FIELD OF INVENTION

The present invention relates to plant molecular biology, genetic engineering, and transgenic plant, in particular to plant promoter isolated from Eucalyptus camaldulensis, wherein the promoter is capable of expressing one or more target genes in plant.

BACKGROUND OF THE INVENTION

Recombinant DNA technology and plant genetic engineering has contributed substantially to the understanding of gene regulation and plant development, in the generation of transgenic organisms such as transgenic plants for widespread usage in agriculture, and has increased the potential uses of crops for industrial and pharmaceutical purposes.

As the application of transgenic plants has widened, it is necessary to develop methods to control the expression of transgene in plant. The availability of a broad spectrum of promoters that differ in their ability to regulate the temporal and spatial expression patterns of the transgenic in transgenic plant can dramatically increase the successful application of transgenic technology.

Various factors need to be taken into account to produce transgenic plant exhibiting improved characteristics. Therefore, it is necessary to refine and improve strategies for the genetic manipulation of plant cell metabolism. Pathway control may be shared among a number of components and require treatment as a quantitative trait. Intricate regulatory networks, mediated by multiple signal transduction pathways, will require dissection and individual manipulation of contributing elements. The effects of the compartmentation of metabolism and metabolic channeling must be considered. All of these require detailed knowledge of relevant gene expression and of the fate of the products of that expression.

In addition to the identification of targets, effective manipulation of the targeted complex metabolic processes requires highly specific promoters capable of regulating gene expression in a highly controlled manner in plant, to ensure that spatial and temporal constraint are met. Promoters are one of the most important elements regulating gene expression in any organism. Promoters are classified into 4 categories viz. constitutive promoters, tissue-specific, inducible promoters, and synthetic promoters.

Various promoters, which are capable of expressing one or more target genes in plant cells have been described in the prior art. There are various constitutive non specific promoters such as nopalaine synthase promoter nopaline synthase (nos) promoter and octopine synthase (ocs) promoters from Agrobacterium tumefaciens, Cauliflower Mosaic Virus (CaMV) 19S or 35S promoter (U.S. Pat. No. 5,352,605), CaMV 35S promoter with a duplicated enhancer (U.S. Pat. Nos. 5,164,316; 5,196,525; 5,322,938; 5,359,142; and 5,424,200), and the Figwort Mosaic Virus (FMV) 35S promoter (U.S. Pat. No. 5,378,619) which can drive the expression of genes in almost all tissues throughout plant development. As these promoters lack temporal and spatial regulation, these promoters can lead to abnormal phenotypes.

Tissue-specific promoters are particularly useful for modification of plant to produce transgenic plants exhibiting desired characteristics, where tissue specific expression and/or developmental timing of gene expression are important factor, or where constitutive expression would be detrimental to the development and physiological function of the transgenic plant. The successful application of biotechnology in crop and plant management and/or improvement is based on the discovery of novel genes and proper means such as use of tissue specific promoters for the control of their expression in resulting transgenic crops.

Numerous prior art has provided a number of plant promoters useful to direct transcription in plants. However, industrial or commercial use of biotechnology in crop improvement programs is severely hindered by the lack of promoters that can drive gene expression in a tissue-specific and/or or temporally controlled manner.

Hauffe at al (Hauffe KD, Paszkowski U, Schulze-Lefert PS, Hahlbrock K, Dangi JL. and Douglas CJ (1991); A Parsley 4CL-1 Promoter Fragment Specifies Complex Expression Patterns in Transgenic Tobacco; The Plant Cell 3: 435-443) describes developmental regulation of the 4CL-1 promoter from Parsley by analyzing the expression of 4CL-1-[beta]-glucuronidase fusions in transgenic tobacco plants.

Deletions past -210 base pairs resulted in drastic decline in [beta]-glucuronidase activity in protoplasts and loss of tissue-specific expression in transgenic tobacco.

An intense GUS histochemical staining was detected in the xylem of stem of transgenic tobacco transformed with Populus tomentosa 4CLI promoter (Lu, H., Zhao, Y. and Jiang, X. (2004) Stable and specific expression of 4- coumarate coenzyme A ligase gene (4CL1) driven by the xylem specific pto4CLI promoter in the transgenic tobacco. Biotechnology letters 26: 1147-1152). The activity of 4CL1 gene under the same promoter was also noticed to be two times higher in stems of transgenic tobacco but no difference was observed in leaf tissues.

US Pat No. 6,83,1208 describes Populus 4-coumarate Co-enzyme A ligase gene promoter that could be used for directing the expression in the xylem of plants.

Osakabe et al (Osakabe, Y., Osakabe, K. and Chiang, VL. (2009) Isolation of 4-coumarate Co-A ligase gene promoter from loblolly pine (Pinus taeda) and characterization of tissue-specific activity in transgenic tobacco. Plant Physiol Biochem 47 (11-12) 1031-6) describe promoter activity of a phenylpropanoid biosynthetic gene encoding 4-coumarate Co-A ligase (4CL), Pta4Clalpha, from Pinus taeda. Deletion analysis of the Pta4Clalpha promoter showed that the region -524 to -252, which has two AC elements, controls the high expression levels in ray-parenchyma cells of older tobacco stems. High activity level of the promoter domain of Pta4CLalpha was also detected in the xylem cells under bending stress.

Many tissue specific promoters and/or inducible promoters are reported in various prior art, however, there is still a need for novel promoters with beneficial expression characteristics. In particular, there is a need for xylem specific promoters that can be activated specifically in tissues involved in xylogenesis and primary and secondary xylem and are capable of directing expression of exogenous genes in tree species. Many previously identified promoters fail to provide the patterns or levels of expression required to fully realize the benefits of expression of selected genes in transgenic tree plants. These types of promoters can be used to selectively modulate the expression of genes involved in secondary cell wall formation in plants, for example, by eliminating or reducing lignification and increasing cellulose deposition in secondary xylem, increasing the volume of particular secondary cell wall layers, and controlling the sites and levels of lignification and cellulose deposition. There is, therefore, a need in the art of plant genetic engineering for novel promoters for use in tree species.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a promoter having the polynucleotide molecule selected from the group consisting of the polynucleotide having at least about 85% sequence identity with the nucleotide sequence as set forth in SEQ ID NO: 3, the nucleotide sequence as set forth in SEQ ID NO: 3, the nucleotide sequence as set forth in SEQ ID NO: 5, the nucleotide sequence as set forth in SEQ ID NO: 6, the nucleotide sequence as set forth in SEQ ID NO: 7, a fragment of the nucleotide sequence as set forth in SEQ ID NO: 3, wherein the fragment consists of at least 250 contiguous nucleotide of SEQ ID NO: 3, and a variant of the nucleotide sequence as set forth in SEQ ID NO: 3, wherein the promoter is capable of regulating the transcription of the operably linked polynucleotide molecule of interest.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

The following drawings form part of the present specification and are included to further illustrate aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

Figure 1 shows gel picture showing amplification of 4CL1 promoter using genome walking with nested PCR for isolation of 4CL1 promoter.

# Lane 1-4 samples amplified with 2054F primer.

1: Dra I library. 2: EcoR V library. 3: Pvu II library. 4: Stu I library.

# Lane 5-8 samples amplified with 2053F primer.

5: Dra I library. 6: EcoR V library. 7: Pvu II library. 8: Stu I library.

Figure 2 shows map of DNA constructs for deletion analysis 4CLI promoter.

Figure 3 shows map of DNA construct containing GUS gene under 4CL1 promoter.

Figure 4 shows map of DNA construct containing F5H gene under 4CL1 promoter.

Figure 5 shows Histochemical staining of 2 weeks old seedling of Arabidopsis transformed with 4CL500; A- wild type root, B-Transgenic root, C-wild type stem, D-Transgenic stem, E-wild type leaf, F-Transgenic leaf.

Figure 6 shows the expression level of various lignin biosynthetic genes such as CAD, CCR, F5H and OMT were studied in comparison with 4CL in stem of eucalyptus using real time qRT PCR.

Figure 7 shows the expression level of various lignin biosynthetic genes such as CAD, CCR, F5H and OMT were studied in comparison with 4CL in stem of eucalyptus using real time qRT PCR.

Figure 8 shows the plants transformed with 4CL1_500 subjected to pricking with needles on leaf surface and leaf sample were collected at 0 hr, 2 hrs and 3 hrs. Relative quantification of |3 glucuronidase transcripts indicated a higher expression after two hours of injury in comparison with leaf collected at the start of experiment.

DETAILED DESCRIPTION OF THE INVENTION

Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

Definition

The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

The term "polynucleotide" or "polynucleotide molecule" or "polynucleotide sequence" used herein refers to the single or double stranded DNA or RNA of genomic or synthetic origin, i.e., a polymer of deoxyribonucleotide or ribonucleotide bases, respectively, read from the 5' (upstream) end to the 3' (downstream) end.

The term "nucleotide sequence" as used herein refers to the sequence of a polynucleotide molecule.

The term "promoter" as used herein, refers to a polynucleotide molecule that is in its native or non native state located upstream or 5' to a translational start codon of an open reading frame (or protein-coding region) and that is involved in recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription.

A "plant promoter" is a native or non-native promoter that is functional in plant cells. Constitutive plant promoters are functional in most or all tissues of a plant throughout plant development. Any plant promoter can be used as a 5' regulatory element for modulating expression of a particular gene or genes operably associated thereto. When operably linked to a transcribable polynucleotide molecule, a promoter typically causes the transcribable polynucleotide molecule to be transcribed in a manner that is similar to that of which the promoter is normally associated.

