# Patent

## Title
Polynucleotides encoding enzymes from the jute lignin biosynthetic pathway

## Abstract

Disclosed are polynucleotides encoding polypeptides that comprise the biosynthetic pathway for lignin in the jute plant. The present invention relates generally to the field of plant lignin biosynthesis genes, polypeptides encoded by such genes, and the use of such polynucleotide and polypeptide sequences for controlling plant lignin production. Also disclosed are methods for using the polynucleotides and polypeptides to influence the quality and amount of fiber produced by jute.

## Background
RELATED APPLICATIONS
This application is a 371 national stage application of PCT/US12/034980, filed Apr. 25, 2012, which claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 61/480,668 filed Apr. 29, 2011. The entire contents of each of these applications are hereby incorporated by reference.


FIELD OF THE INVENTION
The invention relates to the identification and characterization of various portions of the jute lignin biosynthetic pathway. More specifically, the invention relates to polynucleotides from jute plants that encode enzymes responsible for lignin synthesis, and methods for using these polynucleotides and enzymes for gene regulation and manipulation of lignin production to give fibers with desired lignin content and other characteristics.
BACKGROUND OF THE INVENTION
Lignin is a collective name for a complex aromatic heteropolymer of monolignol (hydroxycinnamyl alcohols), usually derived from phenylalanine in a multistep process. (Whetten, R. and Sederoff, R., (1995) Lignin Biosynthesis, Plant Cell, 7, pp. 1001-1013). These polymers, deposited primarily in cell walls, ensure the necessary mechanical strength of plant stems and most importantly, the hydrophobicity of a plant's vascular tissues. (Vanholme, R. et al. (2010) Lignin biosynthesis and structure, Plant Physiol, 153, pp. 895-905). Due to its hydrophobic nature, lignin serves as a major component of the vascular tissues and plays an essential role in water transport. In addition to its structural and transport-oriented role, lignin is a key component of a plant's defense system. (Goujon, T. et al. (2003) Genes involved in the biosynthesis of lignin precursors in Arabidopsis thaliana, Plant Physiology and Biochemistry, 41, pp. 677-687). Not surprisingly, environmental conditions influence the amount of lignin deposited. (Boerjan, W. et al. (2003) Lignin biosynthesis, Annu Rev Plant Biol, 54, pp. 519-546). For example, lignin biosynthesis is induced in response to various stress conditions like wounding, abiotic stress, and pathogen infection. Lignin limits pathogen invasion and protects the cell wall polysaccharides against microbial degradation. (Vanholme et al., 2010).
A large part of our current understanding of lignin biosynthesis comes from the complete understanding of this pathway in A. thaliana and P. trichocarpa. (Goujon, et al., 2003; Shi, et al. (2010) Towards a systems approach for lignin biosynthesis in Populus trichocarpa: transcript abundance and specificity of the monolignol biosynthetic genes, Plant Cell Physiol, 51, pp. 144-163). There are three basic monolignol monomers: p-coumaryl, coniferyl, and sinapyl alcohols. These monolignols are incorporated into the three lignin units, or building blocks: p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S). See FIG. 1. These monolignols differ in the number of methoxy groups. P-hydroxyphenyl (H) has no methoxy group, guaiacyl (G) has one methoxy group, and syringyl (S) has two methoxy groups. (Goujon et al., 2003). However, in addition to these three monolignols, a few other phenylpropanoids, such as hydroxycinnamyl aldehydes, hydroxycinnamyl esters, and hydroxycinnamyl acetates may also be incorporated. (Boerjan et al., 2003).
After the biosynthesis of these basic lignin building blocks, they are transported to lignifying zones. In the lignifying zones, polymerization occurs by oxidative free-radical-based coupling by peroxidases or laccases and a mesh-like structure is formed by cross-linking with cellulose and hemicellulose. (Boerjan et al., 2003; Vanholme, R. et al. (2008) Lignin engineering, Curr Opin Plant Biol, 11, pp. 278-285). Lignification occurs in different phases during the secondary thickening of the cell wall when the polysaccharide matrix formation is complete. Lignin deposition is influenced by the nature of the polysaccharide matrix. In the primary cell wall, it is found as spherical structures; whereas in the secondary cell wall, it forms lamellae. (Boerjan et al., 2003).
Notwithstanding the indispensable role of lignin in the life of a plant, it is a major limiting factor in the cost-effective/efficient use of plant material in the pulp and biofuel industries. Lignin also limits the use of biomass for fiber, chemical, and energy production. Removal of lignin is a very expensive process and these industries would benefit from access to biomass having less lignin, or a lignin that is easy to degrade. In the last few decades, some understanding of the lignin biosynthetic pathway has been achieved, although portions of the process are not fully understood.
Despite the importance of lignin synthesis to the overall welfare of the jute plant, as well as its impact on several aspects of fiber quality, at present there is no available information detailing lignin biosynthesis in jute. Therefore, a need exists to identify, isolate and utilize genes and enzymes from the jute plant that are involved in the biosynthesis of lignin. The present invention addresses this need.
SUMMARY OF THE INVENTION
One aspect of the invention is an isolated nucleic acid molecule having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 16, 18, 20, 22, 24, 25, 26, 28, 29, 31, 33, 35, 37, 39, 40, 42, 44, 45, 47, 49, and 51.
