# Patent

## Title
Rubisco promoters and uses thereof

## Abstract

The present invention is related to a family of novel spatiotemporally active Rubisco promoters (SEQ ID NO: 1, 2, 3) obtainable from light grown Brassica seedlings. Furthermore the invention is related to transgene expression in specific plant organs or at specific stages of plant development. DNA constructs and expression cassettes comprising at least one of the promoter sequences functionally fused in frame with genes encoding desired gene products are disclosed. Seeds from transformed homologous and herterologous plants and from subsequent generation of the transformed plants are collected and used for efficient production of desired gene products, especially in contained conditions.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This non-provisional patent application claims priority date of the U.S. provisional patent application No. 60/484,707 filed on Jul. 3rd, 2003.


SEQUENCE DATA
This application contains sequence data provided on a computer readable diskette and as a paper version. The paper version of the sequence data is identical to the data provided on the diskette.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to transgenic plants. More specifically, it relates to transgene expression at a specific stage of plant development. Even more specifically the invention relates to novel Rubisco promoters of Brassica species, and DNA constructs or expression cassettes comprising at least one of the promoters for transformation of homologous or heterologous plants for efficient production of gene products, particularly for contained use.
2. Description of Related Art
Assimilation and conversion of atmospheric carbon dioxide via the reaction with ribulose-1,5-bisphosphate into phosphoglycerate strictly depends on the activity of Rubisco enzyme. Structurally it consists of eight small subunits (SSU) and eight large subunits (LSU). The SSU proteins are encoded by several genes located in plant nuclear genome, while LSU genes are found in plastid genome. The number of Rubisco SSU genes in different plants varies form four copies up to fifteen copies or more in some polyploidy genomes. There are at least four copies of Rubisco SSU genes in Arabidobsis thaliana and twelve or even more copies in wheat.
Based on their structure and function these nuclear genes may form multigenic families. The structure of these families is extensively studied for example in Arabidopsis and tomato plants. In tomato there are five Rubisco SSU (rbcS) genes located in three chromosomal loci, one of these genes being situated in chromosome 3, and the other four in chromosome 2. Moreover, three of the genes are known to be organized in tandem array within a 10 kb region. The same situation is known in Arabidopsis thaliana rbcS gene family.
In Brassica napus coding sequences, 5′ and 3′ regulatory regions of three rbcS genes have been cloned and sequenced (accession numbers X75334, X55937, X61097). It has been suggested that Brassica napus contains no more than three rbcS genes.
There are also cDNA sequences obtained from mRNA of Brassica napus rbcS genes (one of them has been published with accession number X07367).
There have also been attempts to clarify the fine structure of the promoters of Brassica napus rbcS genes. Essential regulatory elements, like TATA, G-, GS, and I-boxes, necessary for basic activity and light regulation of the promoters has been described, and also putative silencer elements in one of the promoters has been studied.
Clearly, however the information available on gene structure and activity does not enable identification of differently expressing members of Brassica rbcS gene family in different plant tissues or development stages and under various environmental conditions.
Transgenic plants are used increasingly for production of various desired proteins and other gene products. An important aspect in designing transgenic plants is how to obtain significant levels of transgene expression in desired plant tissues or at desired plant development phases. The role of promoters is essentially important in this aspect and there is a clear need for new plant promoters.
Outchkourov et al. (2002) cloned an abundantly transcribed rbcS1 of the Rubisco small-subunit gene family of Chrysanthemum species (Chrysantemum morifolium Ramat.). Outchkourov et al. showed that tobacco plants transformed with a gene cassette containing uidA gene under the control of rbsS1-promoter provided GUS levels up to 10% of total soluble proteins in the leaves.
Even if the Chrysanthemum Rubisco promoter cloned by Outchkourov gives high protein expression levels in tobacco leaves it may not fit for purposes where protein production is needed at a specific stage of development, such as protein production in seedlings or in germinating seeds.
Plant seeds and cotyledons are particularly advantageous for production because at early cotyledon development, nutritional sources from seeds, including amino acids and oils are abundantly available as raw material for de novo synthesis and the recovery of the expressed gene products from the substrate solution is easier and more efficient than from harvested leaves. Production of transgene expression products in germinating seeds is an approach that can be realized in contained manner in a suitable laboratory. For such purposes a promoter being active during seed germination or cotyledon development is essential and the published rbcS promoters are not applicable.
For purposes of producing transgenic expression products in developing sprouts there is a need for promoters expressing strongly at late stages of cotyledon development, because then the leaf size is bigger than at early cotyledon development and the material needed for compound collection is easier and more efficient to harvest.
Moreover, another important prerequisite for foreign protein production in plant tissues is high expression level of the proteins, and therefore there is a clear need for new promoters giving high protein content at a specific development stage and/or in a specific organ of a plant.
A promoter, which is active during seed germination or cotyledon development, is of particular importance in the production of transgene products in contained conditions in a suitable laboratory. None of the rbcS promoters so far published, are however applicable for said purpose. New promoters for different new applications are therefore clearly needed.
SUMMARY OF THE INVENTION
The present invention provides a solution for the problems encountered by industry seeking for plant promoters giving high expression rates and being specific for a certain development stage or a certain organ.
An objective of the present invention is to obtain a spatiotemporally targeted high expression level of desired gene product or protein.
The present invention provides new rbcS promoters, which are obtainable from a selection of rbcS gene sequences identified by their abundant expression in light-grown cotyledons of Brassica rapa species.
The present invention provides new promoters according to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.
The present invention provides fusion constructs comprising new promoters functionally linked to reporter genes. The promoters used in the expression cassettes, and whole constructs provide both homologous and heterologous systems, which are useful for transforming homologous and heterologous plants and confer the capacity of efficient production of homologous and heterologous proteins.
The present invention provides transgenic seedlings of various plant species for production of foreign proteins and peptides.
