding sequences were designed in a way that no changes are introduced in the encoded amino acid sequence (of selected polypeptides from Table 1, Example 1) while using codons preferred for expression in dicotyledonous plants mainly Arabidopsis, Canola and Soya; and monocotyledonous plants such as maize. Such optimized sequences promote better translation rate and therefore higher protein expression levels. To the optimized sequences flanking additional unique restriction enzymes sites were added-SalI, XbaI, BamHI, SmaI at the 5′ end and Sad at the 3′ end (except one gene-BDL-1, in which the SmaI site was excluded). The genes for which codon optimized synthetic (artificial) sequences were prepared are: BDL-1 (SEQ ID NO:1040), BDL-4 (SEQ ID NO:1041), BDL-11 (SEQ ID NO:1042), BDL-17 (SEQ ID NO:1043), BDL-20b (SEQ ID NO:1044), BDL-24 (SEQ ID NO:1045), BDL-30 (SEQ ID NO:1046). The artificial optimized polynucleotide sequences were synthesized by a commercial supplier [GeneArt, GmbH, (Hypertext Transfer Protocol://World Wide Web (dot) geneart (dot) com/)].
Generation of Binary Vectors Comprising BDL Genes and Plant Functional Promoters for Driving Expression of Same
The plasmid pPI was constructed by inserting a synthetic poly-(A) signal sequence, originating from pGL3 basic plasmid vector (Promega, Acc No U47295; by 4658-4811) into the HindIII restriction site of the binary vector pBI101.3 (Clontech, GenBank Accession. No. U12640). In some cases the backbone binary plasmid used was pGI which is similar to pPI but the GUS gene was replaced by the GUS-Intron gene (Vancanneyt. G, et al MGG 220, 245-50, 1990). pGI was used to clone part of the polynucleotide sequences, initially under the control of 35S promoter [Odell, J T, et al. Nature 313, 810-812 (28 Feb. 1985); SEQ ID NO:921]. Additional sequences were cloned into pMBLArt under the control of 35S promoter.
Some polynucleotide sequences were cloned under other preferential promoter as described below. The promoter, named Napin originated from Brassica napus which is characterized by a seed specific promoter activity [Stuitje A. R. et. al. Plant Biotechnology Journal 1 (4): 301-309], was amplified by direct PCR on genomic DNA extracted from leaf tissue using the DNAeasy kit (Qiagen Cat. No. 69104) using the following primers:









Napin F Hind III (Enzyme HindII)-









(SEQ ID NO: 1013)









5′-ATAAGCTTATTGATTCCTTTAAAGACTTATGTT



 



Napin R Sal I (Enzyne Sal I)-









(SEQ ID NO: 1014)









5′-TCGTCGACGGGTGTATGTTTTTAATCTTGTTT.






The following genes were cloned downstream of the Napin promoter sequence: BDL-2, BDL-3, BDL-4, BDL-6, BDL-12, BDL-14, BDL-15, BDL-17, BDL-18, BDL-21, BDL-23, BDL-25, BDL-27, BDL-28, BDL-29, BDL-32b, Wrinkle1. For control purposes, the β-glucuronidase enzyme (GUS, SEQ ID NO:1051) encoded by the uid A gene (GUS-Intron, SEQ ID NO:1049).
Example 4
Producing Transgenic Arabidopsis Plants Expressing the Seed Oil Genes
Materials and Methods
Plant transformation was performed according to (Clough S J, Bent A F. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16(6): 735-43, Desfeux C, Clough S J, Bent A F. 2000. Female reproductive tissues are the primary targets of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method. Plant Physiol. 123(3): 895-904.).
The Arabidopsis thaliana var Columbia (T0 plants) were transformed according to the Floral Dip procedure described by Clough S J, Bent A F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16(6): 735-43; and Desfeux C, Clough S J, Bent A F. (20000 Female reproductive tissues are the primary targets of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method. Plant Physiol. 123(3): 895-904) with minor modifications. Briefly, Arabidopsis thaliana Columbia (Col0) T0 Plants were sown in 250 ml pots filled with wet peat-based growth mix. The pots were covered with aluminum foil and a plastic dome, kept at 4° C. for 3-4 days, then uncovered and incubated in a growth chamber at 18-24° C. under 16/8 hours light/dark cycles. The T0 plants were ready for transformation six days before anthesis.
Single colonies of Agrobacterium carrying the binary vectors harboring the seed oil genes were cultured in LB medium supplemented with kanamycin (50 mg/L) and gentamycin (50 mg/L). The cultures were incubated at 28° C. for 48 hours under vigorous shaking and centrifuged at 4000 rpm for 5 minutes. The pellets comprising Agrobacterium cells were resuspended in a transformation medium which contained half-strength (2.15 g/L) Murashige-Skoog (Duchefa); 0.044 μM benzylamino purine (Sigma); 112 μg/L B5 Gambourg vitamins (Sigma); 5% sucrose; and 0.2 ml/L Silwet L-77 (OSI Specialists, CT) in double-distilled water, at pH of 5.7.
Transformation of T0 plants was performed by inverting each plant into an Agrobacterium suspension such that the above ground plant tissue was submerged for 3-5 seconds. Each inoculated T0 plant was immediately placed in a plastic tray, then covered with clear plastic dome to maintain humidity and kept in the dark at room temperature for 18 hours to facilitate infection and transformation. Transformed (transgenic) plants were then uncovered and transferred to a greenhouse for recovery and maturation. The transgenic T0 plants were grown in the greenhouse for 3-5 weeks until siliques were brown and dry, then seeds were harvested from plants and kept at room temperature until sowing
For generating T1 and T2 transgenic plants harboring the genes, seeds collected from transgenic T0 plants were surface-sterilized by soaking in 70% ethanol for 1 minute, followed by soaking in 5% sodium hypochlorite and 0.05% triton for 5 minutes. The surface-sterilized seeds were thoroughly washed in sterile distilled water then placed on culture plates containing half-strength Murashig-Skoog (Duchefa); 2% sucrose; 0.8% plant agar; 50 mM kanamycin; and 200 mM carbenicylin (Duchefa). The culture plates were incubated at 4° C. for 48 hours then transferred to a growth room at 25° C. for an additional week of incubation. Vital T1 Arabidopsis plants were transferred to a fresh culture plates for another week of incubation. Following incubation the T1 plants were removed from culture plates and planted in growth mix contained in 250 ml pots. The transgenic plants were allowed to grow in a greenhouse to maturity. Seeds harvested from T1 plants were cultured and grown to maturity as T2 plants under the same conditions as used for culturing and growing the T1 plants.
Example 5
Identification of Additional Sequences with Highest Probability to Confer Similar Favorable Effects in the Transgenic Plants
Methods for the search and identification of homologues of seed yield polypeptide or polynucleotide would be well within the realm of a person skilled in the art. The search and identification of homologous genes involves the screening of sequence information available, for example, in public databases, that include but are not limited to the DNA Database of Japan (DDBJ), Genbank, and the European Molecular Biology Laboratory Nucleic Acid Sequence Database (EMBL) or versions thereof or the MIPS database. A number of different search algorithms have been developed, including but not limited to the suite of programs referred to as BLAST programs. There are five implementations of BLAST, three designed for nucleotide sequence queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology: 76-80, 1994; Birren et al., Genome Analysis, I: 543, 1997). Such methods involve alignment and comparison of sequences. The BLAST algorithm calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information. Other such software or algorithms are GAP, BESTFIT, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.
The homologous genes may belong to the same gene family. The analysis of a gene family may be carried out using sequence similarity analysis. To perform this analysis one may use standard programs for multiple alignments e.g. Clustal W. A neighbour-joining tree of the proteins homologous to the genes in this invention may be used to provide an overview of structural and ancestral relationships. Sequence identity may be calculated using an alignment program as described above. It is expected that other plants will carry a similar functional gene (orthologue) or a family of similar genes and those genes will provide the same preferred phenotype as the genes presented here. Advantageously, these family members may be useful in the methods of the invention. Example of other plants are included here but not limited to, barley (Hordeum vulgare), Arabidopsis (Arabidopsis thaliana), maize (Zea mays), cotton (Gossypium), Oilseed rape (Brassica napus), Rice (Oryza sativa), Sugar cane (Saccharum officinarum), Sorghum (Sorghum bicolor), Soybean (Glycine max), Sunflower (Helianthus annuus), Tomato (Lycopersicon esculentum), Wheat (Triticum aestivum)
The above-mentioned analyses for sequence homology is preferably carried out on a full-length sequence, but may also be based on a comparison of certain regions such as conserved domains. The identification of such domains, would also be well within the realm of the person skilled in the art and would involve, for example, a computer readable format of the nucleic acids of the present invention, the use of alignment software programs and the use of publicly available information on protein domains, conserved motifs and boxes. This information is available in the PRODOM (Hypertext Transfer Protocol://World Wide Web (dot) biochem (dot) ucl (dot) ac (dot) uk/bsm/dbbrowser/protocol/prodomqry (dot) html), PIR (Hypertext Transfer Protocol://pir (dot) Georgetown (dot) edu/) or Pfam (Hypertext Transfer Protocol://World Wide Web (dot) sanger (dot) ac (dot) uk/Software/Pfam/) database. Sequence analysis programs designed for motif searching may be used for identification of fragments, regions and conserved domains as mentioned above. Preferred computer programs include, but are not limited to, MEME, SIGNALSCAN, and GENESCAN.
A person skilled in the art may use the homologous sequences provided herein to find similar sequences in other species and other organisms. Homologues of a protein encompass, peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. To produce such homologues, amino acids of the protein may be replaced by other amino acids having similar properties (conservative changes, such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a-helical structures or 3-sheet structures). Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company). Homologues of a nucleic acid encompass nucleic acids having nucleotide substitutions, deletions and/or insertions relative to the unmodified nucleic acid in question and having similar biological and functional activity as the unmodified nucleic acid from which they are derived.
Genes identified in publicly available sequence databases as sharing high sequence homology to the arabidopsis genes identified herein are summarized in Table 18 below. Those genes are expected to possess similar functions when exogenously introduced into plants, as the arabidopsis genes identified. Homolog genes sequences are also provided.