Plant promoters can include promoters produced through the manipulation of known promoters to produce artificial, chimeric, or hybrid promoters. Such promoters can also combine cis-elements from one or more promoters, for example, by adding a heterologous regulatory element to an active promoter with its own partial or complete regulatory elements. Thus, the design, construction, and use of chimeric or hybrid promoters comprising at least one cis-element of SEQ ID NO: 3 for modulating the expression of operably linked polynucleotide sequences is encompassed by the present invention.

As used herein, the term "cis-element" refers to a cis-acting transcriptional regulatory element that confers an aspect of the overall control of gene expression. A cis-element may function to bind transcription factors, trans-acting protein factors that regulate transcription. Some cis-elements bind more than one transcription factor, and transcription factors may interact with different affinities with more than one cis-element.

The promoter of the present invention desirably contains cis-elements that can confer or modulate gene expression. Cis-elements can be identified by various known techniques, including deletion analysis, i.e., deleting one or more nucleotides from the 5' end or internal to a promoter; DNA binding protein analysis using DNase I footprinting, methylation interference, electrophoresis mobility-shift assays, in vivo genomic footprinting by ligation-mediated PCR, and other conventional assays; or by DNA sequence similarity analysis with known cis-element motifs by conventional DNA sequence comparison methods. The fine structure of a cis-element can be further studied by mutagenesis (or substitution) of one or more nucleotides or by other conventional methods. Cis-elements can be obtained by chemical synthesis or by isolation from promoters that include such elements, and they can be synthesized with additional flanking nucleotides that contain useful restriction enzyme sites to facilitate subsequence manipulation.
The promoter of the present invention comprises multiple cis-elements each of which confers a different aspect to the overall control of gene expression. The cis-elements from the polynucleotide molecules of SEQ ID NO: 3 are identified using computer programs designed specifically to identify cis-element, domains, or motifs within sequences. Cis-elements may either positively or negatively regulate gene expression, depending on the conditions. The present invention therefore encompasses cis-elements of the disclosed promoter.

As used herein, the phrase "substantially homologous" refers to polynucleotide molecules that generally demonstrate a substantial percent sequence identity with the promoter disclosed herein. Of particular interest are polynucleotide molecules wherein the polynucleotide molecules function in plants to direct transcription and have at least about 70% sequence identity, at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, specifically including about 73%, 75%, 78%, 83%, 85%, 88%, 92%, 94, 95%, 96%, 97%, 98%, 99% or greater sequence identity with the polynucleotide of the promoters described herein, having nucleotide sequence as set forth in SEQ ID NO: 3.

Polynucleotide molecules that are capable of regulating transcription of operably linked transcribable polynucleotide molecules and are substantially homologous to the polynucleotide sequences of the promoter provided herein are encompassed within the scope of this invention.

The term "percent sequence identity" as used herein refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference polynucleotide molecule (or its complementary strand) as compared to a test polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide insertions, deletions, or gaps totaling less than 20% of the reference sequence over the window of comparison).

Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as BLAST, CLUSTAL W, CLUSTAL X, T-COFFEE etc.

The term "homology" As used herein, refers to the level of similarity or percent identity between polynucleotide sequences in terms of percent nucleotide positional identity, i.e., sequence similarity or identity. The term homology also refers to the concept of similar functional properties among different polynucleotide molecules, e.g., promoters that have similar function may have homologous cis-elements.

Polynucleotide molecules are homologous when under certain conditions they specifically hybridize to form a duplex molecule. Under these conditions, referred to as stringency conditions, one polynucleotide molecule can be used as a probe or primer to identify other polynucleotide molecules that share homology.

The terms "Recombinant DNA expression cassette" and "recombinant DNA molecule" used herein can be used interchangeably.

The term "stringent conditions" as used herein is functionally defined with regard to the hybridization of a nucleic-acid probe to a target nucleic acid (i.e., to a particular nucleic-acid sequence of interest) by the specific hybridization procedure known in the art for example Southern hybridization, Dot Blot, Solution hybridization etc.

The term a "variant" is a promoter containing changes in which one or more nucleotides of an original promoter is deleted, added, and/or substituted, preferably while substantially maintaining promoter function. For example, one or more base pairs may be deleted from the 5' or 3' end of a promoter to produce a "truncated" promoter. One or more base pairs can also be inserted, deleted, or substituted internally to a promoter. In the case of a promoter fragment, variant promoters can include changes affecting the transcription of a minimal promoter to which it is operably linked. Variant promoters can be produced, for example, by standard DNA mutagenesis techniques or by chemically synthesizing the variant promoter or a portion thereof.

A minimal or basal promoter is a polynucleotide molecule that is capable of recruiting and binding the basal transcription machinery. One example of basal transcription machinery in eukaryotic cells is the RNA polymerase II complex and its accessory proteins.

As used herein the term "marker gene" refers to any transcribable polynucleotide molecule whose expression can be screened for or scored in some way.

As used herein, the term "gene of agronomic interest" refers to a transcribable polynucleotide molecule that includes but is not limited to a gene that provides a desirable characteristic associated with plant morphology, physiology, growth and development, yield, nutritional enhancement, disease or pest resistance, or environmental, physical strength or chemical tolerance.

The present invention relates to plant molecular biology, in particular to polynucleotide of promoter regulating gene expression in plants. In particular, the present invention relates to promoter isolated from Eucalyptus camaldulensis that are useful for regulating gene expression of heterologous polynucleotide molecules in plants. The present invention also relates to expression constructs and transgenic plants containing the heterologous polynucleotide molecules. coumarate ligase 1 (4CL1) is one of the widely conserved lignin biosynthetic pathway genes and it catalyses the formation of CoA esters of various cinnamate derivatives at the divergence point of phenyl propaniod metabolism. 4CL1 is one of the key enzymes involved in lignin biosynthetic pathway and it is positioned on the branching point of the whole network.

Ferulate 5-hydroxylase (F5H) is a cytochrome P450-dependent monooxygenase that catalyses the hydroxylation of ferulic acid, coniferaldehyde and coniferyl alcohol for syringyl (S unit) lignin biosynthesis. Over expression of Ferulate 5 hydroxylase shown to increase the syringyl to guacyl ratio higher in poplar and hence could be mean for increasing the S/G ratio in eucalypts. The increased S/G ratio leads to less usage of chemicals during pulping thus leading to less degradation of cellulose and hence increased pulp recovery apart from making the paper making process less polluting.

There has been considerable interest in isolating tissue specific promoters for localized expressions of particular gene of interest. 4 coumarate Ligase is one of the important and highly conserved enzymes in lignin biosynthetic pathway. Chromosome walking was carried out to isolate the promoter region of 4CL1 gene from E. camaldulensis. Genome walker library was screened with primary followed by nested PCR to get unique and specific band for isolation purpose. An amplicon size of 1060 bp was excised from gel, purified, and subjected to sequencing. The analysis of sequence data revealed that the sequence showed 100 bp reading in to the sequence known for 4CL1 cDNA indicating the other remaining coming from prior to coding sequence of 4CL1. Altogether, there was 1068 bases prior to start codon of 4CL1 gene. This 4CL1 promoter region was subsequently subjected to PLACE (Plant Cis Acting Elements) software to characterize the various motifs in the sequence.

The data indicated that there was considerable number of sequence signals indicating the promoter could be vascular specific, light and wound inducible (Table 1). Sequence also had various signature sequences indicating the possibility of getting activated by various stress factors. The characteristic features of enzymes in phenyl propaniod pathway were also noticed in 4CL1 promoter region like MYBPLANT.

To characterize the promoter, there was a need to clone the promoter/ deletion constructs in a binary vector so that it could be expressed in plant. For that purpose, promoter region was first cloned in blunt end vector pTZ57R with Hindll and Xbal sites, and the vector (pBI121) was digested with Hindll and Xbal Finally the pBI121 with out 35S promoter was fused with released 4CLI fragment from pTZ57R to get the construct of pBI121:4CLlp. Similarly other deletion constructs were made by excising 35S promoter and replacing with respective promoter fragments.

All the constructs including the construct with F5H cDNA were mobilized in to Agrobacterium strain LBA 4404 cells. The expression of GUS under various promoter fragments was studied in Arabidopsis as the model plant.

In accordance with the present invention in one of the preferred embodiment there is provided a promoter having the polynucleotide selected from the group consisting of the polynucleotide having at least about 80% sequence identity with the polynucleotide sequence as set forth in SEQ ID NO: 3; the polynucleotide sequence as set forth in SEQ ID NO: 3; a fragment, variants, or regions of the polynucleotide sequence as set forth in SEQ ID NO: 3, wherein the fragment consists of at least 250 contiguous nucleotide of SEQ ID NO: 3; wherein the promoter is capable of regulating the transcription of the operably linked polynucleotide of interest.

In another embodiment of the present invention there is provided a promoter having the polynucleotide having at least about 70% sequence identity, at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, specifically including about 73%, 75%, 78%, 83%, 85%, 88%, 92%, 94, 95%, 96%, 97%, 98%, 99% or greater sequence identity with the polynucleotide of the promoters described herein, having nucleotide sequence as set forth in SEQ ID NO: 3.

One of the embodiment of the present invention provides a promoter having the polynucleotide selected from the group consisting of the polynucleotide having at least about 85% sequence identity with the polynucleotide sequence as set forth in SEQ ID NO: 3; the polynucleotide sequence as set forth in SEQ ID NO: 3; a fragment of the polynucleotide sequence as set forth in SEQ ID NO: 3, wherein the fragment consists of at least 250 contiguous nucleotide of SEQ ID NO: 3; wherein the promoter is capable of regulating the transcription of the operably linked polynucleotide of interest, wherein the promoter is a xylem specific promoter.