Furthermore, the present invention provides transgenic Camelina plants, particularly Camelina sativa plants producing high levels of desired proteins in germinating seedlings.
The present invention provides transgenic plants producing high levels of gene products encoded by naturally isolated genes or synthetic or semisynthetic genes in germinating seedlings. Such gene products may for example be Human Serum Albumin (HSA), antibodies and medically active proteins.
The DNA constructs or cassettes are used for transforming host plants, which are exemplified by Brassica and Camelina species. The transformed zero generation plant comprises one or more of the expression cassettes according to the present disclosure and seeds of the zero generation plants may be used for providing further generations of transgenic plants but the seeds may also be used directly for production of the desired gene products in the seedlings during seed germination and cotyledon development.
Therefore, the present invention is also related to transformed plants, subsequent generations thereof as well as seeds and seedlings carrying at least one expression cassette having at least one of the novel promoters.
The present invention also discloses a method for producing further promoters having properties which are substantially similar to those of the family of Rubisco promoters disclosed here. The method comprises the step of evaluating the expression in light grown seedlings, identifying the most highly expressed genes and selecting from said genes promoters having the capability to direct gene expression into developing cotyledons.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates relative quantities of different 3′UTR-type rbcS-genes in germinating seeds of Brassica rapa. 
FIG. 2A illustrates alignments of cloned Brassica rapa rbcS promoters (300 bp). Global DNA alignment. Reference molecule rbcS-300 nt. Region 1 to 300. Sequences 5. Scoring matrix: Linear (Mismatch 2, OpenGAp 4m ExtGap 1). Sequence view: Similarity Format, Color areas of high matches at the same base position.
FIG. 2B illustrates alignments of cloned Brassica rapa rbcS-4A (SEQ ID NO: 1) and rbcS-4B promoters (SEQ ID NO: 2) promoters (300 bp), in which the horizontal boxes represent homologous regions. Global DNA alignment. Reference molecule: rbcS-300 nt. Region 1 to 300. Sequences 5. Scoring matrix: Linear (Mismatch 2, OpenGAp 4m ExtGap 1). Sequence view: Similarity Format, Color areas of high matches at the same position.
FIG. 2C illustrates alignments of promoter (upper part) and 3′UTRs (two lowermost alignings) of Brassica rapa rbcS-2 (SEQ ID NO: 3) and B. napus rbcS (X61097). Global DNA alignment. Reference molecule: rbcS-300 nt. Region 1 to 300. Sequences 5. Scoring matrix: Linear (Mismatch 2, OpenGAp 4m ExtGap 1). Sequence view: Similarity Format, Color areas of high matches at the same base position.
FIG. 3 illustrates alignments of 1 kb sequences of Rbcs-4A promoter (SEQ ID NO: 1) (upper line) and Brassica napus Rubisco promoter published with access number X61097 (lower line). The sequences show 52% dissimilarity.
FIG. 4 illustrates alignment of 1 kb sequences of rbcS-4A promoter (SEQ ID NO: 1) (upper line) and Chrysanthemum rbcS1 promoter (lower line) published with access number AY163904. The sequences show 57% dissimilarity.
FIG. 5 illustrates sequences of the forward and reverse primers, specific for unique parts of different rubisco 3′UTR types. The primers show that it is possible to discriminate between the different 3′UTR species in real-time PCR. Forward primers are divided in two parts (underlined italics and bold): the left part of each primer corresponds to the last few nucleotides of relevant Rubisco coding regions, and the right part corresponds to specific 3′UTR sequence. All the reverse primers anneal to specific 3′UTR regions. All the amplicon sizes vary in 80 to 100 nt rage.
FIG. 6 is a scheme of consensus regulatory elements found in rbcS-2 (SEQ ID NO: 3) and rbcS-4A (SEQ ID NO: 1) promoters.
FIG. 7 demonstrates the amount of mRNA from rbcS-2, rbcS-4 and UidA (GUS) using real-time PCR in transgenic Brassica plants transformed with rbcS-2-GUS and rbcS-4A-GUS (Rubisco promoter sequences SEQ ID NO:3 and SEQ ID NO:1, respectively).
FIG. 8 illustrates real-Time PCR data indicating the amount of HSA mRNA molecules in RNA samples of transgenic tobacco plants transformed with RbcS-4A-HSA and rbcS-2-HSA.
FIG. 9 illustrates Real-Time PCR data of the expression levels of different Rubisco promoters in Brassica napus seeds, germinated for various times (0 to 4 days). Data represents numbers of molecules per a sample (100 ng of total RNA).
FIG. 10 illustrates the GUS expression in transgenic tobacco plants transformed with rbcS-4B UidA (Rubisco promoter sequence SEQ ID NO: 2). Plants transformed with 35Sp-GUS were used as controls.
FIG. 11 illustrates the GUS expression under Rubisco promoter rbcS-4A (SEQ ID NO: 1) during germination of Brassica seeds in constant light at 24° C. or 30° C.
FIG. 12 illustrates Northern data, showing HSA mRNA content in transgenic Camelina plants.
FIG. 13 illustrates HSA mRNA content in germinating seeds of transgenic Brassica napus plants at various times.
FIG. 14 illustrates the amount of HSA protein in transgenic Brassica napus (B.n.), Camelina sativa (C.s.) and tobacco plants (N.T) calculated as % TSP (total soluble protein). Averages with minimum and maximum values are presented.
FIG. 15 illustrates the GUS activity in transgenic tobacco plants transformed with constructs containing GUS as the reporter gene and either full length rbcS-2 ((SEQ ID NO:3) promoter (1.6 kb) or a truncated versions of the promoter. Plants transformed with 35Sp-GUS construct were used as a positive control.
FIG. 16A is a Northern blot showing the synthesis of Rubisco SSU mRNA in Brassica seedling after sprouting in an airlift tank for 12 to 168 hours.
FIG. 16B shows unlabelled Rubisco RNA of germinating Brassica seedlings produced by in vitro transcription when loaded on the same filter as the control. The amount of control RNA is indicated in pg.
FIG. 17 illustrates homological regions of the sequence of Rbcs-4A (SEQ ID NO: 1) promoter and Brassica sequence published in Brassica genome project with accession number BH 484651 and cDNA sequence (accession number CD811761) of Brassica napus. 
FIG. 18A illustrates quantitative GUS-activity data for transgenic Camelina and tobacco plants transformed with RbcS-2-GUS or RbcS-4-GUS constructs. Plants transformed with 35Sp-GUS are used as positive controls.
FIG. 18B illustrates Northern blot data, obtained from transgenic Camelina and tobacco plants carrying TNFR-constructs. Rbcs-2-TNFR-Fc-56UTRshort contains rbcS-2 (SEQ ID NO:3) promoter, TNFR part (489 nt) (SEQ ID NO:19), linked to the part of IgG1 heavy chain constant region (CH2+CH3 domains), and terminator from natural rbcS-4 gene (0.5 kb in length). Rbcs-2-TNFR-FcKDEL-56UTRshort is the same construct, but there is also KDEL signal (12 nt) (SEQ ID NO:24) after Fc region (just before STOP codon). Rbcs-4-TNFR-Fc-56UTRlong contains rbcS-4A (SEQ ID NO:1) promoter, TNFR part (SEQ ID NO: 19), linked to the part of IgG1 heavy chain constant region (CH2+CH3 domains), and terminator from natural rbcS-4 gene (2 kb length). Rbcs-4-TNFR-FcKDEL-56UTRlong is the same as previous construct, but there is also KDEL signal (12 nt) (SEQ ID NO:24) after Fc region Oust before STOP codon).
FIG. 19 depicts an artificial HSA gene (SEQ ID NO: 15). The sequence of natural human gene (cDNA, i.e. only exons and no introns) was codon-optimized.
FIG. 20 depicts an artificial light chain (anti-hevein 1C2) coding region (SEQ ID NO: 16). The sequence consists of three parts:

