ARABIDOPSIS

FIELD OF INVENTION

[0001] The present disclosure relates to the field of plant biology. In particular, the present disclosure relates to a DNA fragment comprising of a core sequence capable of driving expression of an operably linked transcribeable gene.

BACKGROUND OF THE INVENTION

[0002] Plants are subjected to various biotic and abiotic stresses and have developed vivid mechanisms for their adaptation to these stresses. The growth and development of organisms is a complex network of different gene products which are controlled by upstream regulatory elements of genes or promoters which induce spatial and temporal specificity to the expression pattern of respective genes.

[0003] Transgenic plants with a strong constitutive expression of stress responsive genes often suffer from undesirable phenotypes like growth retardation, severe reduction in seed production (Yamaguchi-Shinozaki K and Shinozaki K, Norvartis Found Symp, 2001, 236: 176-186; Liu Q et al., Plant Cell, 1998, 10: 1391-1406). The need for identification of highly specific nematode-responsive promoters is thus desired (Ross MN et al., Annu Rev Phytopathol, 2009, 47: 201-232). The expression of transgene using inducible promoters or tissue specific promoters to regulate expression of the transgene has demonstrated to alleviate the adverse effects observed, when transgene genes were constitutively expressed (Liu Q et al., Plant Cell, 1998, 10: 1391-1406).

[0004] Promoters are regulatory elements that play a pivotal role in the transcriptional regulation of eukaryotic genes. Both constitutive and regulated promoters are used to direct gene expression in transgenic organisms including plants. Constitutive promoters direct expression in most or all tissues, and are useful when high levels of production of a gene product are desired, lite 35Spromoter from cauliflower mosaic vims (CMV) is frequently used to direct constitutive expression. Regulated promoters, such as tissue-specific and inducible promoters, are used to direct spatially or temporally specific expression or expression in response to environmental factors. The vast range of gene expression observed in cell, tissue or organ-specific manner or in response to developmental or environmental clues points to the complex organization of promoter elements. Therefore, there is emphasis on analysis of promoters for understanding various regulatory elements and for use in precise control of transgene expression.

[0005] Wang et al, (2009, BMC Bioinformatics, 10; 1 (S-29)) discloses comprehensive analysis of bidirectional gene pairs through the whole Arabidopsis tha!iana genome and identified 2471 bidirectional gene pairs. ). Based on in silico studies, it was found that out of total 2471 bi- directional gene pairs identified in the Arabidopsis thaliana genome, 27 divergent gene pairs were regarded as having bidirectional promoters, because their intergenic regions were found to be specifically enriched in regulatory elements The analysis shows that bidirectional genes are often co expressed and tend to be involved in the same biological function.

[0006] Wang et al. (2008, Journal of Plant Biology, 51: 108) describes a bi-directional promoter, cloned from melon . Transient expression in cucumber leaves, stems, and fruits as well as in tobacco leaves and stems showed that the bi-directional promoter drives transcription to a much higher levels in both directions.

[0007] Shin et al. (2003, Plant Cell Physiology, 44: 549) describes a bi-directional promoter, cloned from Capsicum annuum.

[0008] Bi-directional promoters have the potential application for tissue-specific co- expression of gene pairs coding for subunits of antibodies for other proteins, which may be needed in equal quantities, or for engineering synthetic pathways. Use of bidirectional promoters would ensure simultaneous expression of two genes and also halve the number of promoters needed for expressing transgenes.

[0009] Mitra et al. (2009, Planta, 229: 1015) reported bi-directional promoter activity in the intergenic region of chlorophyll binding proteins cabl (Atlg29930) and cab2 (Atlg29920). This 1258 bp intergenic region contains a number of environmental stress responsive and tissue specific c/s-regulatory elements.

[0010] Bondino et al. (2009, BMC Mol Biol 10: 1471 ) showed that the intergenic region between At5g06290 (2-Cys peroxiredoxin B) and At5g06280 (protein of unknown function) was tissue specific and shows stress inducible bi-directional promoter activity.

[0011] In another study, a methyl jasmonate-inducible bi- directional promoter was isolated from poplar (Zheng et al) Although synthetic bi-directional promoters can be generated, natural bi-directional promoters are preferred as they have evolved to be effective under natural physiological conditions