In one embodiment, an isolated nucleic acid molecule is selected from the group consisting of: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, and 15.
In one embodiment, an isolated nucleic acid molecule is selected from the group consisting of: SEQ ID NO: 16, 18, and 20.
In one embodiment, an isolated nucleic acid molecule is selected from the group consisting of: SEQ ID NO: 22, 24, 25, 26, 28, and 29.
In one embodiment, an isolated nucleic acid molecule is selected from the group consisting of: SEQ ID NO: 31.
In one embodiment, an isolated nucleic acid molecule is selected from the group consisting of: SEQ ID NO: 33.
In one embodiment, an isolated nucleic acid molecule is selected from the group consisting of: SEQ ID NO: 35, 37 and 39.
In one embodiment, an isolated nucleic acid molecule is selected from the group consisting of: SEQ ID NO: 40 and 42.
In one embodiment, an isolated nucleic acid molecule is selected from the group consisting of: SEQ ID NO: 44, 45, and 47.
In one embodiment, an isolated nucleic acid molecule is selected from the group consisting of: SEQ ID NO: 49.
In one embodiment, an isolated nucleic acid molecule is selected from the group consisting of: SEQ ID NO: 51.
One aspect of the invention is an isolated polypeptide molecule having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 17, 19, 21, 23, 27, 30, 32, 34, 36, 38, 41, 43, 46, 48, 50, and 52.
In one embodiment, a pair of forward and reverse primers useful for the amplification of cDNA selected from the group consisting of SEQ ID NO 53 and SEQ ID NO 54; SEQ ID NO 55 and SEQ ID NO 56; SEQ ID NO 57 and SEQ ID NO 58; SEQ ID NO 59 and SEQ ID NO 60; and SEQ ID NO 61 and SEQ ID NO 62.
In certain embodiments, the present invention relates to any one of the aforementioned polynucleotide sequences or polypeptide sequences, wherein said sequence has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the sequences identified by a SEQ ID NO.
One aspect of the invention is an expression vector comprising an isolated nucleic acid molecule of the present invention.
One aspect of the invention is an isolated antibody or antigen binding fragment thereof that specifically binds to a polypeptide molecule of the present invention.
One aspect of the invention is a transfected plant cell transfected by a vector of the present invention.
One aspect of the invention is a material derived from a transgenic plant of the present invention.
One aspect of the invention is a seed from a plant transfected by a vector of the present invention.
One aspect of the invention is a method for making a transgenic plant, comprising the steps of transfecting at least one plant cell with a vector of the present invention, and growing at least one plant cell into a plant.
One aspect of the invention is a method of improving growth, fiber yield, fiber strength, disease resistance, or water utilization in a jute plant, comprising incorporating in to a jute plant a non-native nucleic acid sequence of the present invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Proposed monolignol biosynthesis pathway of jute.
FIGS. 2a and 2b: Protein sequence alignment of ColCAD1, ColCAD2, ColCAD3, ColCAD4, ColCAD5, ColCAD6, and ColCAD7 with plant CAD protein sequences. FIGS. 2a and 2b disclose SEQ ID NOS 2, 4, 63-64, 6, 65, 10, 66, 12, 67, 14, 68, 8 and 69, respectively, in order of appearance.
FIG. 3: Protein sequence alignment of ColCCoAOMT1, ColCCoAOMT2 and ColCCoAOMT3 with plant CCoAOMT protein sequences. FIG. 3 discloses SEQ ID NOS 19, 70-71, 17, 21 and 72, respectively, in order of appearance.
FIG. 4: Protein sequence alignment of Col4CL1, Col4CL4 and Col4CL6 with plant 4CL protein sequences. FIG. 4 discloses SEQ ID NOS 23, 73, 27, 74, 30 and 75, respectively, in order of appearance.
FIG. 5: Protein sequence alignment of Col6HCT1 with plant 6HCT protein sequences. FIG. 5 discloses SEQ ID NOS 32 and 76-77, respectively, in order of appearance.
FIG. 6: Protein sequence alignment of ColC3H with plant C3H protein sequences. FIG. 6 discloses SEQ ID NOS 78-79, 34 and 80, respectively, in order of appearance.
FIG. 7: Protein sequence alignment of ColC4H1 and ColC4H2 with plant C4H protein sequences. FIG. 7 discloses SEQ ID NOS 81-82, 36 and 38, respectively, in order of appearance.
FIG. 8: Protein sequence alignment of ColPAL1 and ColPAL2 with plant PAL protein sequences. FIG. 8 discloses SEQ ID NOS 43, 83, 41 and 84, respectively, in order of appearance.
FIG. 9: Protein sequence alignment of ColCCR2 with plant CCR protein sequences. FIG. 9 discloses SEQ ID NOS 85-86, 46, and 87, respectively, in order of appearance.
FIG. 10: Protein sequence alignment of ColCCR3 with plant CCR protein sequences. FIG. 10 discloses SEQ ID NOS 48 and 88-89, respectively, in order of appearance.
FIG. 11: Protein sequence alignment of ColF5H with plant F5H protein sequences. FIG. 11 discloses SEQ ID NOS 90, 50 and 91, respectively, in order of appearance.
FIG. 12: Protein sequence alignment of ColCOMT with plant COMT protein sequences. FIG. 12 discloses SEQ ID NOS 52 and 92-93, respectively, in order of appearance.
FIG. 13: DNA gel of ColCAD2. Lane 1 shows the isolated ColCAD2 DNA. Lane 2 shows a molecular weight marker.
FIG. 14: DNA gel of ColCCoAOMT1. Lane 2 shows the isolated ColCCoAOMT1 DNA. Lane 1 shows a molecular weight marker.
FIG. 15: DNA gel of Col4CL1. Lane 1 shows the isolated Col4CL1 DNA. Lane 2 shows a molecular weight marker.
FIG. 16: DNA gel of ColCCR3. Lane 1 shows the isolated ColCCR3 DNA. Lane 2 shows a molecular weight marker.
FIG. 17: DNA gel of ColF5H. Lane 1 shows the isolated ColF5H DNA. Lane 2 shows a molecular weight marker.