    a) 66 nt length sequence coding for mouse signal peptide (22 amino acids). The sequence has a 100% similarity with the partial sequence having the accession number AF078548;
    b) 324 nt long light chain anti hevein 1C2 antigen variable region isolated from a phage display library obtained from VTT, Espoo, Finland). Genbank accession number AB095291 shows 100% similarity to the 16–305 nt region of SEQ ID NO:16); and
    c) 324 nt long kappa light chain constant region, which has a 100% similarity with the sequence having accession number BC063599.


FIG. 21 depicts and artificial rbcS-4 terminator sequence (SEQ ID NO:17), which was originally cloned from the genome of Brassica rapa by Genome Walking techniques. Similar sequences are published in the Brassica genome Project. Accession number BH691838 has a partial similarly of 88%.
FIG. 22 depicts an artificial heavy chain (anti-hevein 1C2) coding region (SEQ ID NO:18). The sequence consists of three parts:

    a) 57 nt long sequence coding for mouse signal peptide (17 amino acids); A part of the sequence having accession number X67210 has a similarity of 100%;
    b) 387 long heavy chain anti hevein 1C2 antigen variable region made by phage display techniques (obtained from VTT, Espoo, Finland). Genebank accession number AB067222 has 95% similarity to the 1–295 nt region of the variable region of SEQ ID NO: 18; and
    c) 990 nt long IgG1 heavy chain constant region. A sequence having accession number BC024289 has similarity of 99%.


FIG. 23 depicts an artificial signal signal sequence (1–69 nt in SEQ ID NO: 19)) and the TNFR part (70–489 nt in SEQ ID NO:19). A sequence having accession number NM001066 has similarity of 100%.
FIG. 24 depicts an artificial part of the Arabidopsis VSP1 (vegetative storage protein-1 gene) (nt 1–226 in SEQ ID NO:20) and a part of the rbcS-4-terminator shown in FIG. 21, starting form behind the cleavage site and being 350 nt long (nt 227–576 in SEQ ID NO:20).