SUMMARY OF THE INVENTION

[0012] In an aspect of present disclosure, there is provided a DNA construct comprising a polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 1 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0013] In an aspect of present disclosure, there is provided a DNA construct comprising a polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 2 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0014] In an aspect of present disclosure, there is provided a DNA vector comprising a DNA construct, wherein said DNA construct comprises polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 1 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0015] In an aspect of present disclosure, there is provided a DNA vector comprising a DNA construct, wherein said DNA construct comprises polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 2 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0016] In an aspect of present disclosure, there is provided a recombinant host cell comprising a DNA construct, wherein said DNA construct comprises DNA construct comprises polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 1 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0017] In an aspect of present disclosure, there is provided a recombinant host cell comprising a DNA construct, wherein said DNA construct comprises DNA construct comprises polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 2 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0018] In an aspect of present disclosure, there is provided a recombinant host cell comprising a DNA vector, wherein said DNA vector comprises DNA construct, wherein said DNA construct comprises polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 2 and said polynucleotide fragment is capable of expressing said gene of interest in a cell. [0019] In an aspect of present disclosure, there is provided a recombinant host cell comprising a DNA vector, wherein said DNA vector comprises DNA construct, wherein said DNA construct comprises polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 1 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0020] In an aspect of present disclosure, there is provided a method of expressingat least one heterologous gene of interest in a plant, wherein said method comprises: (a) obtaining a DNA construct as described herein; (b) obtaining an explant from a plant; (c) transforming said explant with said DNA construct to obtain transformed plant cells; and (d) regenerating said transformed plant cells into a transgenic plant expressing said gene of interest.

[0021] In an aspect of present disclosure, there is provided a method of expressingat least one heterologous gene of interest in a plant, wherein said method comprises: (a) obtaining a DNA vector as described herein; (b) obtaining an explant from a plant; (c) transforming said explant with said DNA construct to obtain transformed plant cells; and (d) regenerating said transformed plant cells into a transgenic plant expressing said gene of interest.

[0022] In an aspect of present disclosure, there is provided a method of expressingat least one heterologous gene of interest in a plant, wherein said method comprises: (a) obtaining a recombinant host cell as described herein; (b) obtaining an explant from a plant; (c) transforming said explant with said DNA construct to obtain transformed plant cells; and (d) regenerating said transformed plant cells into a transgenic plant expressing said gene of interest.

[0023] In an aspect of present disclosure, there is provided a DNA fragment capable of driving expression of an operably linked heterologous gene of interest, wherein said DNA fragment comprises a contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 1. [0024] In an aspect of present disclosure, there is provided a DNA fragment capable of driving expression of an operably linked heterologous gene of interest, wherein said DNA fragment comprises a contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 2.

[0025] In an aspect of present disclosure, there is provided a transgenic plant or parts thereof, including seeds, comprising a polynucleotide fragment linked to a gene of interest, wherein said polynucleotide fragment is capable of driving the expression of said gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises a contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 1.

[0026] In an aspect of present disclosure, there is provided a transgenic plant or parts thereof, including seeds, comprising a polynucleotide fragment linked to a gene of interest, wherein said polynucleotide fragment is capable of driving the expression of said gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises a contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 2.

[0027] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0028] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein. [0029] Figure 1 (a-c) depicts the expression of GUS in different localization in the GUS-650 promoter trap line, in accordance with an embodiment of present disclosure.

[0030] Figure 2 depicts the expression of GUS in different development stages of the ovule in GUS-650 line, in accordance with an embodiment of the present disclosure.

[0031] Figure 3 (a-c) depicts the characterization of the T-DNA insertion site in the GUS-650 line, in accordance with an embodiment of the present disclosure.

[0032] Figure 4 depicts the diagrammatic representation of the organization At3gl7140 and AT3gl7150 genes in the genome of A. thaliana, along with the insertion site of the T-DNA in the GUS-650 line, in accordance with an embodiment of the present disclosure.

[0033] Figure 5 depicts the nucleotide sequence of the 672 bp promoter fragment having SEQ ID NO: l, wherein each of the cis regulatory element is marked in different colors, in accordance with an embodiment of the present disclosure.

[0034] Figure 6 depicts the nucleotide sequence of the 672 bp promoter fragment having SEQ ID NO:2, wherein each of the cis regulatory element is marked in different colors, in accordance with an embodiment of the present disclosure.

[0035] Figure 7 (a) depicts the RT-PCT results to determine the transcripts of genes AT3gl7150 and AT3gl7140 in flowers of GUS-650 homozygous line and wild type control, in accordance with an embodiment of the present disclosure.

[0036] Figure 7b depicts the expression of AT3gl7150 gene in different plant parts, in accordance with an embodiment of the present disclosure.

[0037] Figure 8 (a-d) depicts the results of the histochemical assay of the transgenic Arabiodopsis plants showing bi-directional nature of the intergenic region, in accordance with an embodiment of the present disclosure.

[0038] Figure 9 (a-b) depicts the graphical representation of the results to measure the strength of promoter AT3gl7140 and AT3gl7150 in plant cell, in accordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION [0039] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure 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 or more of such steps or features.

Definitions:

[0040] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0041] The articles "a", "an" and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0042] The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as "consists of only".

[0043] The terms "induce" and "activate" are used in the context of promoter activity, and may be used interchangeably throughout the specification.