In another embodiment of the present invention there is provided a promoter having the polynucleotide selected from the group consisting of the polynucleotide having at least about 85% sequence identity with the polynucleotide sequence as set forth in SEQ ID NO: 3; the polynucleotide sequence as set forth in SEQ ID NO: 3; and a fragment of the polynucleotide sequence as set forth in SEQ ID NO: 3, wherein the fragment consists of at least 250 contiguous nucleotide of SEQ ID NO: 3; wherein the promoter is capable of regulating the transcription of the operably linked polynucleotide of interest, wherein the promoter is light and wound inducible promoter.

Another embodiment of the present invention provides a promoter having the polynucleotide molecule selected from the group consisting of the polynucleotide having at least about 85% sequence identity with the nucleotide sequence as set forth in SEQ ID NO: 3, the nucleotide sequence as set forth in SEQ ID NO: 3, a fragment of the nucleotide sequence as set forth in SEQ ID NO: 3, wherein the fragment consists of at least 250 contiguous nucleotide of SEQ ID NO: 3 and a variant of the nucleotide sequence as set forth in SEQ ID NO: 3, wherein the promoter is capable of regulating the transcription of the operably linked polynucleotide molecule of interest.

Another embodiment of the present invention there is provided a recombinant DNA expression cassette comprising the promoter having the polynucleotide selected from the group consisting of the polynucleotide having at least about 85% sequence identity with the polynucleotide sequence as set forth in SEQ ID NO: 3; the polynucleotide sequence as set forth in SEQ ID NO: 3; a fragment of the polynucleotide sequence as set forth in SEQ ID NO: 3, wherein the fragment consists of at least 250 contiguous nucleotide of SEQ ID NO: 3; wherein the promoter is capable of regulating the transcription of the operably linked polynucleotide of interest, wherein the promoter is operably linked to a polynucleotide of interest.

Another embodiment of the present invention provides the polynucleotide of interest, wherein the polynucleotide of interest is selected from a group consisting of a gene of agronomic interest, a marker gene, and a reporter gene such as genes involved in lignin biosynthetic pathway, cellulose biosynthetic pathway, and common transcription factors.

One of the preferred embodiments of the present invention provides a recombinant vector comprising a promoter having the polynucleotide selected from the group consisting of the polynucleotide having at least about 85% sequence identity with the polynucleotide sequence as set forth in SEQ ID NO: 3; the polynucleotide sequence as set forth in SEQ ID NO: 3; a fragment of the polynucleotide sequence as set forth in SEQ ID NO: 3, wherein the fragment consists of at least 250 contiguous nucleotide of SEQ ID NO: 3; wherein the promoter is capable of regulating the transcription of the operably linked polynucleotide of interest.

Another embodiment of the present invention provides a recombinant vector comprising a recombinant DNA expression cassette comprising the promoter having the polynucleotide selected from the group consisting of the polynucleotide having at least about 85% sequence identity with the polynucleotide sequence as set forth in SEQ ID NO: 3; the polynucleotide sequence as set forth in SEQ ID NO: 3; a fragment of the polynucleotide sequence as set forth in SEQ ID NO: 3, wherein the fragment consists of at least 250 contiguous nucleotide of SEQ ID NO: 3; wherein the promoter is capable of regulating the transcription of the operably linked polynucleotide of interest, wherein the promoter is operably linked to a polynucleotide of interest.

An another embodiment of the present invention, there is provided a method of directing the expression of a polynucleotide molecule of interest in plant tissue, wherein the said method comprising transforming a plant with the recombinant DNA expression cassette as disclosed herein or the recombinant vector comprising as of the present invention, selecting a transgenic plant cell comprising the polynucleotide molecule of interest and generating from the transgenic plant cell a plant that expresses polynucleotide molecule of interest.

Yet another embodiment of the present invention, there is provided a method of directing the expression of a polynucleotide molecule of interest in plant tissue, wherein the said method comprising transforming a plant with the recombinant DNA expression cassette as disclosed herein or the recombinant vector comprising as of the present invention, selecting a transgenic plant cell comprising the polynucleotide molecule of interest and generating from the transgenic plant cell a plant that expresses polynucleotide molecule of interest, wherein the plant tissue is xylem.

Still yet another embodiment of the present invention, there is provided a method of directing the expression of a polynucleotide molecule of interest in plant tissue, wherein the said method comprising transforming a plant with the recombinant DNA expression cassette as disclosed herein or the recombinant vector comprising as of the present invention, selecting a transgenic plant cell comprising the polynucleotide molecule of interest and generating from the transgenic plant cell a plant that expresses polynucleotide molecule of interest, wherein light or wound induces expression of the polynucleotide molecule of interest in plant.

Still yet another embodiment of the present invention, there is provided a method of directing the expression of a polynucleotide molecule of interest in plant tissue, wherein the said method comprising transforming a plant with the recombinant DNA expression cassette as disclosed herein or the recombinant vector comprising as of the present invention, selecting a transgenic plant cell comprising the polynucleotide molecule of interest and generating from the transgenic plant cell a plant that expresses polynucleotide molecule of interest, wherein the plant tissue is xylem, wherein light or wound induces expression of the polynucleotide molecule of interest in plant.

Another embodiment of the present invention provides a method of directing the expression of a polynucleotide of interest in xylem tissue of a plant, wherein the said method comprising transforming a plant with the recombinant DNA expression cassette as disclosed herein or the recombinant vector as disclosed herein, regenerating a plant cell expressing the gene of interest in the xylem tissue and selecting transgenic plant exhibiting the expression of the gene of interest in the xylem tissue.

Another embodiment of the present invention provides a method of transformation of a plant, wherein the method is selected from a group consisting of Agro bacterium mediated transformation method, particle gun bombardment method, in-plantar transformation method, liposome mediated transformation method, protoplast transformation method, microinjection and microinjection.

One preferred embodiment of the present invention provides a transgenic plant, plant cell, tissue, comprising the recombinant DNA expression cassette of the present invention.

Another preferred embodiment of the present invention provides a transgenic plant, plant cell, tissue, comprising the recombinant DNA expression cassette of the present invention, wherein the transgenic plant exhibits expression of the polynucleotide of interest in xylem tissue.

Still another preferred embodiment of the present invention provides a transgenic plant, plant cell, tissue, comprising the recombinant DNA expression cassette of the present invention, wherein light, or wound induces expression of the polynucleotide molecule of interest in plant.

Still another preferred embodiment of the present invention provides a transgenic plant, plant cell, tissue, comprising the recombinant DNA expression cassette of the present invention, wherein the transgenic plant exhibits expression of the polynucleotide of interest in xylem tissue, wherein light, and/or wound induces expression of the polynucleotide molecule of interest in plant.

Another embodiment of the present invention provides a transgenic plant, plant cell, tissue, comprising the recombinant DNA expression cassette as disclosed herein, wherein the transgenic plant exhibits expression of the polynucleotide of interest.

Another embodiment of the present invention provides a transgenic plant, plant cell, tissue, comprising the recombinant DNA expression cassette as disclosed herein, wherein the transgenic plant exhibits expression of the polynucleotide of interest in xylem tissue.

Another embodiment of the present invention provides a transgenic plant, plant cell, tissue, comprising the recombinant DNA expression cassette as disclosed herein, wherein the transgenic plant exhibits expression of the polynucleotide of interest, wherein light, or wound induces expression of the polynucleotide.

Another embodiment of the present invention provides a transgenic seed or progeny thereof obtained from the transgenic plant as disclosed herein, wherein the seed transgenic or the progeny comprises the recombinant DNA expression cassette as disclosed herein, and exhibits expression of the polynucleotide of interest in xylem tissue.

Another embodiment of the present invention provides a transgenic plant comprising the recombinant DNA expression cassette as disclosed herein, wherein the transgenic plant is monocotyledonous or dicotyledonous plant.

Further embodiment of the present invention provides a transgenic plant comprising the recombinant DNA expression cassette as disclosed herein, wherein the transgenic plant is selected from a group consisting of Eucalyptus, poplar, oak, bamboo, tobacco, tomato, pea, soybean, Brassica, chickpea, pigeon pea, rice, maize, wheat, barley and sorghum, potato, casuarina, Subabul, acacia, Teak apple and woody plant.

Still further embodiment of the present invention provides a transgenic seed or progeny thereof obtained from the transgenic plant as disclosed in the present invention, wherein the transgenic seed or the progeny comprises the recombinant DNA expression cassette of the present invention.

One embodiment of the present invention provides a promoter having the polynucleotide molecule selected from the group consisting of the polynucleotide having at least about 85% sequence identity with the nucleotide sequence as set forth in SEQ ID NO: 3, the nucleotide sequence as set forth in SEQ ID NO: 3, the nucleotide sequence as set forth in SEQ ID NO: 5, the nucleotide sequence as set forth in SEQ ID NO: 6, the nucleotide sequence as set forth in SEQ ID NO: 7, a fragment of the nucleotide sequence as set forth in SEQ ID NO: 3, wherein the fragment consists of at least 250 contiguous nucleotide of SEQ ID NO: 3, and a variant of the nucleotide sequence as set forth in SEQ ID NO: 3, wherein the promoter is capable of regulating the transcription of the operably linked polynucleotide molecule of interest.

The promoter as disclosed in the present invention is xylem specific. Further, the promoter is light and/or wound inducible.

Another embodiment of the present invention provides the polynucleotide molecule of interest selected from a group consisting of a gene of agronomic interest, a marker gene and a reporter gene, genes involved in lignin biosynthetic pathway, cellulose biosynthetic pathway, and common transcription factors..

Yet another embodiment of the present invention provides a recombinant vector comprising the recombinant DNA expression cassette as disclosed in the present invention.