DETAILED DESCRIPTION OF THE INVENTION
The present disclosure is related to transgene expression in germinating seedlings and sprouts. According to the present disclosure a strong protein expression is achieved by fusing the gene coding for the desired gene products with novel Rubisco promoters cloned from Brassica rapa. The novel promoters are selected from a group of Rubisco promoters derivable from rbcS genes, which have been selected from abundantly expressed rbcS genes in light-grown cotyledons of Brassica rapa. 
The novel promoters consist essentially of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The promoter sequences are derived from native Rubisco promoters, but similar sequences can be prepared by other means including synthetic and semisynthetic methods.
The promoters according to the present disclosure were obtained by selecting and identifying genes, which were highly expressed in the developing cotyledons of light grown seedlings of Brassica species. 3′-UTRs of said highly expressed gene were isolated and characterized. Using said method three strong Rubisco promoters essentially comprising the sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 were obtained and the capability of these promoters to direct gene expression in a spatiotemporal manner in developing cotyledons was characterized.
The Rubisco promoters are useful for designing recombinant DNA constructs or expression cassettes comprising SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 functionally fused in frame with reporter genes. In the present disclosure the term reporter gene means genes coding for homologous or heterologous proteins or other metabolic gene products. Reporter gene may code for any selected product, which is desired to be expressed in transgenic plant.
The reporter genes are exemplified by GUS encoding gene (uidA) and by gene coding for human serum albumin (HSA). Also a synthetic gene consisting of part of immunoglobulin (Ig) G heavy chain and extra cellular domain of Tumor Necrosis Factor Receptor (TNFR) with or without ER-retention signal KDEL was used to exemplify a reporter gene. Furthermore, an antibody reporter gene is exemplified by heavy and light chains of human antibodies directed against hevein 1C2 antigen. One skilled in the art understands that these reporter genes are only examples and that any other reporter gene coding for a desired product may as well be used.
The recombinant constructs or expression cassettes according to the present disclosure are useful in transforming homologous and heterologous plants. The plant species are exemplified in this disclosure by Brassica sp, Nicotiana tabacum and Camelina sativa. Plant transformation procedures are familiar to those skilled in the art and therefore any other plant species can be transformed as well with the constructs according to the present disclosure. Applicable transformation systems include, but are not limited for example to the conventional Agrobacterium mediated transformation system. Especially transformation of Camelina plants according to a novel transformation system described in WO02/38779 and U.S. Ser. No. 10/416,091 is included and hereby incorporated by reference.
The host plants were transformed with one or more of the above described DNA constructs or expression cassettes. Seeds from the transformed host plants, representing a zero generation, are collected and used for production of subsequent plant generations providing transgenic seeds. The transgenic seeds can be used for production of the desired proteins or gene products by allowing the seeds to germinate. When using the seeds for production in contained system, the germination can take place for example on buffered agar plates or in aerated vessels, such as appropriate fermentation equipment. The transgenic seeds provide an excellent nutritional source and the transgenic seed may germinate into seedlings in a solution comprising mainly water, which may be appropriately buffered and contain growth hormones and other advantageous growth and germination promoting ingredients. Such cultivation enables production under sterile conditions and an easy recovery of the gene products.
In the present invention a method for producing the Rubisco promoters SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 is also provided. The method may be used to provide further promoters having similar useful properties as the Rubisco promoters of the present invention. The method thus enables production of new useful promoters for production of desired gene products from transformed seed in contained conditions. In the method the expression of light grown seedlings is evaluated and genes, which are highly expressed during the development of the cotyledon, are identified and their promoters are characterized. Promoters, which are capable of direction expression in cotyledons during their development, are selected for designing DNA constructs and expression cassettes.
The following examples are meant to be descriptive and by no means limiting the various embodiments of the present invention.
EXAMPLE 1
Rubisco mRNA Types Expressed in Cotyledons of Germinating Brassica rapa (campestris) Seeds
A cDNA library was constructed in order to identify the most abundant types of Rubisco mRNA to be expressed in cotyledons of germinating Brassica rapa (campestris) seeds.
Total RNA was isolated from four days old Brassica seedlings, and a mRNA fraction was isolated from the total RNA preparations using oligo(d)T cellulose. A first strand cDNA was synthesized using oligo(d)T with M-MLV (Point mutant) reverse transcriptase. The next PCR step was carried out with a forward primer e3a 5′-CAUCAUCAUCAUCAACCGTCAAGTCCAGTGCATCAGTTTCAT-3′ (SEQ ID NO: 4) specific to the 3rd exon of Rubisco SSU coding region and the reverse primer atu 5′-CUACUACUACUATTTTTTTTTTTTTTT-3′ (SEQ ID NO: 5), an oligo(d)T derivative, specially designed according to CloneAmp procedure (Life Technologies). Both primers comprised on their 5′-terminal end several dUMP residues, which were destroyed by the enzyme UDG (Uracil DNA Glycosylase). The PCR step was carried out in 2 cycles, and subsequently the PCR product was digested by UDG and directly inserted into the linearized pAMP1 vector (Life Technologies) containing special protruding 3′-terminal ends compatible with protruding 3′-terminal ends of the RT-PCR products. The insert containing vector was transformed into competent E. coli strain XL-1. One hundred plaques were selected and analyzed. Inserts from plasmid DNA were amplified by PCR and resulting PCR-products were sequenced. Relative number of separate colonies containing inserts of each type was calculated by the aid of sequence analysis.
Referring to FIG. 1 sequence analysis of the cloned 3′UTRs showed striking quantitative differences between different Rubisco mRNA species. The sequence named ‘56’ comprised 56% of all the Rubisco mRNA cloned. The other sequence named ‘29’ comprised 29% of all the Rubisco mRNA. The rest 15% of the clones corresponded to the other types of Rubisco mRNAs.
The 29-type and 56-type sequences received from the cDNA library were compared to published sequences. The sequence alignments indicated that these Rubisco mRNAs are expressed from novel Rubisco promoters. The 29-type sequences are called rbcS-2 and the 56-type sequences are called as rbcS-4, respectively.
EXAMPLE 2
Cloning Rubisco Promoters Obtained from Brassica rapa 
Based on the ‘56’ and ‘29’ type of sequences reverse primers were designed to be used in subsequent steps of promoter cloning.
Cloning of rbcs-2 Promoter
An EST-library was constructed first. The most common UTR found was UTR2, which was used to design reverse primers for Genome Walking step. Genomic DNA of Brassica rapa was digested by EcoR V, DraI, HincII, PvuII, SmaI and SspI and ligated to adapters (5′-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT (SEQ ID NO:6) and 5′-p-ACCAGCCC-NH2 .3′(SEQ ID NO:7) to get six DNA libraries.
The next PCR amplifications (first and nested) were performed with adapter primer AP1 5′-GTAATACGACTCACTATAGGGC-3′ (SEQ ID NO:8) and UTR2-specific L1 primer 5′-GGCCACACTTGACAATCCGATATAACATGCCTCA-3′ (SEQ ID NO:9).
Nested PCR was conducted with AP2 primer 5′-ACTATAGGGCACGCGTGGT-3′ (SEQ ID NO:10) and nested UTR2-specific L2 primer 5′-CAAATGGAAATGAAATGAGGTAG-3′ (SEQ ID NO:11).
The longest 900 bp product was obtained by using a DraI DNA library. This fragment was cloned into a pGEM3Zf(+) vector and sequenced. The sequence was compared with the sequences in Gen Bank database. The most homologous sequence found was B. napus rbcS (accession number X61097).
Near the 5′-end of one of the clones received (Rud3) was a 22 nt long stretch lacking from B. napus rbcS (beginning from 1037 nt of B. napus rbcS). Two reverse primers, RbNco and RbSiB, downstream from the putative transcription initiation site (based on the homology with X61097) and two forward primers, BNRb1 and BNRb3, based on X61097 homology, were designed. Full-length rbcS-2-gene was amplified using BNRb1 as a forward primer and UTR2-L2 as a reverse primer. Subsequently, two promoters of different length were amplified in nested PCRs using combinations of BNRb3 as a forward primer and RbSiB (with signal peptide) as a reverse primer, or BNRb3 as a forward primer and RbNco as a reverse primer (without signal peptide).
Cloning of rbcS-4 Promoter
Promoter cloning was conducted in several steps. Two reverse primers (for the first and nested PCRs) matching with the same sequences on the beginning of the first exons of three published Rubisco genes were used for the first step of Genome Walking.
Genomic DNA was isolated from Brassica rapa leaves and divided into six fractions. Each fraction was digested by one of six restricting enzymes (EcoR V, DraI, PvuII, StuI, SspI, XmnI) and ligated with Genome Walking adapters (Clontech) mentioned above. Each restriction-ligation mixture represents a genomic DNA library.
The next step included two successive PCRs (first and nested) using adapter-specific AP1 and AP2 (forward) and gene-specific (reverse) primers. The PCR was started by using three different reverse primers, annealed to different parts of the first exon of Rubisco SSU gene in order to get the overlapping PCR products listed below.








(SEQ ID NO: 12)









(RbcS-RN: 5′-ACCCGGGCCCAGGAGAGCATAGAGGAAGCC-3′,



 








(SEQ ID NO: 13)









RbcS-R1: 5′-CGGTGAATGGAGCGACCATCGTGGCTTGAG-3′,



 








(SEQ ID NO: 14)









RbcS-R2: 5′-CTGTGAATGGAGCAACCATGGCCGCTTGAG-3.