[0044] DNA (T-DNA) promoter trap technique is a powerful tool to functional and cryptic promoters. In this approach, transgenics are generated using a T-DNA vector having a selectable marker gene under the control of a constitutive promoter, and a promoter-less reporter gene placed at the right border for visualizing the trapped promoter (Pratibha et al., Gene, 2013, 524:22-27). Transgenics are first selected on the basis of trait conferred by the selectable marker gene and then screened for the reporter gene expression. Expression of the reporter gene in the transforrnant indicates that the T-DNA has trapped a promoter. Apart from tissue- specific, developmental stage-specific and stress-induced promoters, this approach has also led to the identification and isolation of several cryptic promoter elements.

[0045] The intergenic region between transcription start sites (TSS) of two genes that are arranged in head-to-head configuration and transcribed in opposite directions is termed as the bidirectional promoter. Bioinformatics studies have indicated genome wide occurrence of bi-directional promoters in invertebrates, vertebrates and several eukaryotes (Human, plants, yeast). Usually TSS of such genes lies within 1000 base pairs in the case of human genome (Trinklein et al., Genome Res, 2004, 14:62-66) but may exceed 1 kb in plants (Mitra et al., Planta, 2009, 229: 1015-1022). Bi-directional promoters have also been reported from melon (Wang et al. J Plant Biol, 51: 108- 1152008) and Capsicum annuum (Shin et al. Plant cell Physiol, 2003, 44:549-554). Although synthetic bi-directional promoters can be generated, natural bi-directional promoters are preferred as they have evolved to be effective under natural physiological conditions.

[0046] Among the many bi-directional promoters characterized so far, to the best of our knowledge, none shows specific activity in the embryo sac. The disclosure reports the identification and characterization of a 672 bp A. thaliana fragment which shows bi-directional promoter activity. This bi-directional promoter drives gene expression specifically in the female gametophyte and is the promoter identified to show expression in embryo sac.

[0047] In an objective of present disclosure, there is provided a embryo sac specific promoter. This promoter will help to manipulate reproductive pathways in plants, which will help in seed development pathways like apomixes etc.

[0048] Bi-directional promoters have the potential application for tissue specific co- expression of gene pairs coding for subunits of antibodies for other proteins, which may be needed in equal quantities, or for engineering synthetic pathways. The use of bi-directional promoter would ensure simultaneous expression of two genes and also halve the number of promoters needed for expressing transgenes and thus minimize the amount of foreign DNA in transgenic plants. The bi-directional promoters are capable to drive expression of genes in both sense and anti sense orientation. Further, homology-dependent gene silencing due to repeated use of a promoter can be avoided through the use of bi-directional promoter. The promoter identified the present disclosure is bidirectional, specific for expression in ovules and developing seeds therefore it finds application in improving nutritional quality of seeds through engineering of metabolic pathways. Further the ability of the promoter to drive gene expression in the embryo sac during early stages of differentiation could be deployed to manipulate reproductive pathways in plants.

[0049] A person skilled in the art can use the promoter disclosed in the instant application for raising transgenic plants expressing the desired gene of interest under the promoter disclosed here. The transgenic plants can be such as of rice, wheat, sorgrum, maize, mustard, cotton, tobacco.

[0050] Throughout this specification, unless the context requires otherwise the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0051] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0052] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.

Sequences:

[0053] SEQ ID NO: 1 depicts full length sequence of promoter driving the expression of AT3gl7150 gene in A. thaliana [0054] SEQ ID NO : 2 depicts full length sequence of promoter driving the expression of AT3gl7140 gene in A. thaliana .

[0055] SEQ ID NO: 2 is complimentary to SEQ ID NO: 1

[0056] In an embodiment of present disclosure, there is provided a DNA construct comprising a polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 1 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0057] In an embodiment of present disclosure, there is provided a DNA construct comprising a polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 90% identity to sequence as set forth in SEQ ID NO: 1 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0058] In an embodiment of present disclosure, there is provided a DNA construct comprising a polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 95% identity to sequence as set forth in SEQ ID NO: 1 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0059] In an embodiment of present disclosure, there is provided a DNA construct comprising a polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 99% identity to sequence as set forth in SEQ ID NO: 1 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0060] In an embodiment of present disclosure, there is provided a DNA construct comprising a polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having sequence as set forth in SEQ ID NO: 1 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0061] In an embodiment of present disclosure, there is provided a DNA construct comprising a polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 2 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0062] In an embodiment of present disclosure, there is provided a DNA construct comprising a polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 90% identity to sequence as set forth in SEQ ID NO: 2 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0063] In an embodiment of present disclosure, there is provided a DNA construct comprising a polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 95% identity to sequence as set forth in SEQ ID NO: 2 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0064] In an embodiment of present disclosure, there is provided a DNA construct comprising a polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 99% identity to sequence as set forth in SEQ ID NO: 2 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0065] In an embodiment of present disclosure, there is provided a DNA construct comprising a polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having sequence as set forth in SEQ ID NO: 2 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0066] In an embodiment of present disclosure, there is provided a DNA construct as described herein, wherein said polynucleotide fragment expresses the said gene of interest in plant female reproductive parts.