DETAILED DESCRIPTION OF THE INVENTION
Ten known enzyme families are associated with monolignol biosynthesis. (Goujon et al., 2003). The families are PAL (phenylalanine ammonia-lyase), C4H (cinnamate-4-hydroxylase), 4CL (4-coumarate:CoA ligase), HCT (p-hydroxycinnamoyl-CoA:shikimate/quinate p-hydroxycinnamoyl transferase), C3H (4-coumarate 3-hydroxylase), CCoAOMT (caffeoyl-CoA O-methyltransferase), CCR (cinnamoyl-CoA reductase), F5H (ferulate 5-hydroxylase), COMT (caffeic acid O-methyltransferase), and CAD (cinnamyl alcohol dehydrogenase). A proposed schematic of the monolignol biosynthetic pathway in Jute is shown in FIG. 1.
The lignin biosynthesis pathway in jute owes its complexity, in part, to the presence of several multifunctional enzymes, and to the constituent enzymes spanning several diverse gene families. The first enzyme of the phenylpropanoid pathway is PAL (phenylalanine ammonia-lyase), which causes the deamination of phenylalanine, producing cinnamic acid. The second enzyme of the pathway, C4H (cinnamate 4-hydroxylase), converts cinnamic acid to 4-hydroxycinnamic acid, which is followed by subsequent hydroxylation and methylation steps as the pathway becomes branched. The enzyme 4CL catalyzes the CoA ligation of hydroxycinnamic acids, generating activated phenolic precursors for lignin biosynthesis. (Hu et al. (1999) Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees, Nat Biotech, 17, pp. 808-812.).
The next enzyme in the monolignol pathway (HCT) catalyses the production of p-coumaroyl-shikimate/quinate esters, which are the substrates for C3H. HCT was shown to transfer the acyl group of p-coumaroyl-CoA to shikimate or quinate. (Hoffman et al. (2005) Plant Biosystems, v. 139, No. 1, pp. 50-53). The hydroxylation steps at C3 and C5 are performed by two cytochrome P450 enzymes, 4-coumarate 3-hydroxylase (C3H) and ferulate 5-hydroxylase (F5H), respectively. The methylation steps are performed by CCoAOMT (caffeoyl-coenzyme A (CoA) O-methyltransferase) and COMT (caffeic-O-methyltransferase). CCoAOMT is a bifunctional enzyme which converts caffeoyl-CoA to feruloyl-CoA and 5-hydroxyferuloyl-CoA to sinapoyl-CoA and plays a role in the synthesis of feruloylated polysaccharides. (Inoue et al., 1998). CCoAOMT has been shown to be involved in lignin biosynthesis in the differential tracheary elements of Zinnia elegans. (Ye, Z. H. and Varner J. E. (1995) Differential expression of two O-methyltransferases in lignin biosynthesis in Zinnia elegans, Plant Physiol. 108, pp. 459-467). CCoAOMT is involved in the reinforcement of the plant cell wall, and is also involved in responses to wounding or pathogen challenge by the increased formation of cell wall-bound ferulic acid polymers.
Additional enzymes involved in the monolignol biosynthesis pathway are cinnamoyl coenzyme A reductase (CCR) and cinnamyl alcohol dehydrogense (CAD). CCR catalyzes the reduction of the hydroxycinnamoyl CoA esters to produce cinnamaldehydes, whereas CAD catalyzes their reduction to cinnamyl alcohols. (Goujon et al., 2003).
One of the last enzymes involved in the monolignol pathway is cinnamyl alcohol dehydrogenase (CAD), which catalyzes the NADPH dependent conversion of coniferaldehyde, 5-hydroxy-coniferaldehyde, and sinapaldehyde to the corresponding alcohols. (Kim, S. J. et al. (2004) Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis, Proc. Natl. Acad. Sci. USA, 101, pp. 1455-60). In Arabidopsis, single mutants of the CAD genes AtCAD-C and AtCAD-D were found to have lower CAD activities, and the double-mutant, obtained by crossing the two mutants, had a 40% decrease in stem lignin content, thus demonstrating that these are the main CAD genes involved in stem lignin synthesis. (Sibout, R. et al. (2005) Cinnamyl Alcohol Dehydrogenase-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis, Plant Cell, 17, pp. 2059-76).
Two enzymes are specific to the monolignol biosynthesis pathway. They are caffeic acid O-methyltransferase (COMT) and cinnamoyl coenzyme A reductase (CCR). COMT was first identified in angiosperms. COMT is capable of converting caffeic acid to ferulic acid, as well as converting 5-hydroxyferulic acid to sinapic acid. (Dixon, R. A., et al. (2001) The biosynthesis of monolignols: a “metabolic grid,” or independent pathways to guaiacyl and syringyl units? Phytochemistry, 57, pp. 1069-1084). Down regulation of the COMT gene in maize (Zea mays) has been shown to cause a significant reduction of COMT activity (a fall of 70 to 85%), resulting in the modification of lignin content and composition, indicating that this enzyme is a key enzyme for lignin synthesis.