The six genomic DNA libraries described above produced amplification products after nested PCR. These products were directly cloned into pGEM-T-Easy vector (Promega) by TA-cloning. Colonies were screened by PCR using M13-universal and reverse primers. Colonies carrying plasmid DNA with insert were grown in liquid cultures and plasmid DNA isolated was used for sequencing analysis.
A total number of about ninety plasmid DNA insert-containing clones were analyzed. Based on data obtained from the sequencing analysis the sequences were divided into five groups according to sequence similarities. Three promoters were identified to be similar to the ones published in GenBank. Moreover, PCR using specially designed forward primers, specific to the cloned promoter regions, and reverse primers, specific for the ‘56’-type of 3′UTR (rbcS-4 type of 3′UTR) allowed identification of putative promoters having the 56 type of 3′UTR (rbcS-4 type of 3′UTR) in the genome. This promoter was called ‘56A’.
Based on the obtained sequences new reverse primers were designed to make next PCR set with the same forward primers (AP1, AP2) and the new reverse primers and using the same genomic DNA libraries. This procedure was repeated four times. The sequences obtained after the fourth PCR cycle the resulting sequences allowed us to design promoter-specific forward primers. The reverse primer was designed to include a special site for BpiI to create an NcoI-compatible restriction site. PCR using these primers and HiFi KOD polymerase enabled identification of the ‘56’ type of promoter rbc-4A (SEQ ID NO:1) among other sequences. By means of Genome Walking techniques another promoter with the ‘56’ type 3′UTR was found bound in the genome. This rbcS-4B promoter (SEQ ID NO:2) was 98% similar to rbcS-4A on the length of about 230 nt region in (1953–2175 nt SEQ ID NO:1 and 794–1016 nt in SEQ ID NO:2), but distal parts of rbcS-4A and rbcS-4B showed less than 40% similarity. FIG. 2B gives an alignment of −267 to +33 nt regions of these two promoters. RbcS-4B promoter was also cloned with proof-reading KOD polymerase and its functional activity was studied further.
Using the same approach totally four steps of Genome Walking were applied to clone the rbcS-4A promoter and two steps were applied to clone rbcS-1, rbcS-3 and rbcS-5 promoters (SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, respectively). After the final step of Genome Walking whole length promoters were cloned using the proof-reading Pfu enzyme. The 3′-terminal ends of the cloned promoter sequences were designed so that they can be ligated with reporter genes. Genebank BLAST system was used to analyze the promoter sequences obtained. Brassica promoters having accession numbers X55937 and X75334 showed similarity of up to 98–99% with rbcS-3 and rbcS-5 promoters, respectively. All the promoters cloned and known were compared to each other by computer alignment program. This analysis showed that the promoters have similar parts located mostly in about 300 nt region (c.f. FIGS. 2A and 2B).
Alignment of 300 bp length proximal parts of these rbcS promoters (excluding rbcS-4B (see below) is presented in FIG. 2A. The Genome Walking data showed that there were two partially different rbcS-4 (called rbcS-4A and rbcS-4B) promoters connected to the same 3′UTRs and being very similar on the last 230 bp on their 3′-terminal ends (FIG. 2B) (rbcS-4A is SEQ ID NO:1; rbcS-4B is SEQ ID NO:2). On the other hand, the resting (distal) parts of the promoters show the same low level of homology (40%) as they show in alignment with other Rubisco promoters.
Alignment of one of the published Brassica napus Rubisco promoter (accession number X61097) with rbcS-2 (SEQ ID NO:3), demonstrates some differences (91% similarity) between them (FIG. 2C). There are also differences in 3′UTR regions. Therefore these two promoters are not the same ones and probably diverged during evolution or selection process of rbcS gene family in Brassica species.
Referring to FIG. 3 alignment of rbcS-4A promoter sequence and published Brassica rubisco promoter (X61097) revealed dissimilarity of 52%.
Similarly, referring to FIG. 4 alignment of rbcS-4A promoter sequence with the published Chrysanhetmum rbcS-1 promoter (AY163904) revealed dissimilarity of 57%.
Markedly, there are only three stretches in rbsC-4A promoter (1007–1440 nt, 1776–1950 nt and 1959–2175 nt) that have a quite high homology (similarity of about 93%) with Brassica genome project database (FIG. 17). Accession number BH484651 represents genomic clone of Brassica oleracea and CD811761 represents cDNA clone of Brassica napus. No other parts of the rbcS-4A promoter sequence are found in any database including Brassica genome project.
Clearly the nucleotide sequences of rbcS-2 (SEQ ID NO: 3) and rbcS-4A (SEQ ID NO: 1) and rbcS-4B (SEQ ID NO: 2) are novel and useful as described in this disclosure.
EXAMPLE 3
Fusion-Constructs rbcS-4A-GUS, rbcS-4A-HSA, rbcS-4B-GUS, rbcS-2-GUS, rbcS-2-HSA, rbcS-2-Ab(L+H)-1C2, rbcS-4A-Ab(L+H)-1C2, rbcS-2, TNFR-Fc and rbcS-4-TNFR-Fc
The promoters were amplified with reverse primers to get NcoI-compatible restriction site on their 3′-terminal ends. Vector pCAMBIA1301 (CAMBIA) containing GUS gene with NcoI site on its 5′-terminal end was used. HSA fusion constructs were designed in a pBIN19-based plasmid pGPTV with an inserted HSA gene (SEQ ID NO:15) (FIG. 19). RbcS-4A and rbcS4B were cut out by BpiI, HindIII. RbcS-2 was cut out by NcoI, HindIII. RbcS-4A, rbcS-4B, and rbcS-2 were cloned into pCAMBIA1301 or pGPTV vectors opened by NcoI, HindIII. The terminators used for these constructs were as follows: nos-terminator in GUS-containing pCAMBIA1301 vector, and rbcS-4 type of 3′UTR plus part of known Brassica rapa rbcS terminator from GenBank was used in HSA-containing pGPTV plasmids.
Constructs Rbcs-2-Ab(L+H)-1C2 and RbcS-4-Ab(L+H)-1C2 contain the same antibody regions and the same terminator (polyA) signal from the natural Brassica rubisco RbcS-4 gene (directly from the genome). The antibody protein molecule was originally developed against hevein 1C2 antigen. RbcS-2-Ab(L+H)-1C2 consists of RbcS-2 promoter, light chain (anti-hevein 1C2) (SEQ ID NO: 16) coding region as shown in FIG. 20, rbcS-4 terminator (SEQ ID NO:17) as shown in 21, another RbcS-2 promoter, heavy chain (anti hevein 1C2) (SEQ ID NO:18) coding region as shown in FIG. 22, and another Rbcs-4 terminator. The RbcS-4-Ab(L+H)-1C2 construct consists of Rbcs-4 promoter, light chain (anti-hevein 1C2) (SEQ ID NO:16) coding region, RbcS-4 terminator, another RbcS-4 promoter, heavy chain (anti-hevein 1C2) coding region (SEQ ID NO: 18), and another RbcS-4 terminator.
For the constructs Rbcs-2-Ab(L+H)-1C2 and RbcS-4-Ab(L+H)-1C2 rbcS-2 and rbcS-4 promoters were cut by SalI, HindIII and ligated with pVK1-CHC(constant heavy chain)-rbcS-4-terminator, digested with SalI, and HindIII providing the pVK1-RbcS-2(Rbcs-4A)-promoter-CHC-RbcS-4-terminator. RbcS-4 terminator was originally cloned with CHC by BsiWI, EcoRI. Variable heavy chain region of 1C2 antibody (VH-1C2) was cut out by BpiI, Bsp120I and cloned into a pVK1-Rbcs-2(Rbcs-4)-promoter-CHC-RbcS-4-terminator vector by the same sites. The resulting plasmid was the plasmid containing whole H (heavy) chain unit. The same strategy was used to get the whole L (light) chain unit. L chain unit was then cloned into pCAMBIA1301 vector from where 35SUidA gene was removed. This was pCAMBIA1301-L-chain. In the final step the H-chain unit was inserted into pCAMBIA1301-L-chain vector to get the final pCAMBIA1301-H-L. The plasmid was used for plant transformation using Agrobacterium-mediated strategy.