[0067] In an embodiment of present disclosure, there is provided a DNA construct as described herein, wherein said polynucleotide fragment expresses the said gene of interest in embryo sac

[0068] In an embodiment of present disclosure, there is provided a DNA construct as described herein, wherein said polynucleotide fragment is capable to drive the expression of two genes linked to the sense and antisense orientation of the said polynucleotide fragment.

[0069] In an embodiment of present disclosure, there is provided a DNA vector comprising a DNA construct, wherein said DNA construct comprises polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 1 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0070] In an embodiment of present disclosure, there is provided a DNA vector comprising a DNA construct, wherein said DNA construct comprises polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 90% identity to sequence as set forth in SEQ ID NO: 1 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0071] In an embodiment of present disclosure, there is provided a DNA vector comprising a DNA construct, wherein said DNA construct comprises polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 95% identity to sequence as set forth in SEQ ID NO: 1 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0072] In an embodiment of present disclosure, there is provided a DNA vector comprising a DNA construct, wherein said DNA construct comprises polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having sequence as set forth in SEQ ID NO: 1 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0073] In an embodiment of present disclosure, there is provided a DNA vector comprising a DNA construct, wherein said DNA construct comprises polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 2 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0074] In an embodiment of present disclosure, there is provided a DNA vector comprising a DNA construct, wherein said DNA construct comprises polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 90% identity to sequence as set forth in SEQ ID NO: 2 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0075] In an embodiment of present disclosure, there is provided a DNA vector comprising a DNA construct, wherein said DNA construct comprises polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having at least 95% identity to sequence as set forth in SEQ ID NO: 2 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0076] In an embodiment of present disclosure, there is provided a DNA vector comprising a DNA construct, wherein said DNA construct comprises polynucleotide fragment linked to at least one heterologous gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises contiguous stretch of nucleotides having sequence as set forth in SEQ ID NO: 2 and said polynucleotide fragment is capable of expressing said gene of interest in a cell.

[0077] In an embodiment of present disclosure, there is provided a recombinant host cell comprising a DNA construct as described herein.

[0078] In an embodiment of present disclosure, there is provided a recombinant host cell comprising a DNA vector as described herein.

[0079] In an embodiment of present disclosure, there is provided a recombinant host cell as described herein, wherein said host cell is selected from the group consisting of bacterial, fungal, and plant.

[0080] In an embodiment of present disclosure, there is provided a recombinant host cell is E.coli.

[0081] In an embodiment of present disclosure, there is provided a recombinant host cell is Agrobacterium tumefaciens.

[0082] In an embodiment of present disclosure, there is provided a method of expressingat least one heterologous gene of interest in a plant, wherein said method comprises: (a) obtaining a DNA construct as described herein; (b) obtaining an explant from a plant; (c) transforming said explant with said DNA construct to obtain transformed plant cells; and (d) regenerating said transformed plant cells into a transgenic plant expressing said gene of interest.

[0083] In an embodiment of present disclosure, there is provided a method of expressingat least one heterologous gene of interest in a plant, wherein said method comprises: (a) obtaining a DNA construct as described herein; (b) obtaining an explant from a Arabiodopsis thaliana plant; (c) transforming said explant with said DNA construct to obtain transformed plant cells; and (d) regenerating said transformed plant cells into a transgenic plant expressing said gene of interest.

[0084] In an embodiment of present disclosure, there is provided a method of expressingat least one heterologous gene of interest in a plant, wherein said method comprises: (a) obtaining a DNA vector as described herein; (b) obtaining an explant from a plant; (c) transforming said explant with said DNA vector to obtain transformed plant cells; and (d) regenerating said transformed plant cells into a transgenic plant expressing said gene of interest.

[0085] In an embodiment of present disclosure, there is provided a method of expressingat least one heterologous gene of interest in a plant, wherein said method comprises: (a) obtaining a DNA vector as described herein; (b) obtaining an explant from A. thaliana plant; (c) transforming said explant with said DNA vector to obtain transformed plant cells; and (d) regenerating said transformed plant cells into a transgenic plant expressing said gene of interest.

[0086] In an embodiment of present disclosure, there is provided method of expressingat least one heterologous gene of interest in a plant, wherein said plant cell is a transformed by a method selected from the group consisting of particle gun bombardment method, in-planta transformation method, liposome mediated transformation method, protoplast transformation method, microinjection, and macroinjection.