Ferulic acid generated by COMT can be hydroxylated by ferulate 5 hydroxylase (F5H), which is a cytochrome P450-dependent monooxygenase, to form 5-hydroxy-ferulic acid. F5H is also capable of hydroxylating coniferaldehyde and coniferyl alcohol to form 5-hydroxy-coniferaldehyde and 5-hydroxy-coniferyl alcohol, respectively. (Meyer, K. et al. (1996) Ferulate-5-hydroxylase from Arabidopsis thaliana defines a new family of cytochrome P450-dependent monooxygenases, Proc. Natl. Acad. Sci. USA, 93, pp. 6869-74). F5H is believed to be a rate limiting step in syringyl lignin biosynthesis, a proposal supported by the observation that an Arabidopsis mutant deficient in F5H expression is also affected at the level of sinapate esters accumulation in siliques and seeds. (Ruegger, M. et al. (1999) Regulation of ferulate-5-hydroxylase expression in Arabidopsis in the context of sinapate ester biosynthesis, Plant Physiol., 119, pp. 101-10).
The second enzyme specifically involved in lignol biosynthesis, CCR, catalyzes the conversion of feruloyl CoA and 5-hydroxy-feruloyl CoA into coniferaldehyde and 5-hydroxy-coniferaldehyde, respectively. This step leads directly to the biosynthesis of G (coniferaldehyde) and S (5-hydroxy-coniferaldehyde) lignin units. (Ma et al., 2005). In tobacco, down regulation of the CCR gene using antisense constructs, produced plants with abnormal development and reduced growth, as well as abnormal leaf morphology and collapsed vessels. There was also an associated reduction in the level of G lignin compounds. (Ralph, J. et al. (1998) NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamylalcohol dehydrogenase and cinnamoyl-CoA reductase, Proc. Natl. Acad. Sci USA, 95, pp. 12803-8).
Computational Identification of Genes and Transcripts
Remarkably, we have determined the sequences of the jute enzymes involved in lignin biosynthesis. The pathway of lignin biosynthesis has been well characterized and each enzyme is encoded by a gene family in most of the plant species. A total of 106 gene sequences of A. thaliana and P. trichocarpa were retrieved from NCBI and P. trichocarpa genome database (Goujon et al., 2003; Shi et al., 2010). Jute monolignol biosynthesis genes were identified from the gene models of the Corchorus olitorius genome assembly and transcriptome data of C. olitorius and C. capsularis using the program BLASTN with the e-value cut-off at 1e-20. (Altschul, S. F., et al. (1990) Basic local alignment search tool, J Mol Biol, 215, pp. 403-410). The resulting gDNA contigs were subjected to gene model prediction using the software AUGUSTUS. (Stanke, M. et al. (2004) AUGUSTUS: a web server for gene finding in eukaryotes, Nucleic Acids Research, 32, W309-W312). The gene models and the isotigs from the transcriptome data of C. olitorius and C. capsularis were searched against the NCBI nr (non-redundant) database for further confirmation.
An amino acid sequence alignment of putative proteins encoded by the ColCAD genes with other CAD proteins available in the NCBI database, using the CLUSTAL W program, is shown in FIGS. 2a and 2b. The following is a list of the proteins aligned with the putative ColCAD proteins, with the GeneBank Accession Numbers in parentheses: PtcCADL4 (Populus tricocarpa cinnamyl alcohol dehydrogenase-like protein, CADL4, gi224138226); RcoCAD (Ricinus communis alcohol dehydrogenase, putative, gi25558709); FraCAD (Fragaria×ananassa, cinnamyl alcohol dehydrogenase, gi13507210) (Chandler et al. (2002) Cloning, expression and immunolocalization pattern of a cinnamyl alcohol dehydrogenase gene from strawberry (Fragaria×ananassa), J. Exp. Bot., 53 (375), pp. 1723-1734); GhiCAD5 (Gossypium hirsatum, cinnamyl alcohol dehydrogenase 5, gi268528129); PtcCAD (Populus tricocarpa, gi183585165) ((2010) Towards a systems approach for lignin biosynthesis in Populus trichocarpa: transcript abundance and specificity of the monolignol biosynthetic genes, Plant Cell Physiol., 51 (1), pp. 144-163); GhiCAD3 (Gossypium hirsatum, gi229368450) (Genes of phenylpropanoid pathway cloning and expression in developing cotton fibre); and GhiCAD (Gossypium hirsatum, gi166865124) ((2009) Molecular and biochemical evidence for phenylpropanoid synthesis and presence of wall-linked phenolics in cotton fibers, J Integr Plant Biol, 51 (7), pp. 626-637).
An amino acid sequence alignment of putative proteins encoded by the ColCCoAOMT genes with other CCoAOMT proteins available in the NCBI database, using the CLUSTAL W program, is shown in FIG. 3. The following is a list of the proteins aligned with the putative ColCCoAOMT proteins, with the GeneBank Accession Numbers in parentheses: PtrCCoAOMT (Populus tremuloides, gi3023436); GhiCCoAOMT2 (Gossypium hirsatum, gi229368460); and GhiCCoAOMT1 (Gossypium hirsatum, gi253509567).