Further embodiment of the present invention provides a method of directing the expression of a polynucleotide molecule of interest in plant tissue, wherein the said method comprising transforming a plant with the recombinant DNA expression cassette comprising the promoter molecule having the polynucleotide molecule selected from the group consisting of the polynucleotide having at least about 85% sequence identity with the nucleotide sequence as set forth in SEQ ID NO: 3, the nucleotide sequence as set forth in SEQ ID NO: 3, the nucleotide sequence as set forth in SEQ ID NO: 5, the nucleotide sequence as set forth in SEQ ID NO: 6, the nucleotide sequence as set forth in SEQ ID NO: 7, a fragment of the nucleotide sequence as set forth in SEQ ID NO: 3, wherein the fragment consists of at least 250 contiguous nucleotide of SEQ ID NO: 3, and a variant of the nucleotide sequence as set forth in SEQ ID NO: 3; or the recombinant vector as disclosed in the present invention, selecting a transgenic plant cell comprising the polynucleotide molecule of interest and generating from the transgenic plant cell a plant that expresses polynucleotide molecule of interest.

Further embodiment of the present invention provides a method of transformation selected from a group consisting of Agro bacterium mediated transformation method, particle gun bombardment method, in-plantar transformation method, liposome mediated transformation method, protoplast transformation method, microinjection and microinjection.

Another embodiment of the present invention provides a transgenic plant, plant cell, tissue, comprising the recombinant DNA expression cassette as disclosed in the present invention.

Another embodiment of the present invention relates to transgenic plants disclosed in the present invention that exhibit expression of the polynucleotide of interest in xylem tissue.

Another embodiment of the present invention relates to transgenic plants disclosed in the present invention, wherein light or wound induces expression of the polynucleotide molecule of interest in plant.

Another embodiment of the present invention relates to transgenic plants disclosed in the present invention, wherein the transgenic plant is monocotyledonous or dicotyledonous plant.

Another embodiment of the present invention relates to transgenic plants disclosed in the present invention, wherein the transgenic plant is selected from a group consisting of Eucalyptus, poplar, oak, bamboo, tobacco, tomato, pea, soybean, Brassica, chickpea, pigeon pea, rice, maize, wheat, barley and sorghum, potato, casuarina, Subabul, acacia, Teak apple and woody plant.

Another embodiment of the present invention relates to a transgenic seed or progeny thereof obtained from transgenic plants disclosed in the present invention wherein the transgenic seed or the progeny comprises the recombinant DNA expression cassette as disclosed in the present invention.

The promoter disclosed herein can be modified. Those skilled in the art can create promoters that have variations in the polynucleotide sequence. The polynucleotide sequences of the promoters of the present invention as shown in SEQ ID NO: 3 may be modified or altered to enhance their control characteristics. One preferred method of alteration of a polynucleotide sequence is to use PCR to modify selected nucleotides or regions of sequences. These methods are well known to those of skill in the art. Sequences can be modified, for example by insertion, deletion, or replacement of template sequences in a PCR-based DNA modification approach.

Based on the promoter molecule as disclosed herein novel chimeric promoters can be designed or engineered by the methods known in the prior art. Many promoters contain cis-elements that activate, enhance, or define the strength and/or specificity of the promoter. For example promoters may contain "TATA" boxes defining the site of transcription initiation and other cis-elements located upstream of the transcription initiation site that modulate transcription levels.

For example, a chimeric promoter may be produced by fusing a first promoter fragment containing the activator cis-element from one promoter to a second promoter fragment containing the activator cis-element from another promoter; the resultant chimeric promoter may cause an increase in expression of an operably linked transcribable polynucleotide molecule.

Promoters can be constructed such that promoter fragments or elements are operably linked, for example, by placing such a fragment upstream of a minimal promoter. The cis-elements and fragments of the present invention can be used for the construction of such chimeric promoters.
In the present invention, the promoter region of 4CL1 gene was isolated by genome walking method from E. camaldulensis and sequencing of the region was carried out. The 4CLI promoter (1068 bp) was cloned in pB121 in place of CaMV 35S promoter.

Methods for construction of chimeric and variant promoters of the present invention include, but are not limited to, combining control elements of different promoters or duplicating portions or regions of a promoter. A person skilled in the art is familiar with the standard resource materials that describe specific conditions and procedures for the construction, manipulation, and isolation of macromolecules and generation of recombinant organisms, producing transgenic plants.

A promoter comprising the polynucleotide as set for the in SEQ ID NO: 3 include any length of said polynucleotide sequence that is capable of regulating an operably linked transcribable polynucleotide molecule. For example, the promoter as disclosed in SEQ ID NO: 3 may be truncated or portions deleted and still be capable of regulating transcription of an operably linked polynucleotide molecule.

A cis-element of the disclosed promoters may confer a particular specificity such as conferring enhanced expression of operably linked polynucleotide molecules in certain tissues and therefore is also capable of regulating transcription of operably linked polynucleotide molecules. Thus, any fragments, portions, or regions of the promoters comprising the polynucleotide sequence shown in SEQ ID NO: 3 can be used as regulatory polynucleotide molecules, including but not limited to cis-elements or motifs of the disclosed polynucleotide molecules. The promoter as disclosed herein encompasses ant type of substitutions, deletions, insertions, or any combination thereof to produce a final construct.

Expression cassette and/or or DNA Constructs as disclosed herein would contain a promoter operably linked to a transcribable polynucleotide molecule operably linked to a 3' transcription termination polynucleotide molecule. In addition, the expression cassette and/or DNA constructs may include but are not limited to additional regulatory polynucleotide molecules from the 3'-untranslated region (3' UTR) of plant genes to increase mRNA stability of the mRNA, such as the PI-II termination region of potato or the octopine or nopaline synthase 3' termination regions. Various 3' UTR plant genes are known in the prior art.

Further, the expression cassette and/or the DNA Constructs as disclosed herein may contain but are not limited to the 5' untranslated regions (5' UTR) of an mRNA polynucleotide which is play an important role in translation initiation.

These additional upstream (5' UTR) and downstream (3' UTR) regulatory polynucleotide molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.

Thus, the expression cassette and/or the DNA Constructs of the present invention comprise promoters foe example as set forth in SEQ ID NO: 3, or modified as described above, operatively linked to a transcribable polynucleotide of interest so as to direct transcription of said transcribable polynucleotide at a desired level and/or in a desired tissue or developmental pattern upon introduction of said construct into a plant cell.

In some cases, the transcribable polynucleotide molecule comprises a protein-coding region of a gene, and the promoter provides for transcription of a functional mRNA molecule that is translated and expressed as a protein product. Constructs may also be constructed for transcription of antisense RNA molecules or other similar inhibitory RNA in order to inhibit expression of a specific RNA molecule of interest in a target host cell.

Exemplary transcribable polynucleotide of interest for incorporation into constructs of the present invention include, for example, DNA molecules or genes from a species other than the target gene species, or even genes that originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods rather than classical reproduction or breeding techniques. Exogenous gene or genetic element is intended to refer to any gene or DNA molecule that is introduced into a recipient cell. The type of DNA included in the exogenous DNA can include DNA that is already present in the plant cell, DNA from another plant, DNA from a different organism, or a DNA generated externally, such as a DNA molecule containing an antisense message of a gene, or a DNA molecule encoding an artificial or modified version of a gene.

The promoters of the present invention can be incorporated into a construct using marker genes as described and tested in transient analyses that provide an indication of gene expression in stable plant systems. Methods of testing for marker gene expression in transient assays are known to those of skill in the art. Transient expression of marker genes has been reported using a variety of plants, tissues, and DNA delivery systems. For example, types of transient analyses can include but are not limited to direct gene delivery via electro oration or particle bombardment of tissues in any transient plant assay using any plant species of interest. Such transient systems would include but are not limited to electro oration of protoplasts from a variety of tissue sources or particle bombardment of specific tissues of interest. The present invention encompasses the use of any transient expression system to evaluate promoters or promoter fragments operable linked to any transcribable polynucleotide molecules, including but not limited to selected reporter genes, marker genes, or genes of agronomic interest. Examples of plant tissues envisioned to test in transients via an appropriate delivery system would include but are not limited to leaf base tissues, callus, cotyledons, roots, endosperm, embryos, floral tissue, pollen, and epidermal tissue. Any scorable or screenable marker gene known in the art can be used in a transient assay such as a GUS gene or a GFP gene. The constructs containing the promoters or promoter fragments operably linked to a marker gene are delivered to the tissues and the tissues are analyzed by the appropriate mechanism, depending on the marker. The quantitative or qualitative analyses are used as a tool to evaluate the potential expression profile of the promoters or promoter fragments when operatively linked to genes of agronomic interest in stable plants.

Thus, in one of the preferred embodiment, a polynucleotide of the present invention as shown in SEQ ID NO: 3 or fragment, variants, or derivatives thereof, capable of regulating transcription, is operably linked to a transcribable polynucleotide molecule that provides for a selectable, screenable, or scorable marker.

Marker genes as described herein include, but are not limited to transcribable polynucleotide molecules encoding beta.-glucuronidase (GUS), green fluorescent protein (GFP), luciferase (LUC), proteins that confer antibiotic resistance, or proteins that confer herbicide tolerance. Useful antibiotic resistance markers, including but not limited to those encoding proteins conferring resistance to kanamycin (nptll), hygromycin B (aph IV), streptomycin or spectinomycin (aad, spec/strep), and gentamycin (aac3 and aacC4) are known in the art. Herbicides include but are not limited to glyphosate, glufosinate, sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohezanedione, protoporphyrionogen oxidase inhibitors, and isoxasflutole herbicides. Polynucleotide molecules encoding proteins involved in herbicide tolerance are known in the art, and include, but are not limited to a polynucleotide molecule encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase); a polynucleotide molecule encoding bromoxynil nitrilase (Bxn); a polynucleotide molecule encoding phytoene desaturase (crtl); a polynucleotide molecule encoding acetohydroxyacid synthase (AHAS, aka ALS); and bar gene.