TABLE 18







Table 18


Polypeptides and polynucleotides encoding same which share high sequence


homology to the identified arabidopsis polypeptides of the invention















Polynucleotide
Polypeptide

Homology to
%
% query




SEQ_ID_NO:
SEQ_ID_NO:
Organism
SEQ_ID_NO:
identity
coverage
Algorithm


















1
369
523
peanut
51
83
48.4
tblastx


2
370

oil_palm
126
78
19.0
tblastx


3
371
524
tobacco
51
88
45.1
tblastx


4
372
525
tobacco
18
85
22.0
tblastx


5
373
526
tobacco
165
85
13.2
tblastx


6
374
527
tobacco
165
77
14.7
tblastx


7
375
528
barley
51
85
45.1
tblastx


8
376
529
barley
117
85
27.6
tblastx


9
377
530
barley
126
85
31.5
tblastx


10
378
531
barley
137
92
25.4
tblastx


11
379
532
barley
150
65
41.0
tblastx


12
380
533
peach
51
90
46.4
tblastx


13
381

thellungiella
42
86
18.9
tblastx


14
382

thellungiella
61
88
13.1
tblastx


15
383
534
thellungiella
66
91
14.7
tblastx


16
384

thellungiella
70
86
20.5
tblastx


17
385
535
thellungiella
18
95
26.4
tblastx


18
386
536
thellungiella
163
92
27.7
tblastx


19
387
537
strawberry
51
83
47.1
tblastx


20
388
538
canola
36
90
28.5
tblastx


21
389

canola
36
88
17.0
tblastx


22
390
539
canola
9
89
64.7
tblastx


23
391
540
canola
29
88
49.9
tblastx


24
392
541
canola
40
87
82.0
tblastx


25
393
542
canola
40
87
79.4
tblastx


26
394
543
canola
40
87
82.0
tblastx


27
395

canola
40
86
46.5
tblastx


28
396
544
canola
41
95
36.5
tblastx


29
397
545
canola
41
93
36.5
tblastx


30
398
546
canola
44
87
41.1
tblastx


31
399
547
canola
51
93
49.0
tblastx


32
400
548
canola
51
97
51.0
tblastx


33
401
549
canola
51
77
64.7
tblastx


34
402
550
canola
54
94
22.3
tblastx


35
403
551
canola
55
93
59.3
tblastx


36
404

canola
56
85
26.0
tblastx


37
405
552
canola
57
94
19.1
tblastx


38
406
553
canola
60
90
23.6
tblastx


39
407
554
canola
61
88
27.7
tblastx


40
408
555
canola
63
92
47.5
tblastx


41
409
556
canola
10
87
49.7
tblastx


42
410
557
canola
66
91
24.9
tblastx


43
411

canola
7
87
31.6
tblastx


44
412

canola
14
92
44.1
tblastx


45
413

canola
14
92
44.1
tblastx


46
414

canola
81
85
36.3
tblastx


47
415
558
canola
35
90
32.4
tblastx


48
416
559
canola
35
88
45.3
tblastx


49
417
560
canola
35
91
45.3
tblastx


50
418
561
canola
91
88
28.9
tblastx


51
419
562
canola
93
95
14.5
tblastx


52
420

canola
101
95
11.3
tblastx


53
421
563
canola
106
84
32.1
tblastx


54
422

canola
107
83
62.3
tblastx


55
423
564
canola
108
94
14.4
tblastx


56
424
565
canola
118
90
20.6
tblastx


57
425
566
canola
118
95
34.4
tblastx


58
426
567
canola
118
95
34.4
tblastx


59
427
568
canola
119
83
57.2
tblastx


60
428

canola
125
84
28.1
tblastx


61
429

canola
135
96
24.6
tblastx


62
430
569
canola
137
90
32.7
tblastx


63
431

canola
18
93
33.4
tblastx


64
432
570
canola
21
84
83.9
tblastx


65
433

canola
140
92
52.2
tblastx


66
434
571
canola
143
92
41.7
tblastx


67
435
572
canola
143
93
41.0
tblastx


68
436
573
canola
145
89
49.1
tblastx


69
437
574
canola
145
91
39.8
tblastx


70
438

canola
153
94
26.4
tblastx


71
439
575
canola
160
89
79.6
tblastx


72
440
576
canola
163
91
27.7
tblastx


73
441
577
canola
164
80
76.6
tblastx


74
442
578
canola
165
85
11.9
tblastx


75
443
579
melon
51
84
47.1
tblastx


76
444
580
sugarcane
137
90
25.7
tblastx


77
445
581
sugarcane
137
88
28.4
tblastx


78
446
582
b_rapa
41
95
36.1
tblastx


79
447
583
b_rapa
57
92
9.5
tblastx


80
448

b_rapa
64
86
45.7
tblastx


81
449
584
b_rapa
10
84
39.0
tblastx


82
450

b_rapa
4
86
40.5
tblastx


83
451
585
b_rapa
35
86
17.2
tblastx


84
452
586
b_rapa
106
78
36.8
tblastx


85
453
587
b_rapa
122
94
71.4
tblastx


86
454
588
b_rapa
126
87
32.6
tblastx


87
455
589
b_rapa
135
86
41.7
tblastx


88
456
590
b_rapa
137
85
17.8
tblastx


89
457
591
b_rapa
18
94
26.0
tblastx


90
458
592
b_rapa
150
82
42.9
tblastx


91
459

b_rapa
152
88
32.6
tblastx


92
460
593
b_rapa
165
85
11.9
tblastx


93
461
594
maize
137
86
24.1
tblastx


94
462
595
maize
137
89
14.0
tblastx


95
463
596
maize
137
86
24.1
tblastx


96
464
597
maize
165
72
15.5
tblastx


97
465
598
almond
18
89
20.8
tblastx


98
466
599
sorghum
123
87
20.6
tblastx


99
466
599
sorghum
124
87
20.5
tblastx


100
467
600
sorghum
123
89
19.8
tblastx


101
467
600
sorghum
124
89
19.6
tblastx


102
468
601
sorghum
137
85
12.6
tblastx


103
469

soybean
126
97
22.4
tblastx


104
470
602
soybean
137
92
20.1
tblastx


105
471
603
soybean
137
92
11.2
tblastx


106
472
604
soybean
137
92
20.1
tblastx


107
473
605
soybean
137
85
13.1
tblastx


108
474
606
soybean
137
87
17.0
tblastx


109
475
607
soybean
137
92
11.2
tblastx


110
476
608
soybean
137
85
32.3
tblastx


111
477
609
soybean
18
85
28.0
tblastx


112
478
610
soybean
18
86
28.0
tblastx


113
479
611
soybean
150
86
52.8
tblastx


114
480
612
soybean
150
86
52.8
tblastx


115
481
613
soybean
150
86
52.8
tblastx


116
482
614
rice
137
92
23.6
tblastx


117
483
615
rice
137
93
20.6
tblastx


118
484
616
rice
137
95
23.6
tblastx


119
485
617
sunflower
150
83
44.0
tblastx


120
486

sunflower
161
90
7.8
tblastx


121
487
618
poplar
51
85
45.1
tblastx


122
488
619
poplar
123
89
22.6
tblastx


123
488
619
poplar
124
89
22.5
tblastx


124
489
620
poplar
137
87
8.3
tblastx