Ig-TNFR (ENBREL) construct contains rbcS-2 or rbcS-4 promoters, TNFR (tumor necrosis factor receptor) part (489 nt) as shown in FIG. 23 (SEQ ID NO: 19) comprising the IgCHC part (CH2 and CH3 domains) and terminators. TNFR part was cloned directly from human mRNA by reverse transcription followed by PCR, ligated into pGEM-T-Easy plasmid by TA-cloning procedure and sequenced from both directions with M13-universal and reverse primers. Ig CHC part was obtained by PCR and sequenced thereafter. Cloning strategy included ligation of Ig CHC part by BsiWI site and introducing promoter into pVK1 plasmid (pUC19 derivative), containing rbcS-4-terminator. Then TNFR part digested by BsmB1 was introduced into this plasmid, and whole the insert was re-cloned into big pCAMBIA1300 or pCAMBIA2300 plasmids.
IgCHC part was obtained in two variants. The first was without any changes in its 3′end and the second one contained KDEL signal in its 3′end. This signal is 12 nt long sequence AAAGACGAGCTG (SEQ ID NO: 24) and is introduced just before the STOP-codon. Several terminators were used in the Ig-TNFR constructs. One was rbcS-4 terminator (about 500 nt) being the same as used in antibody constructs. Another terminator was a longer version of the rbcS 4-terminator (being about 2 kb). Still another terminator used was from Arabidopsisis VSP1 (vegetative storage protein-1 gene), the part situating right behind the STOP codon and before cleavage site was used and was connected with part of rbcS-4 terminator shown in FIG. 24 (SEQ ID NO: 20). In some of the constructs one or two MAR (matrix attachment regions) sequences (about 2 kb) were also introduced. In case the construct contained two MAR sequences they were introduced before the promoter and after the terminator.
EXAMPLE 4
Plant Transformation
To exemplify the functionality of the novel promoters according to this disclosure, we transformed plants of Brassica species, Nicotiana tabacum plants and Camelina sativa plants. One skilled in the art is able to transform plants of other species.
Brassica plants were transformed with A. tumefaciens strain LBA4404 carrying the pCAMBIA1301 or pGPTV-HPT binary vectors by leaf disk inoculation. Tobacco plants Nicotiana tabacum cv. Samsung were transformed with A. tumefaciens strain LBA4404 carrying pGPTV-HPT binary vectors by leaf disc inoculation. Putative transformants were selected on 30 mg/l hygromycin. Positive lines were transferred to the greenhouse for further studies
Camelina plants were transformed with A. tumefaciens strain, C58 (helper plasmid pGV3850) carrying the pCAMBIA1300 binary vectors by leaf disc inoculation. Putative transform ants were selected on 20 mg/l hygromycin. Positive lines were transferred to the greenhouse for further studies.
EXAMPLE 5
Quantitative GUS Assay for Expression Analysis
The assays were carried out with tobacco leaves or Camelina seedlings. Fresh plant material was mechanically disrupted in Tris-buffer, containing 2-ME. Protein concentrating in extracts was determined using Bio-Rad assay. GUS activity was determined in spectrophotometer using methyl-umbelliferyl as a substrate for the enzymatic reaction. Incubation was 30 min at +37° C. and developed color was measured in spectrophotometer at 450 nm wavelength. Non-transgenic plants were used as negative controls.
EXAMPLE 6
Expression Analysis of mRNA with Real-Time RT-PCR and Northern Analysis
Total RNA isolated from cotyledons of germinating Brassica or Camelina seeds or tobacco leaves were reverse transcribed with gene specific reverse primers. The reverse primers were designed for non-similar parts of all the 3′UTR known as well as for HSA, GUS, heavy and light chains of anti-hevein 1C2 antibody and the third exon of Rubisco SSU coding region. cDNA obtained was used for Real-time PCR step using forward and the same reverse primers.
Real-Time procedure was conducted on API7000 machine mainly according to the manual using SYBRgreen quantitative variant of the method. The passive reference dye was ROX. The calibration curves were constructed using PCR products amplified from genome and purified with the same primers as in Real-Time process. The result was expressed in number of molecules per 1 ng of RNA sample originally taken.
For the Northern analysis total RNA was isolated from plant material and run on agarose gel and transferred on to the membrane. Then RNA was cross-linked to the membrane by short exposure to UV light. Next step is hybridization with specific RNA probe, synthesized in vitro from bacterial T7 or SP6 promoters, Hybridization was going on overnight at optimal temperature, specially optimized for every probe. After washing the membrane is undergone to incubation with antibodies recognizing DIG-labels on the probe. The amount of the RNA probes (i.e. specific mRNA) was detected by enhanced luminescence using negative and positive controls (varying concentrations), allowing determination of the amount of specific mRNA in the experimental sample.
EXAMPLE 7
Expression Levels of Rubisco Genes and Total Rubisco mRNA in Germinating Seeds Increases Toward End of Cotyledon Development
The total RBCS mRNA content in constant light conditions increased during the first 3–4 days and remained on a high level for the next 5 to 7 days (FIG. 16).
In order to determine the expression levels of different Rubisco genes and also total Rubisco mRNA production in germinating Brassica napus seeds we measured the amount of total Rubisco mRNA in seeds on 0, 1, 2, 3 and 4 day of germination in constant light conditions by Real-Time PCR. This is illustrated in FIG. 9.
The quantitative data shown in FIG. 9 (first column), demonstrates the amount or number of Rubisco mRNA molecules in 4 ng of total mRNA per an average sample. Clearly, the amount of mRNA molecules increased from day 0 to day 4, showing the highest amounts on the 4th day. On 4th day of germination the amounts of RBCS mRNA determined in most of the samples were about 4–7×107 molecules per 4 ng of total mRNA.
EXAMPLE 8
rbcS-4 Type of RBCS mRNA is the Most Prevalent and Active Type of mRNA at the Stage of Germinating Seeds of Plant Development
The amount of different types of RBCS mRNAs was analyzed by the Real Time process described above. The expression levels of rbcS-2, rbcS-3, rbcS-4 and rbcS-5 were determined on 0–4th day of Brassica napus seed germination by using primers specific to non-similar parts of 3′UTRs of those mRNA species (FIG. 5). Forward primers were designed so that they have longer right part, corresponding to specific 3′UTR type. The shorter left part of each primer corresponds to the end of the RBCS coding region. This left part helps to increase length and therefore Tm of the primer, but does not disturb the specificity of it (FIG. 5).
Data summarized on FIG. 9 (columns 3–6), demonstrates dramatic differences in the expression levels. The most abundant type during the four days of seed germination was rbcS-4 RBCS mRNA, but as we have already noticed above there are at least two rbcS genes driven by the partially similar promoters (rbcS-4A and -B; SEQ ID NO: 1 and SEQ ID NO:2, respectively) and connected to the same 3′UTR (rbcS-4 type of 3′UTR). This may mean that each rbcS-4 gene can contribute to the sum activity of the gene. But according to quantitative GUS expression data obtained from rbcS-4B-GUS transgenic tobacco plants (FIG. 10) the activity of the promoter is very low and doesn't seem to have remarkable influence on total amount of mRNA containing rbcS-4 type of 3′UTR. The data presented here clearly demonstrates the prevalence of rbcS-4 type of RBCS mRNA on the later stage of germinating seeds of plant development.
Referring now to results shown in FIG. 9 Real-Time PCR showed that rbcS-4 promoter was more active at the fourth day of germination than any other Rubisco promoter examined. The expression level of different RBCS genes followed different kinetics: for example at third day rbcS-2 (SEQ ID NO: 3) and rbcS-3 (SEQ ID NO:22) were more active than rbcS-4 and rbcS-5 (SEQ ID NO:23). These characteristics are extremely important when selecting a promoter for a production method of foreign proteins or other desired gene products to be produced in germinating seeds or sprouts.