In one embodiment, a polynucleotide of the present invention as shown in SEQ ID NO: 3 or fragments, variants, or derivatives thereof, capable of regulating transcription, is operably linked to a transcribable polynucleotide molecule that is a gene of agronomic interest. The expression of a gene of agronomic interest is desirable in order to confer an agronomically important trait.

A gene of agronomic interest that provides a beneficial agronomic trait to crop plants includes, but not limited to genes involved in lignin biosynthetic pathway, cellulose biosynthetic pathway, and common transcription factors, genetic elements comprising herbicide resistance, increased yield, insect control, fungal disease resistance, virus resistance, nematode resistance, bacterial disease resistance, starch production modified oils production, high oil production, modified fatty acid content, high protein production, fruit ripening, enhanced animal and human nutrition, biopolymers, environmental stress resistance, improved processing traits, industrial enzyme production, improved flavor, nitrogen fixation, hybrid seed production and biofuel production.

Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible. As such, the spirit and scope of the appended claims should not be limited to the description of the preferred embodiment contained therein.

EXAMPLES

The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure.

Example 1

Isolation and cloning of 4CL1 promoter

The genome walker library constructed from DNA of E. camaldulensis was used for isolating promoter sequence (Figure 1) The PCR reaction consisted of following components 10X buffer (containing 15 raM MgCl2), 2.5ul of 10 mM dNTP, 0.5ul API (lOuM), 0.5ul of GSP (10 mM), 0.5 ul of Advantage 2 polymerase mix (50x) in a 25 ul reaction.The PCR reaction was performed in Eppendorf mastercycler machine with following thermal conditions, 94°C for 25 sec & 72°C for 3 min for 7 cycles followed by 94°C for 25 sec,67°C for 3 min,67°C for 7 min for 32 cycles. After amplification, products were resolved on 1% agarose gel stained with ethidium bromide. This was followed by nested reaction with nested primers and the single amplified product was eluted with Neucleospin extract II kit (Macherey-Nagel). The eluted DNA was ligated with vector pTZ57R by following kit protocol (Fermentas). The reaction products from Ml3 amplification of clones were subjected to sequencing reaction by M13 primers by ABI3730 sequencer at Vimta Lab Genomic Facility in Hyderabad.

The 4CL1 promoter (SEQ ID NO: 3) was amplified with primers as set forth in SEQ ID NO: 1 and SEQ ID NO: 2 having restriction sites, Hind III and Xbal. The PCR product was first cloned into blunt end vector pTZ57R (Fermentas). The recombinant vector was designated as pTZ57R:4CLI. The promoter region contained 1068 bases (SEQ ID NO: 3) which included 5' UTR of 4CL1 gene. The 4CL1 promoter fragment (1068 bp) was released from vector by digesting with Hindlll and Xbal and cloned in intact pBI121 with Hindlll and Xbal by replacing CaMV 35S promoter (Figure 2). The recombinant pBI121 vector comprising the 4CL1 promoter (SEQ ID NO: 3) was designated as IRC005. The recombinant pBI121 vector comprising the 4CL1 promoter (SEQ ID NO: 3) was further transformed in E. coli .Transformation of E. coli (DH5 alpha) cells with ligated product were carried out by the instructions given in the kit (Fermentas). E. coli cells were harvested by spinning at 14000 rpm for a minute at 4°C and cells were immediately resuspended in 300 ul of T solution for 5 minutes. The cells were harvested again at 4°C and resuspended the cells in 120 ul of T solution, allowed to be on ice for 20 minutes. Ligation mixture of 2.5 ul was mixed with 50 ul of resuspended cells and plated on ampicillin Lauria Bertini Agar plates. White colonies were picked up from the plate grown overnight at 37°C and were inoculated to Lauria Broth with ampicillin 50 ug / ml. Cells were harvested and plasmids were isolated by using Qia prep spin Miniprep kit (Qiagen Cat No.27104).The reaction products from Ml 3 amplification of clones was subjected to sequencing reaction by M13 primers by ABI 3730 sequencer at Vimta Lab Genomic Facility in Hyderabad.

The identity of the clones was confirmed by sequencing and BLAST analysis. The bioinformatics analysis indicated the region contains various elements required for xylem specific expression and could be induced by wounds.

The construct was studied for the xylem specific as well as wound inducible expression patterns.

Example 2

Analysis of 4CL1 promoter region by PLACE Software The sequence of 4CL promoter was analysed by online PLACE (Plant Cis-acting Regulatory DNA Elements) to find signals and possible functions in the promoter region (Table 1).

Example 3

Deletion analysis of 4CL1 promoter for minimal promoter analysis

In order to find minimal promoter of 4CLI, various deletion constructs were made (Figure 3). Three more recombinant constructs were made in the vector pBI121 with 250, 500 and 960 fragments named as IRC002, IRC003 and IRC004 respectively.

The pBI121 vector was digested with Hind III and Xbal to release the CaMV35S promoter from the pBI121. The reactions were carried out in 20 ul reaction with one microgram of plasmid DNA with 1 unit each of Xbal and Hindlll enzymes. The 250 bp fragment (SEQ ID NO: 5), 500 bp fragment (SEQ ID NO: 6), and 960 bp fragment (SEQ ID NO: 7) were amplified from pTZ57R:4CLI with following set of primers along with Xbal and Hindlll recognition sequences.

4CL1_250:
IME2196F:
SEQ ID NO: 8 TGATTACGCCAAGCTTGCAACAAGACAAGCGGAGA
IME2196R:
SEQ ID NO: 9 CCGGGGATCCTCTAGACTCAGGTTTGGTTTGAGCT
4CL1_500
IME2197F
SEQ ID NO: 10 TGATTACGCCAAGCTTGTGCCGTTTAATCGGGAAG
IME2196R
SEQ ID NO: 11 CCGGGGATCCTCTAGACTCAGGTTTGGTTTGAGCT
4CL1_960
IME2216F
SEQ ID NO: 12 GGC CAG TGC CAA GCT TAA TTA TTC GGT CCG AGT AAC
IME2216R
SEQ ID NO: 13 TTG TGA TGT ATC TAG ATG TCG TGG CGG GGT GG

All the three fragments were individually cloned in pBI121 using infusion cloning reagents as per the manufactures instructions (Takara). The reaction mixtures were plated on LB plate with Kanamycin as the selection antibiotic. The positive colonies were picked up and were subjected to colony PCR with promoter specific primers.

Example 4

Transformation of Agrobacterium with plasmid comprising 4CL1 promoter (SEQ ID NO: 3) and its fragments (SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7) All the four promoter constructs as described above were mobilized into Agrobactrium tumenfacinces by electroporation with Electromax Agrobacterium LBA4404 cells with the manufactures instructions (Invitrogen). After two days, positive colonies were picked up and were confirmed by inoculating into YM broth with streptomycin and kanamycin. Cultures were stored in -80 in 25% glycerol.

Cloning of F5H under 4CL1 promoter

The cDNA of F5H was ampiified from pJET: F5H with primers containing Xbal site by proof reading polymerase (Pfx by invitrogen). The cDNA was cloned with stop codon to Xbal site of pBI121 by infusion cloning reagents as per the manufactures instructions (Takara). The selection for recombinants was made on LB plate with Kanamycin. The colonies were screened by PCR by using F5H specific primers.

Ferulate 5 hydroxylase gene was isolated from E. camaldulensis cDNA by Rapid Amplification of cDNA (SEQ ID NO: 4) and shown to exhibit 99% identity with E. globulus F5H at amino acid level. F5H cDNA was cloned in pB121 ahead of GUS gene at Xbal site in two ways- One as F5H cDNA with stop codon and other one by abolishing the stop codon to a tryptophan residue. The latter cloning results in a fusion protein of F5H with GUS protein and will be useful for easy screening of positive transformants for F5H by histochemical ways. Both constructs are basically intended to increase the syringyl units in tree crops by xylem specific promoter 4CL1 (Figure 4, pBI121:4CLlp:F5H: GUS).

Functional analysis of 4CL1 promoter fragments in Arabidopsis

The functional analysis of the promoter constructs were carried out by vacuum infiltration method (Bechtold, N., Ellis, J., and Pelletier, G. (1993). In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci. Paris, Life Sciences 316:1194-1199). Arabidopsis plants were grown in pots containing soil covered with cheesecloth. First bolts were clipped to encourage proliferation of many secondary bolts. Plants were ready for transformation after 4-6 days of clipping. Agrobacterium tumefaciens strain carrying gene of interest on a binary vector was grown in a large liquid culture at 30°C temperature in LB with kanamycin and streptomycin. Agrobacterium cells were spun down and were resuspended in 5% Sucrose solution to an OD600 = 0.8. Before dipping, Silwet L-77 was added to a concentration of 0.05% (500ul/L) and mixed well. The above-ground parts of plant were dipped in Agrobacterium solution for 2 to 3 seconds, with gentle agitation. The plants were kept under a dome or cover for 16 to 24 hour under dark conditions. The plants were taken to normal conditions and watered normally and watering was stopped once seeds become mature. Seeds were harvested for screening of transformants.

The screening of transformants was made by using kanamycin as selection marker. Around 2000 seeds were plated on 0.5 x MS/0.8% agar plates with 50 ug/ml kanamycin. The seeds were cold treated for two days and then they were grown in continuous light for 7-10 days. The putative transformants were further grown for histochmical staining of GUS expression under 4CLI promoter.