An amino acid sequence alignment of putative proteins encoded by the Col4CL genes with other 4CL proteins available in the NCBI database, using the CLUSTAL W program, is shown in FIG. 4. The following is a list of the proteins aligned with the putative Col4CL proteins, with the GeneBank Accession Numbers in parentheses: Ccap4CL1 (Corchorus capsularis, gi294514718); Rco4CL (Ricinus communis, gi255565415); and Ptc4CL (Populus tricocarpa, gi224074401).
An amino acid sequence alignment of putative proteins encoded by the Col6HCT genes with other Col6HCT proteins available in the NCBI database, using the CLUSTAL W program, is shown in FIG. 5. The following is a list of the proteins aligned with the putative Col6HCT proteins, with the GeneBank Accession Numbers in parentheses: CycarHCT (Cynara cardunculus, gi:73671233) ((2007) Isolation and functional characterization of a cDNA coding a hydroxycinnamoyltransferase involved in phenylpropanoid biosynthesis in Cynara cardunculus, BMC Plant Biol. 7, 14); and PtcHCT (Poplus tricocarpa, gi183585181).
An amino acid sequence alignment of putative proteins encoded by the ColC3H genes with other C3H proteins available in the NCBI database, using the CLUSTAL W program, is shown in FIG. 6. The following is a list of the proteins aligned with the putative ColC3H proteins, with the GeneBank Accession Numbers in parentheses: EglC3H (Eucalyptus globulus, gi:295413824); PtcC3H (Poplus tricocarpa, gi:224139664); and Pal×PgrC3H (Poplus alba×Populus grandidentata, gi166209291).
An amino acid sequence alignment of putative proteins encoded by the ColC4H genes with other C4H proteins available in the NCBI database, using the CLUSTAL W program, is shown in FIG. 7. The following is a list of the proteins aligned with the putative ColC4H proteins, with the GeneBank Accession Numbers in parentheses: GarC4H (Gossypium arborium, gi9965897) and GarC4H (Gossypium arborium, gi9965899).
An amino acid sequence alignment of putative proteins encoded by the ColPAL genes with other PAL proteins available in the NCBI database, using the CLUSTAL W program, is shown in FIG. 8. The following is a list of the proteins aligned with the putative ColPAL proteins, with the GeneBank Accession Numbers in parentheses: JcoPAL (Jatropha curcas, gi113203757) and PtrPAL (Populus trichocarpa, gi:183585195).
An amino acid sequence alignment of putative proteins encoded by the ColCCR2 genes with other CCR proteins available in the NCBI database, using the CLUSTAL W program, is shown in FIG. 9. The following is a list of the proteins aligned with the putative ColCCR2 proteins, with the GeneBank Accession Numbers in parentheses: AthCCR (Arabidopsis thaliana, gi:15237678); CofCCR (Camellia oleifera gi228480464); and AlyCCR (Arabidopsis lyrata, gi:297793385).
An amino acid sequence alignment of putative proteins encoded by the ColCCR3 genes with other CCR proteins available in the NCBI database, using the CLUSTAL W program, is shown in FIG. 10. The following is a list of the proteins aligned with the putative ColCCR3 proteins, with the GeneBank Accession Numbers in parentheses: RcoCCR (Ricinus communis, gi:255556687) and AthCCR (Arabidopsis thaliana, gi:15226955).
An amino acid sequence alignment of putative proteins encoded by the CoIF5H genes with other F5H proteins available in the NCBI database, using the CLUSTAL W program, is shown in FIG. 11. The following is a list of the proteins aligned with the putative ColF5H proteins, with the GeneBank Accession Numbers in parentheses: EgIF5H (Eucalyptus globules, gi:255970299) and PtcF5H (Poplus tricocarpa, gi:6688937).
An amino acid sequence alignment of putative proteins encoded by the ColCOMT genes with other COMT proteins available in the NCBI database, using the CLUSTAL W program, is shown in FIG. 12. The following is a list of the proteins aligned with the putative ColCOMT proteins, with the GeneBank Accession Numbers in parentheses: GhiCOMT (Gossypium hirsutum, gi:253509569) and EcaCOMT (Eucalyptus camaldulensis, gi:262474806).
Motif Analysis of the Promoter Regions
For each of the predicted gene models, both strands of the upstream regions of 2000 bp were extracted and searched for cis-motif sequences against the PlantCARE database (Lescot, M., et al. (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences, Nucleic Acids Res, 30, pp. 325-327). If any portion of the selected sequences were found to be overlapping with a nearby gene, that part of the upstream region was excluded from further analysis. A list of important motifs was compiled that are known to be involved in the response to various developmental processes and stress (Table 1).