125
490
621
poplar
18
86
15.8
tblastx


126
491
622
poplar
165
85
13.2
tblastx


127
492

b_oleracea
29
92
23.3
tblastx


128
493

b_oleracea
50
90
20.7
tblastx


129
494
623
b_oleracea
51
93
51.6
tblastx


130
495
624
b_oleracea
55
91
43.7
tblastx


131
496

b_oleracea
107
84
62.3
tblastx


132
497
625
b_oleracea
126
88
32.6
tblastx


133
498
626
b_oleracea
136
85
45.0
tblastx


134
499
627
b_oleracea
136
87
75.3
tblastx


135
500
628
grape
51
87
46.4
tblastx


136
501

grape
4
84
23.5
tblastx


137
502
629
grape
143
90
21.9
tblastx


138
503
630
grape
150
93
21.4
tblastx


139
504
631
grape
150
84
39.5
tblastx


140
505
632
wheat
123
92
14.9
tblastx


141
505
632
wheat
124
92
14.8
tblastx


142
506
633
wheat
126
82
32.9
tblastx


143
507
634
wheat
126
87
28.0
tblastx


144
508
635
wheat
126
83
32.1
tblastx


145
509
636
wheat
137
91
24.1
tblastx


146
510
637
wheat
137
89
32.3
tblastx


147
511
638
wheat
137
95
11.2
tblastx


148
512
639
wheat
150
73
53.9
tblastx


149
513
640
wheat
161
86
8.8
tblastx


150
514
641
wheat
161
88
7.8
tblastx


151
515
642
wheat
161
92
7.2
tblastx


152
516
643
flax
18
74
15.3
tblastx


153
517
644
tomato
51
85
45.8
tblastx


154
518
645
tomato
123
92
15.9
tblastx


155
518
645
tomato
124
92
15.8
tblastx


156
519
646
tomato
126
94
25.1
tblastx


157
520
647
cotton
51
87
45.8
tblastx


158
521
648
cotton
51
88
46.4
tblastx


159
522
649
cotton
123
91
18.8
tblastx


160
522
649
cotton
124
91
18.7
tblastx


161
650
786
b_rapa
169
88
73.7
blastp


162
651
787
canola
169
93
62.6
blastp


163
652
788
radish
169
88
77.1
blastp


164
653
789
b_oleracea
174
93
55.7
blastp


165
654
790
b_rapa
179
94
70.4
blastp


166
655
791
canola
179
88
100.0
blastp


167
656
792
canola
183
85
84.9
blastp


168
657
793
canola
186
89
96.8
blastp


169
658
794
canola
191
89
51.4
blastp


170
659
795
b_oleracea
192
88
56.4
blastp


171
660
796
canola
194
85
96.0
blastp


172
661
797
b_rapa
195
90
100.0
blastp


173
662
798
canola
195
91
100.0
blastp


174
663
799
canola
200
90
94.7
blastp


175
664
800
canola
200
90
98.9
blastp


176
665
801
b_oleracea
205
87
100.0
blastp


177
666
802
b_rapa
205
87
69.1
blastp


178
667
803
b_rapa
205
86
73.5
blastp


179
668
804
canola
205
86
61.4
blastp


180
669
805
radish
205
87
76.5
blastp


181
670
806
canola
206
93
100.0
blastp


182
671
807
radish
206
93
100.0
blastp


183
672
808
b_oleracea
209
87
52.6
blastp


184
673
809
b_rapa
209
86
51.9
blastp


185
674
810
canola
209
88
100.0
blastp


186
675
811
apple
216
89
100.0
blastp


187
676
812
apple
216
89
100.0
blastp


188
677
813
avocado
216
85
100.0
blastp


189
678
814
b_juncea
216
97
69.1
blastp


190
679
815
b_juncea
216
98
91.2
blastp


191
680
816
b_juncea
216
97
100.0
blastp


192
681
817
b_rapa
216
97
100.0
blastp


193
682
818
bean
216
88
100.0
blastp


194
683
819
brachypodium
216
85
100.0
blastp


195
684
820
cassava
216
91
100.0
blastp


196
685
821
cassava
216
86
100.0
blastp


197
686
822
castorbean
216
88
100.0
blastp


198
687
823
centaurea
216
86
100.0
blastp


199
688
824
centaurea
216
86
100.0
blastp


200
689
825
citrus
216
89
100.0
blastp


201
690
826
citrus
216
89
100.0
blastp


202
691
827
coffea
216
85
100.0
blastp


203
692
828
cotton
216
88
100.0
blastp


204
693
829
iceplant
216
86
100.0
blastp


205
694
830
ipomoea
216
88
100.0
blastp


206
695
831
lettuce
216
85
100.0
blastp


207
696
832
lettuce
216
85
100.0
blastp


208
697
833
lettuce
216
85
100.0
blastp


209
698
834
lettuce
216
85
100.0
blastp


210
699
835
lotus
216
89
100.0
blastp


211
700
836
medicago
216
88
100.0
blastp


212
701
837
pepper
216
86
100.0
blastp


213
702
838
periwinkle
216
88
100.0
blastp


214
703
839
petunia
216
88
100.0
blastp


215
704
840
potato
216
86
97.1
blastp


216
705
841
radish
216
95
100.0
blastp


217
706
842
radish
216
95
100.0
blastp


218
707
843
radish
216
97
100.0
blastp


219
708
844
rose
216
85
100.0
blastp


220
709
845
safflower
216
85
100.0
blastp


221
710
846
safflower
216
85
100.0
blastp


222
711
847
safflower
216
86
100.0
blastp


223
712
848
soybean
216
91
100.0
blastp


224
713
849
soybean
216
91
100.0
blastp


225
714
850
spurge
216
89
97.1
blastp


226
715
851
strawberry
216
86
100.0
blastp


227
716

thellungiella
216
90
92.6
tblastn


228
717
852
tobacco
216
88
100.0
blastp


229
718
853
radish
219
87
100.0
blastp


230
719
854
radish
219
92
54.8
blastp


231
720
855
b_oleracea
220
93
70.8
blastp


232
721
856
b_rapa
220
93
99.1
blastp


233
722
857
canola
220
93
81.5
blastp


234
723
858
radish
220
93
99.1
blastp


235
724
859
radish
220
93
99.4
blastp


236
725
860
arabidopsis
244
96
99.6
blastp


237
726
861
arabidopsis
244
96
99.3
tblastn


238
727
862
b_rapa
246
86
52.1
blastp


239
728
863
canola
246
85
53.4
blastp


240
729
864
canola
258
87
100.0
blastp


241
730
865
canola
266
86
51.5
blastp


242
731
866
b_oleracea
272
85
97.1
blastp


243
732
867
canola
272
85
97.1
blastp


244
733
868
arabidopsis
273
87
99.0
blastp


245
734
869
b_rapa
273
94
81.1
blastp


246
735
870
b_rapa
273
88
60.8
blastp


247
736
871
b_rapa
273
94
65.2
blastp


248
737
872
radish
273
89
75.4
blastp


249
738
873
b_rapa
274
86
81.0
blastp


250
739
874
canola
274
90
100.0
blastp


251
740
875
arabidopsis
277
85