For histochemical staining, the leaves were placed in microtitre plate and 250-500 ul stain solution (0.1 M Sodium phosphate pH 7.0, lOmM EDTA, 0.1% Triton X-100, 1 mg/ml X- Glue, lOOug/ml chloramphenicol) were poured in the wells. The vacuum was applied for 10-15 minutes in a dessicator. The vacuum was released slowly and plate was covered with paraffin film to 37°C for 24-48 hours. The tissue was cleared by several changes of 70 % ethanol at 37°C. The samples were observed under stereo microscope.

The four constructs namely 250, 500, 960 and 1060 were transformed in to Arabidopsis and plants were subjected to histochemical analysis for P glucuronidase expression. Two week old plant was used for analysis on comparison with wild type plants. Though the plants transformed with 250 bp fragment were also able to show GUS expression, the pattern was distributed throughout the plant without showing any tissue specificity. At the same time, the plant transformed with 500 bp fragment showed a very specific vascular tissue expression as shown in Figure 5. The expression was highest in roots, followed by stem and then leaves in vascular specific manner when two weeks old plant was analyzed. The results of the experiment showed that 4CL with 500 bp fragment is the minimum promoter region required for vascular specificity of 4CL promoter.

The stem of Arabidopsis plants transformed with recombinant constructs 250, 500 960 and 1060 were also subjected for quantitative real time PCR to study the P glucuronidase expression levels by each fragment (Figure 6). The data showed that all the constructs under study were able to drive the expression of GUS and there was a sharp increase in the expression level of GUS gene when 4CL_1060 was used, indicating the additional 100 bp which is coming from 5' UTR of 4CL1 gene can enhance expression level of genes under 4CL promoter.

The expression level of various lignin biosynthetic genes such as CAD, CCR, F5H and OMT were studied in comparison with 4CL in stem of eucalyptus using real time qRT PCR (Figure 7). The avg delta CT which is the normalized value of cDNA in the samples showed the least value in case of 4CL gene indicating abundance of 4CL transcripts in comparison with rest of the genes under study. The data indicated the relative strength of 4CL promoter in comparison with other candidates in the lignin biosynthetic pathway.

The Arabidopsis plant transformed with minimal promoter region for vascular specificity (4CL1500) were subjected for wounding analysis. The plants transformed with 4CL1500 were subjected to pricking with needles on leaf surface and leaf sample were collected at 0 hr, 2 hr and 3 hr. Relative quantification of P glucuronidase transcripts indicated a higher expression after two hours of injury in comparison with leaf collected at the start of experiment (Figure 8). The data showed the 4CL promoter will be able to get induced by injury indicating the possibility for usage in in manipulating crop defense against insect pests.

SEQ ID NO: 1
FP- AGCTAAAGCTTAAATTATTCGGTCCG
SEQ ID NO: 2
RP- ATATATCTAGATGTCGTGGCGGGGT
SEQ ID NO: 3 nucleotide sequence of 4CLI promoter isolated from E.

camaldulensis

AAATTATTCGGTCCGAGTAACATGGAGAAATTTTAACGCCCTCTCTCCAACATGGAGGACG GATGAAAGTGTGGTATGTGGGATTTGACGATAAGGTAGAGGCATGCACGAAAAGGGGCAAA ATCTGGCTCCGATGCCATGTTCAAATGTCTACGTCAAGCCATGTGAGATGAATGGCTGGGC CGATTCATTTTTGTACATTGATTGATGGGTCTTGACATTATCTAGGTGCTCAAAGTGAATG TGTAGATAAGGATTGGACTTATTATTATCATTATTATTATTTTTTGGAAATGAAAAT AT TA GATTGTCGAGCCTAAATGACATGGGTATTTCAGCGCTGTTTTTTTACATATATCAGAATTA ACTAGTGGAATCTGACTAATAGAGAGCATGAATAATTTCGATGCCACGTTCGAATGTCCAG CTCGGGCGATGTGGGGGTGCCGTTTAATCGGGAAGGAGGGCCCGCTTTGGTAGGTGAAATC TACGTGGCACAGCATTGCTGGCCAAAGGTCAAAACTTGGCATGCACTAAAATAATCTCGA AAGGGCACCCAAAAAAAAAAATGGAAAACGAAAAGAAAACGATGGACTTTTGTTTATGCCA CCAAATGTCCCTCACCAACCTCCGATGGCTCCTTCCCTGGCGTGGTAGGTGATGCCCCGAG ACGACCCAATCTCCCCTCCCCTCCCTCCAATCGCAGCACGCCACGTGCGCAACAAGACAAG CGGAGACGCCCCGCGAACTACCGTCAAGGCTAAGCCCGAGGCGGCACGTGGCGGGTCGCGA CCCCTCCCCAAAGGGACAGCCCGGGCGGACTTCTCCACCGCCGTCCCTGGCCAGGG CGATAGGAAAGGACGGAAATGCCCCCCCATCGAACCCCGACTCCCACCAGCTCCTCCTACC CGCCACCAGCTCGTGTATATTACCCGCAACCAAAGCTCAAACCAAACCTGAGGGAAGGGAG GAAGGCGCCACCACCAAACGCTCACCTTCTCATCATCAGCCCTCTCTCTCTGTCTCTGTCT CTGTCTCTCGATTCTCCACCCCGCCACGACA SEQ ID NO: 4 nucleotide sequence of F5H Cdna GATCACATTCGAAAGATAAGTTGCGCTTAAATCCTCTCCAAAAGAGCTAATCCATGGAT ATTTTCTATTTCTATTCCCAACTCCAGTCTCTTGTTCAAACTCAACTCCAGCAATCTCCCA TGACCCTCCTCCTCTCCGTCGTCCCTCTTCTCCTCTTCCTCGGGCTCGTGGCTCGGCTCCG GCGCAAGCCGCCCTTCCCACCGGGCCCGAGGGGCCTCCCGGTCATCGGGAACATGCTCATG GGGGAGCTCACCCACCGCGGCCTCGCGAGTCTGGCGAAGAAGTATGGCGGGATCTTCC ACCTCCGCATGGGCTTCCTGCACATGGTTGCCGTGTCGTCCCCCGACGTGGCCCGCCAGGT CTCCAGGTCCACGACGGGATCTTCTCGAACCGGCCTGCCACCATCGCGATCAGCTACCTC ACGTATGACCGGGCCGACATGGCCTTCGCGCACTACGGCCCGTTCTGGAGGCAGATGCGGA CTGTGCGTGATGAAGCTCTTCAGCCGGAAGCGGGCTGAGTCGTGGGAGTCGGTCCGCGA TGAGGTGGACACGATGGTGCGCACCGTCGCGGGCAGCGAGGGGACCTCCGTGAACATCGGC CTCGTGTTCGAGCTCACGCGGGACATCATCTACCGCGCGGCCTTCGGCACGAGCTCGA GCGAGGGCCAGGACGAGTTCATCAGCATACTGCAGGAGTTCTCCAAATTATTTGGCGCCTT CAACATAGCCGATTTTATCCCGTACCTGAGCTGGATCGATCCGCAAGGGCTCACCGCCAGG CTTGTCAAGGCGCGCCAGTCGCTGGACGGGTTCATCGACCACATTATAGATGATCACATGG ACAAGAAGAGAAACAAGACGAGTTCCGGTGGAGGCGATCAAGATGTCGATACCGACATGGT GGACGATCTGCTGGCCTTCTACTGCGACAAAGCGAAGGTGAACGAGTCCGACGATTTGCAG AACTCGATCAGGCTAACGAGAGACAACATCAAGGCCATCATCATGGACGTGATGTTCGGCG GGACGGAGACTGTGGCGTCAGCTATCGAGTGGGCCATGGCGGAGCTCATGCGAAGCCCCGA ACCTGAAGAAGGTCCAGCAAGAACTCGCGGATGTCGTGGGCTTAGACCGGAGAGTCGAG GAGAGCGACTTCGAGAAGCTGACCTATCTCAAGTGCTGTCTCAAAGAGACCCTCCGCCTCC ACCCGCCGATCCCGCTGCTCCTCCACGAGACGGCAGAGGACGCCGTGATCTCCGGCTACCG TCCCCGCACGGTCTCGGGTCATGATCAATGCATGGGCCATCGGGCGTGACCCCGGCTCG TGGACCGAACCTGACAAGTTCAAACCGTCCCGGTTCCTGGAGCCAGGCATGCCCGACTACA AGGGGAGCAACTTCGAGTTCATCCCTTTCGGGTCGGGCCGGAGGTCGTGCCCAGGGATGCA TCGGGCTCTACGCGCTCGACATGGCCGTGGCCCACCTCCTGCACTGCTTCACGTGGGAA CTGCCCGACGGGATGAAGCCGAGCGAGATGGACATGGGCGACGTCTTCGGGCTCACCGCGC AGGTCCACCCGGCTCGTGGCGGTGCCGACTCCGAGGTTGGTGGGGGCTCTATATTGAGC AAGCAAATGGAGGGTCGGGTTGGGGGGGGGCGCAGGGAGGGGAACGTATTTTCAGCTCC
SEQ ID NO: 5 for 250 bp fragmentGCAACAAGACAAGCGGAGACGCCCCGCGAACTACCGTCAAGGCTAAGCCCGAGGCGGCACG GTCGCGATTGGCCCCCTCCCCAAAGGGACAGCCCGGGCGGACTTCTCCACCGCCG TCCCTGGCCAGGGCGATAGGAAAGGACGGAAATGCCCCCCCATCGAACCCCGACTCCCACC GCTCCTCCTACCCGCCACCAGCTCGTGTATATTACCCGCAACCAAAGCTCAAACCAAACC TGAG SEQ ID NO: 6 for 500 bp fragment