[0087] In an embodiment of present disclosure, there is provided a method of expressingat least one heterologous gene of interest in a plant, wherein said method comprises: (a) obtaining a recombinant host cell as described herein; (b) obtaining an explant from a plant; (c) transforming said explant with said recombinant host cell to obtain transformed plant cells; and (d) regenerating said transformed plant cells into a transgenic plant expressing said gene of interest.

[0088] In an embodiment of present disclosure, there is provided a method of expressingat least one heterologous gene of interest in a plant, wherein said method comprises: (a) obtaining a recombinant host cell as described herein; (b) obtaining an explant from a plant; (c) transforming said explant with the said recombinant host cell to obtain transformed plant cells; and (d) regenerating said transformed plant cells into a transgenic plant expressing said gene of interest. [0089] In an embodiment of present disclosure, there is provided a method of expressingat least one heterologous gene of interest in a plant, wherein said recombinant host cell is Agrobacterium tumefaciens.

[0090] In an embodiment of present disclosure, there is provided a method of expressingat least one heterologous gene of interest in a plant, wherein said method is floral dip method.

[0091] In an embodiment of present disclosure, there is provided a method of expressingat least one heterologous gene of interest in a plant, wherein the said heterologous gene of interest is a nutrition modifying gene.

[0092] In an embodiment of present disclosure, there is provided a method of expressingat least one heterologous gene of interest in a plant, wherein the said heterologous gene of interest is a pathogen resistance gene.

[0093] In an embodiment of present disclosure, there is provided a method of expressingat least one heterologous gene of interest in a plant, wherein the said heterologous gene of interest is a reproductive pathway modifying gene.

[0094] In an embodiment of present disclosure, there is provided a method of expressingat least one heterologous gene of interest in a plant, wherein the said heterologous gene of interest is a abiotic stress tolerance gene.

[0095] In an embodiment of present disclosure, there is provided a DNA fragment capable of driving expression of an operably linked heterologous gene of interest, wherein said DNA fragment comprises a contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 1.

[0096] In an embodiment of present disclosure, there is provided a DNA fragment capable of driving expression of an operably linked heterologous gene of interest, wherein said DNA fragment comprises a contiguous stretch of nucleotides having at least 90% identity to sequence as set forth in SEQ ID NO: 1.

[0097] In an embodiment of present disclosure, there is provided a DNA fragment capable of driving expression of an operably linked heterologous gene of interest, wherein said DNA fragment comprises a contiguous stretch of nucleotides having at least 95% identity to sequence as set forth in SEQ ID NO: 1.

[0098] In an embodiment of present disclosure, there is provided a DNA fragment capable of driving expression of an operably linked heterologous gene of interest, wherein said DNA fragment comprises a contiguous stretch of nucleotides having sequence as set forth in SEQ ID NO: 1.

[0099] In an embodiment of present disclosure, there is provided a DNA fragment capable of driving expression of an operably linked heterologous gene of interest, wherein said DNA fragment comprises a contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 2.

[00100] In an embodiment of present disclosure, there is provided a DNA fragment capable of driving expression of an operably linked heterologous gene of interest, wherein said DNA fragment comprises a contiguous stretch of nucleotides having at least 90% identity to sequence as set forth in SEQ ID NO: 2.

[00101] In an embodiment of present disclosure, there is provided a DNA fragment capable of driving expression of an operably linked heterologous gene of interest, wherein said DNA fragment comprises a contiguous stretch of nucleotides having at least 95% identity to sequence as set forth in SEQ ID NO: 2.

[00102] In an embodiment of present disclosure, there is provided a DNA fragment capable of driving expression of an operably linked heterologous gene of interest, wherein said DNA fragment comprises a contiguous stretch of nucleotides having sequence as set forth in SEQ ID NO: 2.

[00103] In an embodiment of present disclosure, there is provided a DNA fragment as described herein, wherein said DNA fragment drives similar type of gene expression in both sense and reverse orientations.

[00104] In an embodiment of present disclosure, there is provided a DNA fragment as described herein, wherein said DNA fragment drives the cloned gene expression stably in the embryo sac of Arabidopsis and in other plants also in sense and reverse orientations. [00105] In an embodiment of present disclosure, there is provided a DNA fragment as described herein, wherein said DNA fragment drives gene expression of the gene of interest in from FG1 stage of the female gametophyte development.

[00106] In an embodiment of present disclosure, there is provided a DNA fragment as described herein, wherein said DNA fragment drives the expression of gene of the interest in the embryo sac.

[00107] In an embodiment of present disclosure, there is provided a DNA fragment as described herein, wherein said DNA fragment is functional of driving the expression of gene of interest in sense and anti sense orientation.

[00108] In an embodiment of present disclosure, there is provided a DNA fragment as described herein, wherein said DNA fragment is capable to express two genes at a time in the embryo sac.

[00109] In an embodiment of present disclosure, there is provided a DNA fragment as described herein, wherein said DNA fragment does not drive the expression of the gene of interest in the developed seed.