57.7
blastp


252
741
876
canola
277
90
92.8
blastp


253
742
877
radish
277
88
99.1
blastp


254
743
878
b_oleracea
282
87
75.2
blastp


255
744
879
b_rapa
283
94
74.6
blastp


256
745
880
basilicum
283
85
51.7
blastp


257
746
881
canola
283
90
58.1
blastp


258
747
882
canola
284
85
100.0
blastp


259
748
883
arabidopsis
286
88
54.1
blastp


260
749
884
arabidopsis
286
86
98.2
blastp


261
750
885
b_rapa
286
85
59.2
blastp


262
751
886
radish
287
91
100.0
blastp


263
752
887
thellungiella
287
93
94.7
blastp


264
753
888
canola
288
92
60.4
blastp


265
754
889
b_oleracea
297
86
96.1
blastp


266
755
890
canola
297
85
96.1
blastp


267
756
891
canola
297
86
96.1
blastp


268
757
892
b_oleracea
299
85
53.2
blastp


269
758
893
canola
299
85
100.0
blastp


270
759
894
canola
299
85
58.2
blastp


271
760
895
canola
300
94
51.9
blastp


272
761
896
b_rapa
301
85
98.1
blastp


273
762
897
radish
301
86
99.4
blastp


274
763
898
b_rapa
302
85
100.0
blastp


275
764
899
canola
305
92
87.5
blastp


276
765
900
canola
305
92
94.8
blastp


277
766
901
radish
305
92
100.0
blastp


278
767
902
b_rapa
308
91
62.4
blastp


279
768
903
radish
308
91
51.4
blastp


280
769
904
b_rapa
310
94
89.1
blastp


281
770
905
canola
310
93
99.4
blastp


282
771
906
radish
310
92
99.7
blastp


283
772
907
arabidopsis
313
91
99.8
blastp


284
773
908
b_oleracea
317
93
63.9
blastp


285
774
909
canola
317
85
100.0
blastp


286
775
910
arabidopsis
318
85
99.9
blastp


287
776
911
canola
328
85
100.0
blastp


288
777
912
b_oleracea
329
93
100.0
blastp


289
778
913
b_rapa
329
88
100.0
blastp


290
779
914
b_rapa
329
94
100.0
blastp


291
780
915
canola
329
88
100.0
blastp


292
781
916
canola
329
94
100.0
blastp


293
782
917
radish
329
88
54.1
blastp


294
783
918
thellungiella
329
93
88.1
blastp


295
784
919
b_rapa
354
91
100.0
blastp


296
785
920
canola
354
89
67.7
blastp









Example 6
Improved Transgenic Plant Performance
To analyze whether the transgenic plants has performed better, plants were grown in pots with an adequate amount of nutrient and water. The plants were analyzed for their overall size, growth rate, time to inflorescence emergence (bolting) and flowering, seed yield, oil content of seed, weight of 1,000 seeds, dry matter and harvest index (HI-seed yield/dry matter). Transgenic plants performance was compared to control plants grown in parallel under the same conditions. Mock-transgenic plants expressing the uidA reporter gene (GUS-Intron) under the same promoter were used as control.
Parameters were measured as described in Examples 1 and 2.
Statistical Analyses
To identify genes conferring significantly improved plant performance, the results obtained from the transgenic plants were compared to those obtained from control plants. Plant growth rate, plant area, time to bolt, time to flower, weight of 1,000 seeds, seed yield, oil yield, dry matter, and harvest index area data were analyzed using one-way ANOVA. To identify outperforming genes and constructs, results from mix of transformation events or independent events tested were analyzed. For gene versus control analysis T-test was applied, using significance of p<0.05. The JMP statistics software package was used (Version 5.2.1, SAS Institute Inc., Cary, N.C., USA).
Experimental Results
The polynucleotide sequences of the invention were assayed for a number of commercially desired traits.
Tables 19-24 depict analyses of seed yield in plants overexpressing the polynucleotides of the invention under the regulation of a constitutive (35S) or seed specific (napin) promoter. Each Table represents an independent experiment, using at least 5 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.







TABLE 19







Table 19


Genes showing improved plant performance: Seed yield









Seed yield per plant (gr)













SEQ ID NO:


Significance




of
Under
Least
(t-Test



overexpressed
regulation
Sq
compare to
%


Gene Id
polynucleotide
of
Mean
control)
improvement















BDL8
1021
35S
0.264
A
15.9


BDL25
1032
35S
0.239
B
5.2


BDL27
1035
35S
0.238
B
4.8


BDL29
1037
35S
0.235
B
3.4


BDL32a
1038
35S
0.228
B
0.4


CONTROL (GUS_Intron)
1049
35S
0.228
B
0.0
















TABLE 20







Table 20


Genes showing improved plant performance: Seed yield









Seed yield per plant (gr)













SEQ ID NO:


Significance




of
Under
Least
(t-Test



overexpressed
regulation
Sq
compare to
%


Gene Id
polynucleotide
of
Mean
control)
improvement















BDL3
1017
35S
0.447
A
10.9


BDL11
1042
35S
0.420
A
4.2


BDL17
1043
35S
0.426
A
5.8


CONTROL (GUS_Intron)
1049
35S
0.403
A
0.0
















TABLE 21







Table 21


Genes showing improved plant performance: Seed yield









Seed yield per plant (gr)













SEQ ID NO:


Significance




of
Under
Least
(t-Test



overexpressed
regulation
Sq
compare to
%


Gene Id
polynucleotide
of
Mean
control)
improvement















BDL3
1017
Napin
0.492
A
13.4


BDL6
1019
Napin
0.469
B
8.1


BDL28
1036
Napin
0.470
B
8.3


CONTROL (GUS_Intron)
1049
Napin
0.434
B
0.0
















TABLE 22







Table 22


Genes showing improved plant performance: Seed yield









Seed yield per plant (gr)













SEQ ID NO:


Significance




of
Under
Least
(t-Test



overexpressed
regulation
Sq
compare to
%


Gene Id
polynucleotide
of
Mean
control)
improvement















BDL1
1040
35S
0.359
A
23.5


BDL12
1023
35S
0.319
B
9.7


BDL14
1024
35S
0.378
A
30.3


BDL18
1027
35S
0.334
B
15.0


BDL20a
1029
35S
0.325
B
12.0


BDL20b
1044
35S
0.323
B
11.4


BDL26a
1033
35S
0.340
B
17.0


BDL26b
1034
35S
0.318
B
9.7


BDL30
1046
35S
0.340
B
17.2


CONTROL (GUS_Intron)
1049
35S
0.290
B
0.0
















TABLE 23







Table 23


Genes showing improved plant performance: Seed yield









Seed yield per plant (gr)













SEQ ID NO:


Significance




of
Under
Least
(t-Test



overexpressed
regulation
Sq
compare to
%


Gene Id
polynucleotide
of
Mean
control)
improvement















BDL9
1022
35S
0.312
B
10.1


BDL27
1035
35S
0.320
A
13.0


BDL32b
1039
35S
0.334
A
17.8


CONTROL (GUS_Intron)
1049
35S
0.283
B
0.0
















TABLE 24







Table 24


Genes showing improved plant performance: Seed yield









Seed yield per plant (gr)













SEQ ID NO:


Significance




of
Under
Least
(t-Test



overexpressed
regulation
Sq
compare to
%


Gene Id
polynucleotide
of
Mean
control)
improvement















BDL25
1032
Napin
0.41
B
0.1


BDL29
1037
Napin
0.44
B
8.3


BDL32b
1039
Napin
0.46
A
13.0


CONTROL (GUS_Intron)
1049
Napin
0.41
B
0.0









Tables 25-30 depict analyses of oil yield in plants overexpressing the polynucleotides of the invention under the regulation of a constitutive (35S) or seed specific (napin) promoter. Each Table represents an independent experiment, using at least 5 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.







TABLE 25







Table 25


Genes showing improved plant performance: Oil yield









Oil yield per plant (gr)













SEQ ID NO:


Significance




of
Under
Least
(t-Test



overexpressed
regulation
Sq
compare to
%


Gene Id
polynucleotide
of
Mean
control)
improvement















BDL8
1021
35S
0.080
A
17.1


BDL25
1032
35S
0.074
B
8.3


BDL27
1035
35S
0.070
B
2.1


BDL32a
1038
35S
0.069
B
1.1


CONTROL (GUS_Intron)
1049
35S
0.069
B
0.0
















TABLE 26







Table 26


Genes showing improved plant performance: Oil yield









Oil yield per plant (gr)













SEQ ID NO:
Under

Significance
%



of
regula-
Least
(t-Test
im-



overexpressed
tion
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment















BDL3
1017
35S
0.13
A
13.7


BDL11
1042
35S
0.12
A
7.0


BDL17
1043
35S
0.12
A
6.5


CONTROL
1049
35S
0.12
A
0.0


(GUS_Intron)
















TABLE 27







Table 27


Genes showing improved plant performance: Oil yield









Oil yield per plant (gr)













SEQ ID NO:
Under

Significance
%



of
regula-
Least
(t-Test
im-



overexpressed
tion
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment















BDL3
1017
Napin
0.149
A
13.7


BDL6
1019
Napin
0.143
B
9.2


BDL28
1036
Napin
0.138
B
5.3


CONTROL
1049
Napin
0.131
B
0.0


(GUS_Intron)
















TABLE 28







Table 28


Genes showing improved plant performance: Oil yield









Oil yield per plant (gr)













SEQ ID NO:
Under

Significance
%



of
regula-
Least
(t-Test
im-



overexpressed
tion
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment















BDL1
1040
35S
0.108
A*
23.7


BDL12
1023
35S
0.100
B
14.2


BDL14
1024
35S
0.114
A
31.1


BDL18
1027
35S
0.102
B
16.7


BDL20a
1029
35S
0.098
B
12.0


BDL20b
1044
35S
0.098
B
12.1


BDL26a
1033
35S
0.103
B
18.0


BDL26b
1034
35S
0.097
B
11.8


BDL30
1046
35S
0.107
B
22.4


CONTROL
1049
35S
0.087
B
0.0


(GUS_Intron)





*P = 0.07













TABLE 29







Table 29


Genes showing improved plant performance: Oil yield









Oil yield per plant (gr)













SEQ ID NO:
Under

Significance
%



of
regula-
Least
(t-Test
im-



overexpressed
tion
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment















BDL9
1022
35S
0.092
B
6.2


BDL27
1035
35S
0.095
B
9.1


BDL32b
1039
35S
0.101
A
16.4


CONTROL
1049
35S
0.087
B
0.0


(GUS_Intron)
















TABLE 30







Table 30


Genes showing improved plant performance: Oil yield









Oil yield per plant (gr)













SEQ ID NO:
Under

Significance
%



of
regula-
Least
(t-Test
im-



overexpressed
tion
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment















BDL25
1032
Napin
0.12
B
2.2


BDL29
1037
Napin
0.14
A
15.8


BDL32b
1039
Napin
0.15
A
20.6


CONTROL
1049
Napin
0.12
B
0.0


(GUS_Intron)









Tables 31-32 depict analyses of dry matter in plants overexpressing the polynucleotides of the invention under the regulation of a constitutive (35S). Each Table represents an independent experiment, using at least 5 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.







TABLE 31







Table 31


Genes showing improved plant performance: Dry matter











SEQ ID NO:





of

Dry matter per plant (gr)













over-
Under

Significance
%



expressed
regula-
Least
(t-Test
im-



poly-
tion
Sq
compare to
prove-


Gene Id
 nucleotide
of
Mean
control)
ment















BDL6
1019
35S
1.0277
A
7.9


BDL14
1024
35S
1.0444
A
9.7


BDL18
1027
35S
0.985
A
3.4


BDL20b
1044
35S
1.0656
A
11.9


CONTROL
1049
35S
0.9523
A
0.0


(GUS_Intron)
















TABLE 32







Table 32


Genes showing improved plant performance: Dry matter











SEQ ID NO:





of

Dry matter per plant (gr)













over-
Under

Significance
%



expressed
regula-
Least
(t-Test
im-



poly-
tion
Sq
compare to
prove-


Gene Id
nucleotide
of
Mean
control)
ment















BDL3
1017
35S
1.3915
A
3.3


BDL11
1042
35S
1.3638
A
1.2


CONTROL
1049
35S
1.3474
A
0.0


(GUS_Intron)









Tables 33-34 depict analyses of harvest index (HI) in plants overexpressing the polynucleotides of the invention under the regulation of a constitutive (35S) or seed specific (napin) promoter. Each Table represents an independent experiment, using at least 5 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.







TABLE 33







Table 33


Genes showing improved plant performance: harvest index (HI)











SEQ ID NO:





of

HI













over-
Under

Significance
%



expressed
regula-
Least
(t-Test
im-



poly-
tion
Sq
compare to
prove-


Gene Id
nucleotide
of
Mean
control)
ment















BDL3
1017
35S
0.3218
B
7.2


BDL5
1018
35S
0.3094
B
3.0


BDL8
1021
35S
0.3301
B
9.9


BDL11
1042
35S
0.3063
B
2.0


BDL17
1043
35S
0.3526
A
17.5


BDL25
1032
35S
0.3016
B
0.4


CONTROL
1049
35S
0.3002
B
0.0


(GUS_Intron)
















TABLE 34







Table 34


Genes showing improved plant performance: harvest index (HI)









HI













SEQ ID NO:
Under

Significance
%



of
regula-
Least
(t-Test
im-



overexpressed
tion
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment















BDL2
1016
Napin
0.342
B
3.7


BDL3
1017
Napin
0.358
B
8.8


BDL6
1019
Napin
0.365
B
10.9


BDL28
1036
Napin
0.374
A
13.6


CONTROL
1049
Napin
0.329
B
0.0


(GUS_Intron)









Tables 35-38 depict analyses of growth rate in plants overexpressing the polynucleotides of the invention under the regulation of a constitutive (35S). Each Table represents an independent experiment, using at least 5 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.