GTGCCGTTTAATCGGGAAGGAGGGCCCGCTTTGGTAGGTGAAATCCTACGTGGCACAGCAT TGCTGGCCAAAGGTCAAAACTTGGCATGCACTAAAATAATCTCGAAAGGGCACCCAAAAAA AAAAATGGAAAACGAAAAGAAAACGATGGACTTTTGTTTATGCCACCAAATGTCCCTCACC AACCTCCGATGGCTCCTTCCCTGGCGTGGTAGGTGATGCCCCGAGACGACCCAATCTCCCC TCCCCTCCCTCCAATCGCAGCACGCCACGTGCGCAACAAGACAAGCGGAGACGCCCCGCGA TACCGTCAAGGCTAAGCCCGAGGCGGCACGTGGCGGGTCGCGATTGGCCCCCTCCCCAA AGGGACAGCCCGGGCGGACTTCTCCACCGCCGTCCCTGGCCAGGGCGATAGGAAAGGACGG TGCCCCCCCATCGAACCCCGACTCCCACCAGCTCCTCCTACCCGCCACCAGCTCGTGT ATATTACCCGCAACCAAAGCTCAAACCAAACCTGAG SEQ ID NO: 7 for 960bp fragment

AAATTATTCGGTCCGAGTAACATGGAGAAATTTTAACGCCCTCTCTCCAACATGGAGGACG GATGAAAGTGTGGTATGTGGGATTTGACGATAAGGTAGAGGCATGCACGAAAAGGGGCAAA ATCTGGCTCCGATGCCATGTTCAAATGTCTACGTCAAGCCATGTGAGATGAATGGCTGGGC CGATTCATTTTTGTACATTGATTGATGGGTCTTGACATTATCTAGGTGCTCAAAGTGAATG TGTAGATAAGGATTGGACTTATTATTATCATTATTATTATTTTTTGGAAATGAAAATATTA GATTGTCGAGCCTAAATGACATGGGTATTTCAGCGCTGTTTTTTTACATATATCAGAATTA ACTAGTGGAATCTGACTAATAGAGAGCATGAATAATTTCGATGCCACGTTCGAATGTCCAG CTCGGGCGATGTGGGGGTGCCGTTTAATCGGGAAGGAGGGCCCGCTTTGGTAGGTGAAATC TACGTGGCACAGCATTGCTGGCCAAAGGTCAAAACTTGGCATGCACTAAAATAATCTCGA AAGGGCACCCAAAAAAAAAAATGGAAAACGAAAAGAAAACGATGGACTTTTGTTTATGCCA CCAAATGTCCCTCACCAACCTCCGATGGCTCCTTCCCTGGCGTGGTAGGTGATGCCCCGAG ACGACCCAATCTCCCCTCCCCTCCCTCCAATCGCAGCACGCCACGTGCGCAACAAGACAAG CGGAGACGCCCCGCGAACTACCGTCAAGGCTAAGCCCGAGGCGGCACGTGGCGGGTCGCGA CCCCCTCCCCAAAGGGACAGCCCGGGCGGACTTCTCCACCGCCGTCCCTGGCCAGGG ATAGGAAAGGACGGAAATGCCCCCCCATCGAACCCCGACTCCCACCAGCTCCTCCTACC CGCCACCAGCTCGTGTATATTACCCGCAACCAAAGCTCAAACCAAACCTGAGGGAAGGGAG GAAGGCGCCACCACCAAACGCTCACCTTCTCATCATCAGCCCTCTCTCTCTGTCTCTGTCT CTGTCTCTCGATTCTCCACCCCGCCACGACA SEQ ID NO: 8 Forward Primer TGATTACGCCAAGCTTGCAACAAGACAAGCGGAGA SEQ ID NO: 9 Reverse Primer CCGGGGATCCTCTAGACTCAGGTTTGGTTTGAGCT SEQ ID NO: 10 Forward Primer TGATTACGCCAAGCTTGTGCCGTTTAATCGGGAAG SEQ ID NO: 11 Reverse Primer CCGGGGATCCTCTAGACTCAGGTTTGGTTTGAGCT SEQ ID NO: 12 Forward Primer GGC CAG TGC CAA GCT TAA TTA TTC GGT CCG AGT AAC SEQ ID NO: 13 Reverse Primer TTG TGA TGT ATC TAG ATG TCG TGG CGG GGT GG Table 1: Various elements of the 4CL1 promoter

16 BOXLCOREDCPA L (+) ACCWWCC PALI PROMOTER REGION.

17 BS1EGCCR (-) AGCGGG NUCLEAR PROTEIN BINDING SITE; REQUIRED FOR VASCULAR EXPRESSION;

18 CAATBOX1 (+) CAAT "CAAT PROMOTER CONSENSUS SEQUENCE" FOUND IN LEGA GENE OF PEA;

19 CACGTGMOTIF (+) CACGTG "CACGTG MOTIF"; "G-BOX"; BINDING SITE OF ARABIDOPSIS GBF4.

20 CACTFTPPCA1 (+) YACT TETRANUCLEOTIDE (CACT) IS A KEY COMPONENT OF MEM1.

21 CCAATBOX1 (+) CCAAT ACT COOPERATIVELY WITH HSES TO INCREASE THE HS PROMOTER ACTIVITY;

\2 CELLCYCLESC (+) CACGAAAA CRITICAL FOR CYTOKININ-ENHANCED PROTEIN BINDING.

23 CPBCSPOR (+) TATTAG CAN ENHANCE GENE EXPRESSION; INVERTED GAGA.

24 CTRMCAMV35S (+) TCTCTCTCT COPPER-RESPONSE ELEMENT.

25 DOFCOREZM (+) AAAG REQUIRED FOR BINDING OF DOF PROTEINS IN MAIZE.

26 DPBFCOREDCDC3 (-) ACACNNG A NOVEL CLASS OF BZIP TRANSCRIPTION FACTORS.

27 E2FCONSENSUS (+) WTTSSCSS E2F CONSENSUS SEQUENCE" OF ALL DIFFERENT E2F-DP-BINDING MOTIFS.

28 EBOXBNNAPA (+) CANNTG E-BOX OF NAPA STORAGE-PROTEIN GENE OF BRASSICA NAPUS.

29 ELRECOREPCRP1 (-) TTGACC ELRE (ELICITOR RESPONSIVE ELEMENT).

u° EMBP1TAEM (+) CACGTGGC BINDING SITE OF TRANS-ACTING FACTOR EMBP-1.

31 GATABOX (+) GATA REQUIRED FOR HIGH LEVEL, LIGHT REGULATED, AND TISSUE SPECIFIC EXPRESSION

32 GBOX10NT (-) GCCACGTGCC REQUIRED FOR HIGH-LEVEL CONSTITUTIVE EXPRESSION IN SEED, LEAF, ROOT, AXILLARY BUD, ALMOST ALL PARTS OF FLOWER BUDS AND POLLEN;

33 GCCCORE (-) GCCGCC PLAY IMPORTANT ROLES IN REGULATING JASMONATE RESPONSIVE GENE EXPRESSION

34 GT1 CONSENSUS (+) GRWAAW INFLUENCES THE LEVEL OF SA-INDUCIBLE GENE EXPRESSION;

35 GTGANTG10 (+) GTGA SHOWS HOMOLOGY TO PECTATE LYASE AND IS THE PUTATIVE HOMOLOGUE OF THE TOMATO GENE LAT56.

36 HEXMOTIFTAH3H 4 (+) ACGTCA HEXAMER MOTIF" FOUND IN PROMOTER OF WHEAT.

37 IBOX (+) GATAAG I-BOX"; CONSERVED SEQUENCE UPSTREAM OF LIGHT-REGULATED GENES; SEQUENCE FOUND IN THE PROMOTER REGION OF RBCS OF TOMATO.

38 IBOXCORE (+) GATAA CONSERVED SEQUENCE UPSTREAM OF LIGHT-REGULATED GENES OF BOTH MONOCOTS AND DICOTS;

39 IBOXCORENT (+) GATAAGR ASSOCIATED WITH LIGHT-RESPONSIVE PROMOTER REGIONS.

N,0 INRNTPSADB (+) YTCANTYY "INR (INITIATOR)" ELEMENTS FOUND IN THE TOBACCO PSADB GENE PROMOTER WITHOUT TATA BOXES;

41 IR020S (+) CACGTGG OSIR02-BINDING CORE SEQUENCE; "G-BOX PLUS G";

42 LRENPCABE (+) ACGTGGCA "LRE"; A POSITIVE LIGHT REGULATORY ELEMENT IN TOBACCO.

43 LTRECOREATCO R15 (+) CCGAC CORE OF LOW TEMPERATURE RESPONSIVE ELEMENT (LTRE) OF COR15AGENE.

44 MARTBOX (-) TWTWTTWTT MATRIX ATTACHMENT REGION.

45 MNF1ZMPPC1 (-) GTGCCCTT INVOLVED IN LIGHT INDUCTION.

46 MYB1AT (+) WAACCA DEHYDRATION-RESPONSIVE GENE RD22 AND MANY OTHER GENES.

47 MYBCOREATCYC B1 (-) AACGG "MYB CORE" IN THE 18 BP SEQUENCE WHICH IS ABLE TO ACTIVATE REPORTER GENE WITHOUT LEADING TO M-PHASE-SPECIFIC EXPRESSION