[00110] In an embodiment of present disclosure, there is provided a transgenic plant or parts thereof, including seeds, comprising a polynucleotide fragment linked to a gene of interest, wherein said polynucleotide fragment is capable of driving the expression of said gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises a contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 1.

[00111] In an embodiment of present disclosure, there is provided a transgenic plant or parts thereof, including seeds, comprising a polynucleotide fragment linked to a gene of interest, wherein said polynucleotide fragment is capable of driving the expression of said gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises a contiguous stretch of nucleotides having at least 90% identity to sequence as set forth in SEQ ID NO: 1.

[00112] In an embodiment of present disclosure, there is provided a transgenic plant or parts thereof, including seeds, comprising a polynucleotide fragment linked to a gene of interest, wherein said polynucleotide fragment is capable of driving the expression of said gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises a contiguous stretch of nucleotides having at least 95% identity to sequence as set forth in SEQ ID NO: 1.

[00113] In an embodiment of present disclosure, there is provided a transgenic plant or parts thereof, including seeds, comprising a polynucleotide fragment linked to a gene of interest, wherein said polynucleotide fragment is capable of driving the expression of said gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises a contiguous stretch of nucleotides having sequence as set forth in SEQ ID NO: 1.

[00114] In an embodiment of present disclosure, there is provided a transgenic plant or parts thereof, including seeds, comprising a polynucleotide fragment linked to a gene of interest, wherein said polynucleotide fragment is capable of driving the expression of said gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises a contiguous stretch of nucleotides having at least 85% identity to sequence as set forth in SEQ ID NO: 2.

[00115] In an embodiment of present disclosure, there is provided a transgenic plant or parts thereof, including seeds, comprising a polynucleotide fragment linked to a gene of interest, wherein said polynucleotide fragment is capable of driving the expression of said gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises a contiguous stretch of nucleotides having at least 90% identity to sequence as set forth in SEQ ID NO: 2.

[00116] In an embodiment of present disclosure, there is provided a transgenic plant or parts thereof, including seeds, comprising a polynucleotide fragment linked to a gene of interest, wherein said polynucleotide fragment is capable of driving the expression of said gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises a contiguous stretch of nucleotides having at least 95% identity to sequence as set forth in SEQ ID NO: 2. [00117] In an embodiment of present disclosure, there is provided a transgenic plant or parts thereof, including seeds, comprising a polynucleotide fragment linked to a gene of interest, wherein said polynucleotide fragment is capable of driving the expression of said gene of interest, wherein nucleotide sequence of said polynucleotide fragment comprises a contiguous stretch of nucleotides having sequence as set forth in SEQ ID NO:2.

[00118] In an embodiment of present disclosure, there is provided a transgenic plant as described herein, said transgenic plant or parts thereof, including seeds is a monocot or a dicot.

[00119] Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible.

EXAMPLES

[00120] 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. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

Example 1

Materials and methods

[00121] Plant material: Development and screening of transferred DNA (T-DNA) promoter trap population of Arahiodopsis. thaliana (ecotype Columbia) was performed as described by Pratibha et al (Pratibha et al. Gene , 2013, 524:22-27). The line showing β-glucuronidase (GUS) expression in ovules (designated as GUS-650) was identified and used in this study for detailed analysis.

[00122] Reverse transcription and PCR: Total RNA was isolated from plants using total RNA extraction kit (Real Genomics, Taiwan), treated with DNase (Invitrogen, USA) and quantified by Nanodrop spectrophotometer, ND-1000 (Thermo Scientific, USA). First strand cDNA synthesis was done using Superscript III reverse transcriptase (Invitrogen, USA) with oligo dT primer. Double stranded cDNA was amplified with gene specific forward and reverse primers. Finally, uidA transcripts were amplified using GUSF and GUSR primers. The housekeeping GAPC F and GAPC(R) gene was used as the internal reference control. Table 1 lists the primers used.

[00123] Histochemical GUS assay: The assay was performed as described by Jefferson et al. ( Jefferson et al., EMBO J, 1987, 13:3901-3907) using chromogenic substrate X-gluc (5-bromo-4-chloro-3indolylglucuronide) (Biosynth). Tissue was incubated in GUS staining solution, vacuum infiltrated for 10 min and left in staining solution overnight at 37°C. GUS stained tissues were then washed with 70% ethanol twice to remove chlorophyll and cleared in chloral hydrate solution (chloral hydrate: water: glycerol, 8:2: 1) for at least 5 h. Cleared tissues were observed directly under microscope (AXIO imager. Ml, Carl- Zeiss, GmbH, Germany).