TABLE 35







Table 35


Genes showing improved plant performance: Growth rate









Growth rate (cm2/day)













SEQ ID NO:
Under

Significance
%



of
regula-
Least
(t-Test
im-



overexpressed
tion
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment















BDL14
1024
35S
2.48
A
6.4


BDL18
1027
35S
2.41
A
3.5


BDL20a
1029
35S
2.50
A
7.1


CONTROL
1049
35S
2.33
A
0.0


(GUS_Intron)
















TABLE 36







Table 36


Genes showing improved plant performance: Growth rate









Growth rate (cm2/day)













SEQ ID NO:
Under

Significance
%



of
regula-
Least
(t-Test
im-



overexpressed
tion
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment















BDL11
1042
35S
1.80
A
15.4


CONTROL
1049
35S
1.56
A
0.0


(GUS_Intron)
















TABLE 37







Table 37


Genes showing improved plant performance: Growth rate









Growth rate (cm2/day)













SEQ ID NO:
Under

Significance
%



of
regula-
Least
(t-Test
im-



overexpressed
tion
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment















BDL1
1040
35S
1.81
A*
17.1


BDL12
1023
35S
1.58
B
2.0


BDL14
1024
35S
1.95
A
26.3


BDL18
1027
35S
1.59
B
3.1


BDL20b
1044
35S
1.77
B
14.6


BDL26a
1033
35S
1.57
B
1.9


BDL30
1046
35S
1.75
B
13.0


CONTROL
1049
35S
1.55
B
0.0


(GUS_Intron)





*P = 0.06













TABLE 38







Table 38


Genes showing improved plant performance: Growth rate









Growth rate (cm2/day)













SEQ ID NO:
Under

Significance
%



of
regula-
Least
(t-Test
im-



overexpressed
tion
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment















BDL32b
1039
35S
1.19
A
0.8


CONTROL
1049
35S
1.18
A
0.0


(GUS_Intron)









Tables 39-42 depict analyses of rosette area in plants overexpressing the polynucleotides of the invention under the regulation of a constitutive (35S) or seed specific (napin) promoter. Each Table represents an independent experiment, using at least 5 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.







TABLE 39







Table 39:


Genes showing improved plant performance: Rossete area









Rosette area (cm2)













SEQ ID NO:
Under

Significance
%



of
regula-
Least
(t-Test
im-



overexpressed
tion
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment















BDL6
1019
35S
9.73
A
−10.2


BDL7
1020
35S
8.52
A
−21.4


BDL14
1024
35S
11.83
A
9.2


BDL18
1027
35S
11.62
A
7.3


BDL20a
1029
35S
11.90
A
9.9


BDL20b
1044
35S
11.02
B
1.7


BDL24
1045
35S
8.12
A
−25.1


CONTROL
1049
35S
10.83
B
0.0


(GUS_Intron)





Increase in rosette area means better soil coverage and reduced water loss from soil. Decrease in rosette area means more plants could be put per area increasing yield.













TABLE 40







Table 40:


Genes showing improved plant performance: Rossete area









Rosette area (cm2)













SEQ ID NO:
Under

Significance
%



of
regula-
Least
(t-Test
im-



overexpressed
tion
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment















BDL3
1017
35S
11.99
A
−3.6


BDL5
1018
35S
11.36
A
−8.6


BDL8
1021
35S
9.31
B
−25.1


BDL11
1042
35S
14.09
A
13.2


BDL16
1026
35S
10.91
A
−12.3


BDL17
1043
35S
9.97
B
−19.9


BDL25
1032
35S
7.95
B
−36.1


CONTROL
1049
35S
12.44
A
0.0


(GUS_Intron)





Increase in rosette area means better soil coverage and reduced water loss from soil. Decrease in rosette area means more plants could be put per area increasing yield.













TABLE 41







Table 41:


Genes showing improved plant performance: Rossete area









Rosette area (cm2)













SEQ ID NO:
Under

Significance
%



of
re-
Least
(t-Test
im-



overexpressed
gulation
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment















BDL1
1040
35S
9.13
B
12.4


BDL12
1023
35S
7.92
B
−2.5


BDL14
1024
35S
9.96
A
22.7


BDL18
1027
35S
8.63
B
6.3


BDL20a
1029
35S
8.03
B
−1.1


BDL20b
1044
35S
9.14
B
12.6


BDL26a
1033
35S
8.51
B
4.8


BDL26b
1034
35S
7.92
B
−2.5


BDL30
1046
35S
9.28
A
14.2


CONTROL
1049
35S
8.12
B
0.0


(GUS_Intron)





Increase in rosette area means better soil coverage and reduced water loss from soil. Decrease in rosette area means more plants could be put per area increasing yield.













TABLE 42







Table 42:


Genes showing improved plant performance: Rossete area









Rosette area (cm2)













SEQ ID NO:
Under

Significance
%



of
re-
Least
(t-Test
im-



overexpressed
gulation
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment















BDL9
1022
35S
5.05
B
−17.0


BDL21
1030
35S
4.77
B
−21.5


BDL27
1035
35S
5.22
B
−14.2


BDL32b
1039
35S
6.19
A
1.8


CONTROL
1049
35S
6.08
A
0.0


(GUS_Intron)





Increase in rosette area means better soil coverage and reduced water loss from soil. Decrease in rosette area means more plants could be put per area increasing yield.






Tables 43-49 depict analyses of oil % in seed in plants overexpressing the polynucleotides of the invention under the regulation of a constitutive (35S) or seed specific (napin) promoter. Each Table represents an independent experiment, using at least 5 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.







TABLE 43







Table 43


Genes showing improved plant performance: oil % in seed









Oil % in seed
















Signi-




SEQ ID NO:
Under

ficance
%



of
re-
Least
(t-Test
im-



overexpressed
gulation
Sq
compare
prove-


Gene Id
polynucleotide
of
Mean
to control)
ment















BDL8
1021
35S
30.542
A
1.1


BDL25
1032
35S
31.09
A
2.9


BDL32a
1038
35S
30.264
A
0.2


CONTROL
1049
35S
30.21
A
0.0


(GUS_Intron)
















TABLE 44







Table 44


Genes showing improved plant performance: oil % in seed









Oil % in seed
















Signi-




SEQ ID NO:
Under

ficance
%



of
re-
Least
(t-Test
im-



overexpressed
gulation
Sq
compare
prove-


Gene Id
polynucleotide
of
Mean
to control)
ment















BDL6
1019
35S
30.565
B
0.7


BDL14
1024
35S
31.31
B
3.1


BDL18
1027
35S
30.56
B
0.7


BDL20a
1029
35S
31.393
B
3.4


BDL20b
1044
35S
31.928
A
5.2


BDL24
1045
35S
31.02
B
2.2


CONTROL
1049
35S
30.355
B
0.0


(GUS_Intron)
