Unstable transgenic proteins may degrade quite fast because of enhanced protein mobilization capacity of plant cells in tissues of germinating seeds. When using a promoter such as rbcS-4 with delayed kinetic of activity, there are more chances to protect accumulation of transgenic protein product from the action of lytic vacuoles. Moreover, additional benefits of using rbcs-4A in transgenic constructs arise from the fact that this is the strongest promoter out of the four promoters analyzed at later stages of seed germination.
FIG. 13 compares the accumulation of HSA mRNA in germinating B. napus seeds transgenic for rbcS-4-HSA or for (rbcs-2-HSA)×2. (rbcS-2-HSA)×2 is a variant of rbsS-2-HSA where 2 units of rbcS-2 are arranged in tandem. It is evident that HSA mRNA begins to accumulate earlier in the seeds transgenic for (rbcS-2-HSA)×2. On the other hand HSA mRNA in rbcS-4-HSA transgenic plants starts to accumulate later but the amount accumulating is somewhat bigger. As seen from FIG. 9, the kinetics of rbcs-4 promoter activity is more delayed than that of rbcS-2, and therefore it is evident that both of the promoters are functional even when in non-native conditions
EXAMPLE 9
Heterologous and Homologous Transgenic Plants Harboring rbcS-2 and rbcS-4 Promoters for Production of Desired Gene Products
rbcS-2 (SEQ ID NO: 3) and rbcS-4 (SEQ ID NO: 1 and SEQ ID NO: 2) promoters were used for plant transformation experiments with Brassica, tobacco and Camelina plants to determine the ‘promoter strength’ and also to compare the expression levels in homologous and heterologous systems (i.e. plants transformed with a construct containing a promoter from the same or a different species).
The promoters were amplified with reverse primers to get NcoI-compatible restriction site on their 3′ ends. pCAMBIA1301 vector (CAMBIA) containing GUS gene with NcoI site on its 5′ end designed as described in Example 2 were used.
Promoters rbcS-2 (SEQ ID NO:3), rbcS-4A (SEQ ID NO:1) or rbcS-4B (SEQ ID NO:2) containing constructs inserted in the genome of Brassica represent homologous system, and the insertion of the same constructs in tobacco and Camelina plant's genome represent heterologous system. Recombinant constructs containing rbcS-2 or rbcS-4 promoters fused in frame with reporter genes were designed as described in Example 3 and transformed into plants as described in Example 4.
mRNA expression data of transgenic Brassica plants containing rbcS-4-GUS or rbcS-2-GUS is presented in FIG. 7. The mRNA expression level of reporter gene was measured from cotyledons of seeds of transgenic Brassica plants germinated for 4 days.
Referring to data presented in FIG. 7 it can be seed that expression of transgene (GUS) mRNA in both plant transformants is about 5–6 times less than the expression of corresponding native rubisco gene (rbcS-2 or rbcS-4A). Furthermore expression level of native rbcS-4A gene in transgenic plant corresponds to the one in non-transgenic plant (FIG. 9), but the expression level of native rbcS-2 gene in rbcS-2 transgenic plant is less than that in non-transgenic plant. The result probably is related to higher silencing dependency of rbcS-2 promoter in homologous plant.
For tobacco transformation experiments rbcS-2-HSA and rbcS-4A-HSA constructs were used and seven HSA-producing plant lines for each of them were received. The mRNA expression level of HSA gene determined on 5th day of transgenic tobacco seed germination demonstrate about the same level of expression in both types of these plant lines (FIG. 8). The tobacco transformation experiments show clearly that there is no significant difference between the rbcS-2 and rbcS-4 promoters strength, but both of them are expressing in heterologous system.
Transgenic Camelina and tobacco plants harboring rbcS-2-GUS, RbcS-4-GUS, RbcS-2-TNFR-Fc-56UTRshort, RbcS-2-TNFR-FcKDEL-56UTRshort, RbcS-4-TNFR-Fc-56UTRlong, or RbcS-4-TNFR-FcKDEL-56UTRlong constructs were obtained and analyzed further. The results are shown in FIGS. 18A and B. Determination of GUS-activity demonstrates that enzyme activity level in rbcS-2 (rbcS-4)-GUS transgenic plants is higher than in plants carrying conventional 35Sp-GUS construct used here as positive control. Northern data is available for some TNFR-Fc-harboring Camelina and tobacco plants. The expression level for RbcS-4-TNFR-Fc is comparable with that of native rbcS genes (in Brassica about 50–100 pg/ug of total RNA for whole RBCS gene family).
EXAMPLE 10
Protein Expression in Transgenic Plants
A construct comprising GUS gene coding region was linked to the Rubisco promoter rbcS-4A and transformed into an oilseed rape (Brassica rapa) plant using Agrobacterium mediated transformation. Transgenic plants were grown in greenhouse until seeds were produced. Seeds of transgenic plants were allowed to sprout in 20° C. aerated water; 24° C. aerated 20 mM KNO3 water or in 30° C. aerated water. After variable times of cultivation expressed GUS protein was isolated from the sprouts by homogenization in appropriate buffer and centrifugations. Specific GUS activity was determined by spectrophotometer (FIG. 11). Clearly, GUS activity per sprout was highest after 72 hours of cultivation using KNO3 in the growth medium.
Protein expression of transgenic Brassica napus, Camelina sativa and tobacco plants carrying HSA under the control of rbcS-2 or rbcS-4 were also analyzed. Similarly plants carrying tandem construct of RbcS-2-HSA were analyzed. Protein expression was analyzed from sprouts that germinated at constant light and 24° C. temperature for four days. FIG. 15 shows the data as % of total soluble protein. It is evident that plants carrying the tandem construct have higher expression levels of the protein than plants carrying single construct. Furthermore, it is evident that protein expression is higher under rbcs-4 promoter than under rbcS-2 promoter. The tandem construct having two rbcS-2-HSA constructs is an example of a multiple construct according to the present invention and one skilled in the art would be able to transform plants with more than two constructs in tandem as well. Similarly, one skilled in the art would be able to use tandem constructs having rbcS-4 as the driving promoter to obtain higher protein contents.
Protein expression of transgenic Camelina sativa and tobacco plants carrying TNFR constructs was analyzed. The results are shown in FIG. 18 B.
EXAMPLE 11
In Order to Provide Maximal Activity the RbcS Promoter has to be of Full Length
Truncated versions of rbcS-2 promoter were cloned, (0.3 and 0.6 kb in length) in fusion constructs with reporter uid A gene. Tobacco plants were transformed by Agrobacterium carrying these constructs and the GUS activity was measured from leaves of adult tobacco plants. The data obtained was compared to data obtained from the analysis of high-expressing adult tobacco plants carrying rbcS-2 (1.6 kb) or 35S promoters connected to the GUS gene. The results as shown in FIG. 15 clearly demonstrate decrease of registered GUS activity due to reduction of the length of the promoter. Therefore it is evident that distal regions of the rbcS-2 promoter contain essential regulatory elements supporting basal (not-inducible) promoter activity. Comparative analysis in silico of known tomato rbcS-1 promoter and our cloned Brassica rbcS-2 and rbcS-4A promoters enables to find similar consensus regulatory elements in all of them (FIG. 6). It is clear that most of the known boxes are located in the −500–600 nt region. It could be suggested that those distal parts of the promoters may have some cryptic regulatory elements or they may participate in the promoter action because of possible occurring of MAR (Matrix Attachment Region) sites, for example, in 5′ regions of rbcS-4A (computer MAR prediction analysis).