TABLE 1







List of motifs found in the promoter region of jute monolignol biosynthetic genes
























Tran-



Gibberellin-
Low temp




Salicylic
respon-
Myb

scription
Abscisic


responsive
respon-



Gene model
acid
siveness
binding
Wound
enhancer
acid
Ethylene
Auxin
element
siveness





PAL
contig_310473_g31
*












contig_301819_g4


*



contig_302040_g24

*
*


*


*



contig_309920_g2



*
*
*


C4H
contig_311419_g56



contig_307848_g46

*




*



contig_384302_g42


4CL
contig_331398_g112



contig_299467_g73







*
*



contig_337813_g88




*



contig_306165_g86








*
*



contig_309926_g132





*

*



contig_304308_g125
*


*
*



*



contig_305998_g57
*

*


HCT
contig_308231_g128





*


C3H
contig_597141_g180









*


CCoAOMT
contig_306979_g168


*





*



contig_308402_g160



contig_296218_g172
*
*


*



*



contig_311951_g178




*


CCR
contig_310406_g194







*



contig_297048_g201

*




*



contig_294439_g187
*



contig_309320_g191


*


F5H
contig_304192_g207

*



contig_595270_g204





*


COMT
contig_299952_g235

*


CAD
contig_335778_g278






*



contig_95812_g280



contig_321021_g281

*



contig_808760_g282
*



contig_808760_g284





*



contig_356907_g287







*



contig_356907_g288




*

*



contig_355903_g289





*



contig_306706_g300

*


*



*



contig_304576_g307
*
*


*



*









Polynucleotides of the present invention were isolated by high-throughput sequencing of cDNA libraries comprising jute plant tissue collected from Corchorus olitorius L. Some of the polynucleotides of the present invention may be partial sequences, in that, they do not represent a full-length gene encoding a full-length polypeptide. Such partial sequences may be extended by analyzing and sequencing various DNA libraries using primers and/or probes and well known hybridization and/or PCR techniques. Partial sequences may be extended until an open reading frame encoding a polypeptide, a full-length polynucleotide, a gene capable of expressing a polypeptide, or another useful portion of the genome is identified.
Identification of genomic DNA and heterologous species DNA can be accomplished by standard DNA/DNA hybridization techniques, under appropriately stringent conditions, using all or part of a polynucleotide sequence as a probe to screen an appropriate library. Alternatively, PCR techniques using oligonucleotide primers that are designed based on known genomic DNA, cDNA, or protein sequences can be used to amplify and identify genomic and cDNA sequences.
Polypeptides of the present invention may be produced by inserting a polynucleotide sequence of the present invention encoding the desired polypeptide into an expression vector and expressing the polypeptide in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art could be employed. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a polynucleotide molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, and higher eukaryotic cells.
Polypeptides that comprise the lignin biosynthetic pathways purified from jute, or produced by recombinant methods, may be used to generate monoclonal antibodies, antibody fragments or derivatives as defined herein, according to known methods. Antibodies that recognize and bind fragments of the polypeptides that comprise the lignin biosynthetic pathways of the invention are also contemplated, provided that the antibodies are specific for polypeptides that comprise the lignin biosynthetic pathway.
The genetic constructs of the present invention may also contain a selection marker that is effective in plant cells, to allow for the detection of transformed cells containing the inventive construct. Such markers, which are well known in the art, typically confer resistance to one or more toxins or create visual signals for its presence under fluorescent microscope. Alternatively, the presence of the desired construct in transformed cells can be determined by means of other techniques well known in the art, such as Southern and Western blots. The genetic construct of the present invention may be linked to a vector having at least one replication system, for example, E. coli or yeast (Saccharomyces cerevisiae), whereby after each manipulation, the resulting construct can be cloned and sequenced.
The genetic constructs of the present invention may be used to transform a variety of plants, such as monocotyledonous (e.g., rice) and dicotyledonous (e.g., jute, Arabidopsis). In a preferred embodiment, the inventive genetic constructs are employed to transform jute. As discussed above, transformation of a plant with a genetic construct of the present invention can be used to produce modified lignin content in the plant.
Techniques for stably incorporating genetic constructs into the genome of target plants are well known in the art and include Agrobacterium tumefaciens mediated introduction, electroporation, injection into meristematic tissue or reproductive organs, injection into immature embryos, and the like. The choice of technique will depend upon the target plant/tissue/host to be transformed.
The term “plant” includes whole plants, shoot vegetative organs/structures (e.g., leaves, stems, and tubers), roots, flowers, and floral organs/structures (e.g., bracts, sepals, petals, stamens, carpels, anthers, and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like) and cells (e.g., guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, bryophytes, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid, and hemizygous.
DNA gels of PCR reactions using forward and reverse primers for several jute enzymes are shown in FIGS. 13-17. In FIG. 13, the DNA gel is of CAD2 from Corchorus olitorius. Lane 1 is the PCR product of CAD2 using cDNA as a template. The forward primer and reverse primer are SEQ ID NO. 53 and 54, respectively. Lane 2 is the 1 Kb+ladder. In FIG. 14, the DNA gel is of CCoAOMT1 from Corchorus olitorius. Lane 1 is the 1 Kb+ladder, and Lane 2 is the PCR product of CCoAOMT1 using cDNA as a template. The forward primer and reverse primer are SEQ ID NO. 55 and 56, respectively. In FIG. 15, the DNA gel of 4CL1 from Corchorus olitorius. Lane 1 is the 1 Kb+ladder, and Lane 2 is the PCR product of 4CL1 using cDNA as a template. The forward primer and reverse primer are SEQ ID NO. 57 and 58, respectively. In FIG. 16, the DNA gel is of CCR3 from Corchorus olitorius. Lane 1 is the 1 Kb+ladder, and Lane 2 is the PCR product of CCR3 using cDNA as a template. The forward primer and reverse primer are SEQ ID NO. 59 and 60, respectively. In FIG. 17, the DNA gel is of F5H from Corchorus olitorius. Lane 1 is the 1 Kb+ladder, and Lane 2 is the PCR product of F5H using cDNA as a template. The forward primer and reverse primer are SEQ ID NO. 61 and 62, respectively.