48 MYBPLANT (+) MACCWAMC PROMOTERS OF PHENYLPROPANOID BIOSYNTHETIC GENES SUCH AS PAL, CHS, CHI, DFR, CL, BZ1

49 MYBPZM (+) CCWACC CORE OF CONSENSUS MAIZE P (MYB HOMOLOG) BINDING SITE;

50 MYCATERD1 (+) CATGTG MYC RECOGNITION SEQUENCE

51 MYCATRD22 (-) CACATG BINDING SITE FOR MYC (RD22BP1) IN ARABIDOPSIS

52 MYCCONSENSUS AT (+) CANNTG MYC RECOGNITION SITE FOUND IN THE PROMOTERS OF THE DEHYDRATION-RESPONSIVE GENE RD22

53 NAPINMOTIFBN (-) TACACAT SEQUENCE FOUND IN 5' UPSTREAM
REGION (-6, -95, -188) OF NAPIN (2S ALBUMIN) GENE IN BRASSICA NAPUS

54 PALBOXAPC (+) CCGTCC BOX A; CONSENSUS; ONE OF THREE PUTATIVE CIS-ACTING ELEMENTS (BOXES P, A, AND L) OF PHENYLALANINE AMMONIA-LYASE

55 PALBOXLPC (+)YCYYACCWAC C CONFERRED ELICITOR OR LIGHT RESPONSIVENESS

56 POLASIG3 (+) AATAAT "PLANT POLYA SIGNAL"; CONSENSUS SEQUENCE FOR PLANT POLYADENYLATION SIGNAL;

57 POLLEN1LELAT5 2 (+) AGAAA ONE OF TWO CO-DEPENDENT REGULATORY ELEMENTS RESPONSIBLE FOR POLLEN SPECIFIC ACTIVATION OF TOMATO
N8 PRECONSCRHSP7 0A SCGAYNRNNNNN
NNNNNNNNNNHD INVOLVED IN INDUCTION OF HSP70A GENE BY BOTH MGPROTO AND LIGHT 59 PYRIMIDINEBOX OSRAMY1A (-) CCTTTT GIBBERELLIN-RESPONS CIS-ELEMENT OF GARE AND PYRIMIDINE BOX ARE PARTIALLY INVOLVED IN SUGAR REPRESSION

60 QELEMENTZMZM 13 (+) AGGTCA INVOLVED IN EXPRESSION ENHANCING ACTIVITY 61 RAV1AAT (+) CAACA BINDING CONSENSUS SEQUENCE OF ARABIDOPSIS 62 REALPHALGLHC B21 (+) AACCAA REQUIRED FOR PHYTOCHROME REGULATION;

63 RHERPATEXPA7 (+) KCACGW ROOT HAIR-SPECIFIC CIS-ELEMENTS 64 ROOTMOTIFTAPO XI (+) ATATT MOTIF FOUND BOTH IN PROMOTERS OF ROLD;

S RYREPEATBNNA PA (+) CATGCA REQUIRED FOR SEED SPECIFIC EXPRESSION 66 RYREPEATLEGU MINBOX (+) CATGCAY LEGUMIN BOX FOUND IN SEED-STORAGE PROTEIN GENES IN LEGUME SUCH AS SOYBEAN (G.M.);
67 SEBFCONSSTPR10 A YTGTCWC BINDING SITE OF THE POTATO SILENCING ELEMENT BINDING FACTOR

68 SEF4MOTIFGM7S (+) RTTTTTR SEF4 BINDING SITE 69 SITEIIATCYT(+) TGGGCY "SITE II ELEMENT" FOUND IN THE PROMOTER REGIONS OF CYTOCHROME GENE

70 SORLIP1AT (+) GCCAC SEQUENCES OVER-REPRESENTED IN LIGHT-INDUCED PROMOTERS

71 SORLIP2AT (+) GGGCC SEQUENCES OVER-REPRESENTED IN LIGHT-INDUCED PROMOTERS 72 SURECOREATSUL TR11 (+) GAGAC CORE OF SULFUR-RESPONSIVE ELEMENT (SURE) FOUND IN THE PROMOTER 73 SV40COREENHAN (+) GTGGWWHG SV40 CORE ENHANCER
74 T/GBOXATPIN2 (-) AACGTG INVOLVED IN JASMONATE (JA)INDUCTION OF THESE GENES 75 TATABOX5 (+) TTATTT TATA BOX"; TATA BOX FOUND IN THE 5'UPSTREAM REGION OF PEA (PISUM SATIVUM) GLUTAMINE SYNTHETASE GENE; A FUNCTIONAL 76 TBOXATGAPB (-) ACTTTG "TBOX" FOUND IN THE ARABIDOPSIS THALIANA 77 TGACGTVMAMY (-) TGACGT "TGACGT MOTIF" FOUND IN THE VIGNA MUNGO 78 TRANSINITDICOT S (-) AMNAUGGC CONTEXT SEQUENCE OF TRANSLATIONAL INITIATION CODON IN DICOTS
79 TRANSINITMONO COTS (-) RMNAUGGC CONTEXT SEQUENCE OF TRANSLATIONAL INITIATION CODON IN MONOCOTS
80 UPRMOTIFIIAT (+)CNNNNNNNNN NNNCCACG CIS-ACTING ELEMENT IN ARABIDOPSIS GENES CODING FOR SAR1B
81 WBBOXPCWRKY 1 (-) TTTGACY A TRANSCRIPTION FACTOR GENE IN ARABIDOPSIS 82 WBOXATNPR1 (+) TTGAC CLUSTER OF WRKY BINDING SITES ACT AS NEGATIVE REGULATORY ELEMENTS FOR THE INDUCIBLE EXPRESSION OF ATWRKY18 83 WBOXHVISOl (+) TGACT SUSIBA2 BIND TO W-BOX ELEMENT IN BARLEY ISOl (ENCODING ISOAMYLASE1) PROMOTER; 84 WBOXNTCHN48 (+) CTGACY NTWRKYS POSSIBLY INVOLVED IN ELICITOR-RESPSONSIVE TRANSCRIPTION OF DEFENSE GENES IN TOBACCO 85 WBOXNTERF3 (+) GACYINVOLVED IN ACTIVATION OF ERF3 GENE BY WOUNDING 86 WRKY710S(+) TGACA TRANSCRIPTIONAL REPRESSOR OF THE DE GIBBERELLIN SIGNALING PATHWAY

I/We Claim:

1. A promoter having the polynucleotide molecule selected from the group consisting of a.

the polynucleotide having at least about 85% sequence identity with the nucleotide sequence as set forth in SEQ ID NO: 3,

b. the nucleotide sequence as set forth in SEQ ID NO: 3,

c. the nucleotide sequence as set forth in SEQ ID NO: 5,

d. the nucleotide sequence as set forth in SEQ ID NO: 6,

e. the nucleotide sequence as set forth in SEQ ID NO: 7,

f. a fragment of the nucleotide sequence as set forth in SEQ ID NO: 3, wherein the fragment consists of at least 250 contiguous nucleotide of SEQ ID NO: 3, and

g. a variant of the nucleotide sequence as set forth in SEQ ID NO: 3, wherein the promoter is capable of regulating the transcription of the operable linked polynucleotide molecule of interest.

2. The promoter as claimed in claim 1, wherein the promoter is xylem specific.

3. The promoter as claimed in claim 1, wherein the promoter is light and/or wound inducible.

4. A recombinant DNA expression cassette comprising the promoter as claimed in claim 1,

wherein the promoter is operably linked to a polynucleotide molecule of interest.

5. The recombinant DNA expression cassette as claimed in claim 4,

wherein the polynucleotide molecule of interest is selected from a group consisting of a gene of agronomic interest, a marker gene and a reporter gene.

6. The recombinant DNA expression cassette as claimed in claim 4,

wherein the polynucleotide of interest is selected from a group consisting of genes involved in lignin biosynthetic pathway, cellulose biosynthetic pathway, and common transcription factors.

7. A recombinant vector comprising the recombinant DNA expression cassette as claimed in claim 4.

8. A method of directing the expression of a polynucleotide molecule of interest in plant tissue, wherein the said method comprising transforming a plant with the recombinant DNA expression cassette as claimed in claim 4 or the recombinant vector as claimed in claim 7, selecting a transgenic plant cell comprising the polynucleotide molecule of interest and generating from the transgenic plant cell a plant that expresses polynucleotide molecule of interest.

9. The method as claimed in claim 8, wherein the plant tissue is xylem.

10. The method as claimed in claim 8, wherein light or wound induces expression of the polynucleotide molecule of interest in plant.

11. The method as claimed in claim 8, wherein the plant is transformed using Agro bacterium mediated transformation method, particle gun bombardment method, in-plantar transformation method, liposome mediated transformation method, protoplast transformation method, microinjection and microinjection.

12. A transgenic plant, plant cell, tissue, comprising the recombinant DNA expression cassette as claimed in claim 4.

13. The transgenic plant as claimed in claim 12, wherein the transgenic plant exhibits expression of the polynucleotide of interest in xylem tissue.

14. The transgenic plant as claimed in claim 12, wherein light or wound induces expression of the polynucleotide molecule of interest in plant.

15. The transgenic plant as claimed in claim 12, wherein the transgenic plant is monocotyledonous or dicotyledonous plant.

16. The transgenic plant as claimed in claim 12, wherein the transgenic plant is selected from a group consisting of Eucalyptus, poplar, oak, bamboo, tobacco,

tomato, pea, soybean, Brassica, chickpea, pigeon pea, rice, maize, wheat, barley and sorghum, potato, casuarina, Subabul, acacia, Teak apple and woody plant.

17. A transgenic seed or progeny thereof obtained from the transgenic plant as claimed in claim 12, wherein the transgenic seed or the progeny comprises the recombinant DNA expression cassette as claimed in claim 4.