## Claims

1. A Rubisco promoter isolated from Brassica rapa light grown seedlings, said promoter having capability to direct gene expression into developing cotyledons and said promoter comprising a nucleotide sequence of SEQ ID NO: 1.

2. An expression cassette comprising a Rubisco promoter of SEQ ID NO: 1 and further being operably linked to a heterologous nucleic acid sequence encoding a desired gene product.

3. The expression cassette according to claim 2, wherein the heterologous nucleic acid sequence is synthetic.

4. The expression cassette according to claim 2, wherein the heterologous nucleic acid sequence encodes human serum albumin.

5. The expression cassette according to claim 2, wherein the heterologous nucleic acid encodes an antibody.

6. The expression cassette according to claim 2, wherein the heterologous nucleic acid encodes a medically active compound.

7. Transgenic seedlings for production of desired products, said transgenic seedlings comprising at least one expression cassette according to claim 2.

8. Transgenic seedlings according to claim 7, wherein said transgenic seedlings comprise two expression cassettes.

9. Transgenic seedlings according to claim 7, wherein the seedlings are Camelina sativa seedlings.

10. A transgenic plant, comprising at least one expression cassette according to claim 2.

11. A transgenic seed comprising at least one expression cassette according to claim 2.

12. An isolated polynucleotide comprising the sequence of SEQ ID NO: 1.