DEFINITIONS
A cell has been “transformed” or “transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. The practice of the present invention contemplates a wide variety of stably transformed plant cells.
An “expression cassette” refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of a RNA and/or polypeptide, respectively. The expression cassette may include a nucleic acid comprising a promoter sequence, with or without a sequence containing mRNA polyadenylation signals, and one or more restriction enzyme sites located downstream from the promoter allowing insertion of heterologous gene sequences. The expression cassette is capable of directing the expression of a heterologous protein when the gene encoding the heterologous protein is operably linked to the promoter by insertion into one of the restriction sites. The recombinant expression cassette allows expression of the heterologous protein in a host cell when the expression cassette containing the heterologous protein is introduced into the host cell. Expression cassettes can be derived from a variety of sources depending on the host cell to be used for expression. For example, an expression cassette can contain components derived from a viral, bacterial, insect, plant, or mammalian source. In the case of both expression of transgenes and inhibition of endogenous genes (e.g., by antisense, or sense suppression) the inserted polynucleotide sequence need not be identical and can be “substantially identical” to a sequence of the gene from which it was derived. Preferably the recombinant expression cassette allows expression at an early stage of infection and/or it allows expression in substantially all cells of an organism, such as a plant. Examples of expression cassettes suitable for transformation of plants can be found in U.S. Pat. Nos. 5,880,333 and 6,002,072; International Patent Publications Nos. WO/1990/002189 and WO/2000/026388; Ainley and Key (1990) Plant Mol. Biol., 14, pp. 949-967; and Birch (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol., 48, pp. 297-326, all of which are herein incorporated by reference.
The term “host cell” refers to a cell from any organism. Preferred host cells are derived from plants, bacteria, yeast, fungi, insects, or other animals. The term “recombinant host cell” (or simply “host cell”) refers to a cell into which a recombinant expression vector has been introduced. It should be understood that the term “host cell” is intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Methods for introducing polynucleotide sequences into various types of host cells are well known in the art. Provided are host cells or progeny of host cells transformed with the recombinant expression cassettes of the present invention. The host cells may be plant cells. Preferably, the plant cells are jute cells.
The term “operably linked” or “operably inserted” means that the regulatory sequences necessary for expression of the coding sequence are placed in a nucleic acid molecule in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence. This same definition is sometimes applied to the arrangement of other transcription control elements (e.g., enhancers) in an expression cassette. Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
The terms “promoter,” “promoter region,” or “promoter sequence” refer generally to transcriptional regulatory regions of a gene, which may be found at the 5′ or 3′ side of the coding region, or within the coding region, or within introns. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. The typical 5′ promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
The term “nucleic acid construct” or “DNA construct” is sometimes used to refer to a coding sequence or sequences operably linked to appropriate regulatory sequences and inserted into an expression cassette for transforming a cell. This term may be used interchangeably with the term “transforming DNA” or “transgene”. Such a nucleic acid construct may contain a coding sequence for a gene product of interest, along with a selectable marker gene and/or a reporter gene. The term “selectable marker gene” refers to a gene encoding a product that, when expressed, confers a selectable phenotype such as antibiotic resistance on a transformed cell. The term “reporter gene” refers to a gene that encodes a product which is easily detectable by standard methods, either directly or indirectly.
A “heterologous” region of a nucleic acid construct is an identifiable segment (or segments) of the nucleic acid molecule within a larger molecule that is not found in association with the larger molecule in nature. When the heterologous region encodes a plant gene, the gene will usually be flanked by DNA that does not flank the plant genomic DNA in the genome of the source organism. In another example, a heterologous region is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein. The term “DNA construct” is also used to refer to a heterologous region, particularly one constructed for use in transformation of a cell.
The term “vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, where additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
“Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 25% sequence identity compared to a reference sequence as determined using the programs described herein; preferably BLAST using standard parameters, as described. Alternatively, percent identity can be any integer from 25% to 100%. More preferred embodiments include polynucleotide sequences that have at least: 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity compared to a reference sequence. These values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.
The term “substantial identity” of amino acid sequences (and of polypeptides having these amino acid sequences) normally means sequence identity of at least 40% compared to a reference sequence as determined using the programs described herein; preferably BLAST using standard parameters, as described. Preferred percent identity of amino acids can be any integer from 40% to 100%. More preferred embodiments include amino acid sequences that have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity compared to a reference sequence. Polypeptides that are “substantially identical” share amino acid sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.
INCORPORATION BY REFERENCE
All of the U.S. patents, U.S. published patent applications, and published PCT applications that designate the U.S. cited herein are hereby incorporated by reference.
EQUIVALENTS
While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto; the invention may be practiced otherwise than as specifically described and claimed.