[00124] Identification of T-DNA flanking sequences: Genome walking approach was employed for the isolation and identification of T-DNA flanking sequences. Genome walker libraries were constructed by digesting DNA with Dral, EcoRV, Pvull or Stul restriction enzyme followed by adapter ligation. PCR amplification was carried out according to the manufacturer's instructions (Clontech, USA). Primary PCR product was diluted (1:250) and used as template for nested PCR amplification using the nested adaptor primer (AP2) and a nested gene-specific primer. Resolved nested PCR product was eluted using GFX PCR DNA gel band purification kit (GE HealthCare, UK), cloned in pGEM-T Easy vector (Promega, USA) and sequenced. The sequence retrieved after removal of the vector backbone was BLAST searched against the Arabidopsis genome database (TAIR, www.arabidopsis.org) to localize the T-DNA insertion site. Table 1 lists the various primers used in the study.

[00125] Table 1

[00126] Construction of promoter-M/ A fusion and plant transformation: The 668 bp intergenic region between At3gl7140 and At3gl7150 genes along with the 4 bp 5' UTR region of At3gl7150 was amplified with the Phusion high fidelity DNA polymerase (Finnzymes, Finland). The forward and reverse primers were designed for cloning in sense and antisense orientation to drive the uidA gene. Sacl and S cII restriction sites were included at the 5' end of the forward and the reverse primers, respectively. The amplified fragments were cloned into pGEM-T Easy vector and sub- cloned into the binary vector pORE-R2 (Coutu et al. Transgenic Res, 2007, 16:771- 781) between Sacl and S cII sites upstream to the uidA reporter gene. The recombinant binary vector was then mobilized into Agrobacterium tumefaciens strain GV 3101 and used for floral dip transformation of Arabidopsis plants (Clough and Bent, Plant J, 1998, 16:735-743). Ti seedlings were selected on kanamycin (50 mg/1) supplemented Murashige and Skoog (1962) medium and transgenic plants were raised in controlled growth chambers at 20-22 °C under 16 h photoperiod. Example 2

Results

[00127] Identification of trapped promoter and T-DNA localization: Screening of promoter trap population generated by using T-DNA vector carrying a promoter-less uidA reporter gene identified a line (designated as GUS-650) showing GUS expression in the ovules. GUS expression was observed in ovules from immature stage (Fig la and lb) to mature seed stage i.e. the abortive ovules in the mature siliques. (Fig. lc). GUS expression was first observed in the cells near the micropylar end after completion of meiosis (FG2 stage of flower development) (Fig. 2a). As the female gametophyte progressed towards gametogenesis, GUS expression extended towards the chalazal end (Fig. 2b and 2c). At maturity (FG8 stage of flower development), GUS staining was found throughout the embryo sac (Fig. 2d). GUS expression was observed in 50% of ovules in plants hemizygous for the T-DNA mutation while almost all the ovules of homozygous mutant plants showed GUS staining. This suggested that the uidA reporter gene in the T-DNA has trapped a promoter region of a gene involved in either ovule or seed development.

[00128] Genome walking approach was used to clone the flanking plant sequences and to identify the T-DNA insertion site (Fig. 3). Primary PCR followed by nested PCR using Dral restriction enzyme based library yielded 500 bp and 1200 bp fragments with primer NRBGUS, and AP2 primer. These fragments were cloned in pGEM-T vector and sequenced. Homology search of the obtained sequence against the Arabidopsis reference genome (TAIR database) revealed alignment of both 500 and 1200 bp fragments with the sequence of chromosome 3, between coordinates of nucleotides 5847001 and 5847261. This indicates that the T-DNA right border is located 8 bp upstream to the start codon of the At3gl7150 gene.

[00129] At3gl7150 is annotated as a gene capable of coding for Pectin Methyl Esterase Inhibitor (PMEI). According to GENEVESTIGATOR analysis, this gene is expressed only in the inflorescence. The predicted 5' UTR of this gene comprises 12 nucleotides. T-DNA insertion placed the uidA gene in sense orientation, 8 nucleotides upstream of the start codon of the At3gl7150 gene. Thus the uidA gene appears to have acquired the promoter of At3gl7150 gene. At3gl7140 and At3gl7150 are a pair of genes arranged in head-to-head orientation on chromosome 3 and are separated by 668 bp intergenic region. Both the genes are predicted to code for PMEI protein. Diagrammatic representation of T-DNA insertion in the GUS-650 line in the 3 chromosome region is depicted in Fig. 4. This arrangement suggests that the 668 bp sequence serves as promoter for both these genes and their expression may be co- regulated.