TABLE 45







Table 45


Genes showing improved plant performance: oil % in seed









Oil % in seed
















Signi-




SEQ ID NO:
Under

ficance
%



of
re-
Least
(t-Test
im-



overexpressed
gulation
Sq
compare
prove-


Gene Id
polynucleotide
of
Mean
to control)
ment















BDL3
1017
35S
29.39
A
2.1


BDL5
1018
35S
29.29
A
1.8


BDL8
1021
35S
28.903
A
0.4


BDL11
1042
35S
29.216
A
1.5


BDL17
1043
35S
28.904
A
0.4


BDL25
1032
35S
29.514
A
2.6


CONTROL
1049
35S
28.78
A
0


(GUS_Intron)
















TABLE 46







Table 46


Genes showing improved plant performance: oil % in seed









Oil % in seed













SEQ ID NO:
Under

Signi-ficance
%



of
re-
Least
(t-Test
im-



overexpressed
gulation
Sq
compare
prove-


Gene Id
polynucleotide
of
Mean
to control)
ment















BDL3
1017
Napin
30.34
A
0.46


BDL6
1019
Napin
30.45
A
0.83


BDL28
1036
Napin
29.49
A
2.35


CONTROL
1049
Napin
30.2
A
0


(GUS_Intron)
















TABLE 47







Table 47


Genes showing improved plant performance: oil % in seed









Oil % in seed













SEQ ID NO:
Under

Significance
%



of
re-
Least
(t-Test
im-



overexpressed
gulation
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment





BDL12
1023
35S
31.30
A
3.7


BDL14
1024
35S
30.27
A
0.3


BDL18
1027
35S
30.39
A
0.7


BDL26a
1033
35S
30.33
A
0.5


BDL26b
1034
35S
30.43
A
0.8


BDL30
1046
35S
31.42
A
4.1


CONTROL
1049
35S
30.19
A
0.0


(GUS_Intron)
















TABLE 48







Table 48


Genes showing improved plant performance: oil % in seed









Oil % in seed













SEQ ID NO:
Under

Significance
%



of
re-
Least
(t-Test
im-



overexpressed
gulation
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment





BDL21
1030
35S
30.55
A
1.8


BDL32b
1039
35S
30.35
A
1.1


CONTROL
1049
35S
30.01
A
0.0


(GUS_Intron)
















TABLE 49







Table 49


Genes showing improved plant performance: oil % in seed









Oil % in seed













SEQ ID NO:
Under

Significance
%



of
re-
Least
(t-Test
im-



overexpressed
gulation
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment





BDL25
1032
Napin
30.34
B
1.5


BDL29
1037
Napin
31.54
A
5.5


BDL32b
1039
Napin
31.69
A
6.0


CONTROL
1049
Napin
29.90
B
0.0


(GUS_Intron)









Tables 50-55 depict analyses of weight of 1000 seeds in plants overexpressing the polynucleotides of the invention under the regulation of a constitutive (35S) or seed specific (napin) promoter. Each Table represents an independent experiment, using at least 5 independent events per gene. Genes not connected by same letter as the control (A, B,) are significantly different from the control.







TABLE 50







Table 50


Genes showing improved plant performance: weight of 1,000 seeds









Weight of 1000 seeds (gr)













SEQ ID NO:
Under

Significance
%



of
re-
Least
(t-Test
im-



overexpressed
gulation
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment















BDL8
1021
35S
0.019
B
9.1


BDL21
1030
35S
0.018
B
0.3


BDL25
1032
35S
0.018
B
0.4


BDL32a
1038
35S
0.019
B
5.5


BDL32b
1039
35S
0.020
A
14.2


CONTROL
1049
35S
0.018
B
0.0


(GUS_Intron)
















TABLE 51







Table 51


Genes showing improved plant performance: weight of 1,000 seeds









Weight of 1000 seeds (gr)













SEQ ID NO:
Under

Significance
%



of
re-
Least
(t-Test
im-



overexpressed
gulation
Sq
compare to
prove-


Gene Id
polynucleotide
of
Mean
control)
ment















BDL6
1019
35S
0.019
B
7.1


BDL7
1020
35S
0.018
B
3.8


BDL14
1024
35S
0.019
B
6.1


BDL18
1027
35S
0.019
B
8.2


BDL20b
1044
35S
0.020
A
14.5


BDL24
1045
35S
0.018
B
4.5


CONTROL
1049
35S
0.018
B
0.0


(GUS_Intron)
















TABLE 52







Table 52


Genes showing improved plant performance: weight of 1,000 seeds









Weight of 1000 seeds (gr)
















Signi-




SEQ ID NO:
Under

ficance
%



of
re-
Least
(t-Test
im-



overexpressed
gulation
Sq
compare
prove-


Gene Id
polynucleotide
of
Mean
to control)
ment















BDL3
1017
35S
0.0214
B
5.8


BDL5
1018
35S
0.0205
B
1.1


BDL11
1042
35S
0.0235
A
15.7


CONTROL
1049
35S
0.0203
B
0


(GUS_Intron)
















TABLE 53







Table 53


Genes showing improved plant performance: weight of 1,000 seeds









Weight of 1000 seeds (gr)
















Signi-




SEQ ID NO:
Under

ficance
%



of
re-
Least
(t-Test
im-



overexpressed
gulation
Sq
compare
prove-


Gene Id
polynucleotide
of
Mean
to control)
ment















BDL2
1016
Napin
0.0290
A
30.7


BDL6
1019
Napin
0.0232
B
4.3


BDL14
1024
Napin
0.0227
B
2.3


BDL28
1036
Napin
0.0224
B
1.0


CONTROL
1049
Napin
0.0222
B
0.0


(GUS_Intron)





















TABLE 54







Table 54


Genes showing improved plant performance: weight of 1,000 seeds









Weight of 1000 seeds (gr)
















Signi-




SEQ ID NO:
Under

ficance
%



of
re-
Least
(t-Test
im-



overexpressed
gulation
Sq
compare
prove-


Gene Id
polynucleotide
of
Mean
to control)
ment





BDL1
1040
35S
0.0235
B
0.6


BDL12
1023
35S
0.0234
B
0.1


BDL30
1046
35S
0.0252
A
7.8


CONTROL
1049
35S
0.0234
B
0.0


(GUS_Intron)
















TABLE 55







Table 55


Genes showing improved plant performance: weight of 1,000 seeds









Weight of 1000 seeds (gr)
















Signi-




SEQ ID NO:
Under

ficance
%



of
re-
Least
(t-Test
im-



overexpressed
gulation
Sq
compare
prove-


Gene Id
polynucleotide
of
Mean
to control)
ment





BDL12
1023
Napin
0.0206
B
0.2


BDL18
1027
Napin
0.0214
B
4.0


BDL25
1032
Napin
0.0208
B
1.1


BDL27
1035
Napin
0.0211
B
2.8


BDL29
1037
Napin
0.0211
B
2.6


BDL32b
1039
Napin
0.0224
A
9.3


CONTROL
1049
Napin
0.0205
B
0.0


(GUS_Intron)