## Claims

1. A nucleic acid molecule comprising a coding nucleotide sequence (cDNA) having 100% sequence identity to a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, and 15, wherein said nucleic acid molecule encodes a cinnamyl alcohol dehydrogenase (CAD).

2. An expression vector comprising the nucleic acid molecule of claim 1.

3. A transgenic plant comprising the expression vector of claim claim 2.

4. A material derived from the transgenic plant of claim 3, wherein the material comprises said expression vector.

5. A seed from the transgenic plant of claim 3, wherein the seed comprises said expression vector.

6. A method for making a transgenic plant, comprising the steps of:
transfecting at least one plant cell with the expression vector of claim 2; and
growing said at least one plant cell into a plant.

7. A method of improving fiber yield, or fiber strength in a jute plant, comprising incorporating in the jute plant the nucleic acid molecule of claim 1.

8. The nucleic acid molecule of claim 1, wherein said nucleic acid sequence is SEQ ID NO: 1.

9. The nucleic acid molecule of claim 1, wherein said nucleic acid sequence is SEQ ID NO: 3.

10. The nucleic acid molecule of claim 1, wherein said nucleic acid sequence is SEQ ID NO: 5.

11. The nucleic acid molecule of claim 1, wherein said nucleic acid sequence is SEQ ID NO: 7.

12. The nucleic acid molecule of claim 1, wherein said nucleic acid sequence is SEQ ID NO: 9.

13. The nucleic acid molecule of claim 1, wherein said nucleic acid sequence is SEQ ID NO: 11.

14. The nucleic acid molecule of claim 1, wherein said nucleic acid sequence is SEQ ID NO: 13.

15. The nucleic acid molecule of claim 1, wherein said nucleic acid sequence is SEQ ID NO: 15.