Taking into account the results obtained using these assays, the following BDL genes, when exogenously introduced into plants, induced a significant improvement in:
1. Seed yield: BDL1, BDL3, BDL8, BDL14, BDL27, BDL32b.
2. Oil yield: BDL1, BDL3, BDL8, BDL14, BDL29, BDL32b.
3. Harvest Index: BDL17, BDL28.
4. Growth rate: BDL1, BDL14.
5. Roseate area: BDL14, BDL18, BDL20a, BDL30.
6. Oil % in seed: BDL20b, BDL29, BDL32b.
7. Weight of 1000 Seeds: BDL2, BDL11, BDL20b, BDL30, BDL32b
Example 7
Increased Oil Content in Leaves
In general, oil is composed mainly of tri acyl glycerols (TAG). Seeds of Arabidopsis and other oilseed contain high amounts of TAG. Usually the TAGs are being degraded into sugars through the germination process. Cermac and Benning (Plant journal 2004; 40, 575-585) in their paper used an assay to quantify TAG production in seedlings grown on sucrose. They used this stage of development since normally seedlings not present TAG in high levels. In their study, they demonstrated the importance of the wrinkled gene in the control of oil production by showing that transgenic seedlings overexpressing the wrinkled cDNA produce high amounts of TAG.
Materials and Experimental Methods
The present inventors used the assay of Cermac and Benning assay (Cermac and Benning, Plant journal 2004; 40, 575-585) with minor changes to qualify the effect of the transgenes identified herein for their ability to increase TAG in seedlings, similar to the wrinkled gene.
For triacylglycerol quantification T2 transgenic seedlings were grown on 1⁄2 MS medium (Murashige and Skoog, 1962 Plant Physiology 15, 473-497), pH 5.9, 2% sucrose and 0.7% agar. Seeds were sterilized by evaporating of 100 ml bleach (10%) and 4 ml HCl (37%) for 90 minutes in close plastic chamber of 5.5 L vol. Glufosinate-ammonium and kanamaycin were added to final concentrations of 20 μg ml−1 for glufosinate-ammonium and 50 μg ml−1 kanamaycin. Follow sterilization, seeds were sown on agar plates. Plates were incubated for 3 days in the dark at 4° C. before placing them in a growth room. The conditions at the growth room were of 24° C., light period of 12 hour and a dark period of 12 hour. Seedlings were grown for 10-11 days.
Equal amount of 11 days old seedlings were ground in 1.5-mL polypropylene test tubes with a glass rod, and lipids were extracted in 50 mL of chloroform:methanol:formic acid (10:10:1, v/v). Following the extraction with 12.5 mL of 1 M KCl and 0.2 M H3PO4 and separation of the organic and aqueous phases by centrifugation at 16,000 g for 5 minutes, the lipids in the lower phase were separated on a silica TLC plate (Si 250 PA, J. T. Baker, Philipsburg, N.J.) developed with 80:20:1, petroleum ether:ethyl ether:acetic acid. Lipids were visualized by staining with iodine vapor.
As positive controls the following were used: The naturally produced TriAcyl Glycerols—extracted from seeds of wild-type arabidopsis (lane 5, FIG. 3); and transgenic seedlings expressing WRINKLED cDNA (SEQ ID NO:1050), which are known to produce significant amounts of TriAcyl Glycerols in leaves (Cernac A and Benning C, The Plant Journal 2004, 40, 575-585). As negative controls the transgenic seedlings expressing GUS-Intron gene (SEQ ID NO:1049) were used.
Experimental Results
FIG. 3 depicts iodine vapor staining of lipids isolated from the transgenic plants of independent events (BDL9, WRINKLED) or pool of events (GUS-Intron) expressing the following genes according to Table 56, hereinbelow. An independent Event represents a single stable transformed plant that resulted from random integration of the transformed construct in the Arabidopsis genome. Progenies of an event harboring the transformed construct were used for the gene evaluation separately as in the case of BDL9 and Wrinkeld genes or as pool of events in case of GUS-Intron.







TABLE 56







Table 56









Lane
Description of plant
Name of upregulated gene


No.
transformation
or control plant





1
Transformed with SEQ ID
BDL9 Event 1



NO: 1022


2
Transformed with SEQ ID
BDL9 Event 2



NO: 1022


3
Transformed with SEQ ID
BDL9 Event 3



NO: 1022


4
Transformed plant with
GUS-Intron



control vector SEQ ID



NO: 1049


5
Untransformed plant
SEED


6
Transformed with SEQ ID
Wrinkled Event 1



NO: 1050


7
Transformed with SEQ ID
Wrinkled Event 2



NO: 1050


8
Transformed with SEQ ID
Wrinkled Event 3



NO: 1050









As shown in FIG. 3, transgenic plants expressing the BDL9 gene (SEQ ID NO:1022) produce a significantly higher oil content as compared to the oil content produced by control plants expressing the GUS-intron (SEQ ID NO:1049). In addition, the amount of oil produced by the BDL9-transgenic plants (e.g., FIG. 3, lane 2) is comparable to that produced by seeds (FIG. 3, lane 5) or by transgenic plants expressing the known Wrinkled gene (FIG. 3, lane 6).
SUMMARY
The present inventors have identified genes from Arabidopsis thaliana, which are important for embryogenesis, seed development and oil synthesis and accumulation. These genes, when over-expressed in plants, can effectively increase oil content in seeds or leaves or any other plant part. Tissue or embryonic specific expression of the genes in plants can result in optimal increase oil content in any plant tissue. Thus, the transgenes can be expressed in certain stages of embryo, seed development or to developmental stages of any target tissue, defined as the oil accumulating tissue. This unique expression profile can be achieved by using specific promoters, such as developmental promoters, seed expressing and seed specific promoters.
The present inventors demonstrated improvement of oil synthesis and accumulation by increasing seed size, which enabled the synthesized oil to be accumulated to larger extent, within a larger volume.
In addition, increase of oil can be achieved by controlling embryogenesis. Oil is accumulated in the embryo of developed seed. Some of the early embryo development genes are directly in charge of the regulation of oil synthesis and storage.
The identified genes of the invention can improve oil yield in general, and more specifically oil synthesis, oil accumulation and seed size. The output of the bioinformatics method described herein is a set of genes highly predicted to improve oil and seed yields by modifying their expression. Although each gene is predicted to have its own impact, modifying the mode of expression of more than one gene is expected to provide an additive or synergistic effect on the plant seed/oil yield performance. Altering the expression of each gene described here alone or set of genes together increases the overall oil yield, hence expects to decrease vegetable oil price, as well as to increase productivity.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.
CD-ROM Content
The following lists the file content of the CD-ROM which is enclosed herewith and filed with the application. File information is provided as: File name/byte size/date of creation/operating system/machine format.
CD-ROM1 (1 file of SEQUENCE LISTING):
1. “40040_ST25.txt”/1,820,000 bytes/Apr. 9, 2008/Microsoft Windows XP Professional/PC.

## Claims

1. A method of increasing oil content, growth rate, biomass, yield and/or vigor of a plant, comprising introducing into the plant an exogenous polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 199, thereby increasing the oil content, growth rate, biomass, yield and/or vigor of the plant.

2. A method of producing oil, comprising:
(a) providing the plant according to claim 1; and
(b) extracting the oil from the plant;
thereby producing the oil.

3. The method of claim 1, wherein said polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 1039 or 34.

4. The method of claim 1, wherein said polynucleotide is set forth in SEQ ID NO: 1039 or 34.

5. The method of claim 1, wherein the oil comprises a seed oil.

6. The method of claim 1, wherein the oil comprises a vegetative portion oil.

7. The method of claim 1, wherein the plant is selected from the group consisting of maize, canola, soybean, cotton, rice and wheat.

8. The method of claim 1, wherein said exogenous polynucleotide is comprised in a nucleic acid construct under the regulation of a promoter selected from the group consisting of a constitutive promoter, a tissue-specific, a developmental and an embryo-specific promoter.

9. A transgenic plant having increased oil content, growth rate, biomass, yield and/or vigor when compared to a wild type plant of the same species which is grown under the same growth conditions, comprising a nucleic acid construct, which comprises an exogenous polynucleotide encoding the polypeptide set forth in SEQ ID NO:199, and a promoter for directing transcription of said polynucleotide in the plant.

10. The transgenic plant of claim 9, wherein said exogenous polynucleotide is set forth in SEQ ID NO:1039 or 34.

11. The method of claim 1, further comprising selecting a plant having the increased oil content, growth rate, biomass, yield and/or vigor as compared to a wild type plant of the same species which is grown under the same growth conditions.