[00130] In silico analysis of the identified region: The 672 bp sequence (668 bp intergenic + 4 bp 5' UTR) was analyzed in both orientations for the presence of known c/s-regulatory elements (CREs) using Plant CARE (Lescot et al. Nucleic Acid Research, 2002, 30:325-327) and PLACE (Higo et al. Nucleic acid Research, 1999, 27: 297-300) softwares. Figure 5 and 6 depict the different cis regulatory elements identified in the bi-directional promoter by in-silico analysis. In- silico analysis showed presence of 21 TATA boxes and seven CAAT boxes (Figure 5). A number of elements responsible for tissue specific expression pattern such as endosperm specific Skn-1 motif (GTCAT) (at -204), four endosperm specific motifs (AACA) (at -68, - 179, -248 and -255) , and five GATA boxes (at -46, -212, -232, -291 and -488) were identified in the sequence (Figure 6). These elements might contribute to ovule specific expression observed in the GUS-650 line. In A. thaliana, AACA is a positive regulatory element in the endosperm and functions as a negative regulatory element in other tissues (Bhalothia et al. Am J Plant Sci, 2013, 4:549-554). The CCAAT box elements interact with transcription factor NF-Y for efficient transcription (Zanotto et al. 2007). Another conserved element CAAACAC (at -181) (Figure 5), often found in seed storage protein gene promoters, was also found in this intergenic sequence. Besides, a W-box element (TTGACC) at -76, a T-box element (AACGTT element) at -515 and six YACT elements (at -27, -198, -371, -573, -591 and -613) were also found (Figure 5). CArG motifs (CTATTTAATG) involved in binding with embryo specific transcriptional regulator AGL15 were found at -353 and -476 and AAAG motifs (at -148, -158, -302 and -381) involved in embryo specific DOF protein binding were found in the 5'-3' orientation (Figure 5). Additionally, four ACGT elements which are known binding sites for bZIP family transcription factor were also found at positions - 107 , -455 , -514 and -543.

[00131] Figure 6 depicts the cis regulatory elements identified in the sequence complementary to SEQ ID NO: l. 31 TATA boxes and 6 CAAT boxes were found. Elements like TTGACC (at -622), GATA (at -247,-442, -587, -646 and -653), AACA (at -134, -187, - 310, -339 and -557), CAACAC (at -136), AACGTT (at -171), YACT (at -69, -111, - 456, - 542, -545, -555 and -651), CArG motif ( at -214 and -337), AAAG (at -19, -177, -224, -408, - 488 and -606) and ACGT (at -141, -170, -229 and -577) were also found. Thus, the 668 bp intergenic region contains a number of motifs, enhancer-like elements and tissue specific elements in both orientations in addition to the necessary basal elements which are required for transcription of a gene. Based on the above analysis, the intergenic region between At3gl 7140 and At3gl 7150 genes was predicted to function as promoter in both orientations.

[00132] Table 2 lists the various c/s-acting elements analysis of 5'-upstream regulatory elements and their positions in bi-directional promoter (672 bp) identified by PLACE and PLANTCARE.

[00133] Table 2

responsiveness.

Box-1 TTTCAAA -345 Light responsive Pisum

element. sativum

Box-Ill CATTTACACT -552 Protein binding Pisum

site. sativum

Box-Wl TTGACC +605, -617 Fungal elicitor Petroselinum responsive crispum element.

CATT motif GCATTC +216 Part of a light Zea mays responsive

element.

CGTCA-motif CGTCA +569, -224 C¾-acting Hordeum regulatory vulgare element involved

in the MeJA- responsiveness.

ERE ATTTCAAA -345 Ethylene- Dianthus responsive caryophyllus element.

G-Box CACGTA -137 C¾-acting Antirrhinum regulatory majus element involved

in light

responsiveness.

G-Box GTACGTG +136, +137, C¾-acting Oryza sativa,

TACGTG -225 regulatory Daucus

CACGTC element involved carota, in light Zea mays responsiveness.

GA-motif AAAGATGA -12 Part of a light Helianthus responsive annuus element.

Skn-1 -motif GTCAT +477 C¾-acting Oryza sativa regulatory

element required

for endosperm

expression.

Spl CC(G/A)CCC -357 Light responsive Zea mays element.

TC-rich repeats GTTTTCTTAC +661 C¾-acting Nicotiana element involved tabacum in defense and

stress

responsiveness.

TGACG-motif TGACG +224, -569 C¾-acting Hordeum regulatory vulgare element involved

in the MeJA- responsiveness.

W box TTGACC +605, -617 - Arabidopsis thaliana

WUN-motif TCATTACGAA -627 Wound- Brassica responsive oleracea element.

as-2-box GATAatGATG -1, -7, -4 Involved in shoot- Nicotiana specific tabacum expression and

light

responsiveness.

2SSEEDPROTBAN CAAACAC +500, -130 Conserved in Brassica APA many storage- napus

protein gene

promoters; May

be important for

high activity of

the napA

promoter.

ABRELATERD 1 ACGTG +138 ABRE-like Arabidopsis sequence (from - thaliana 199 to -195)

required for

etiolation-induced

expression of erdl

(early responsive

to dehydration)

in Arabidopsis

ACGTABREMOTI ACGTGKC +226 Experimentally Oryza sativa, FA20SEM determined Arabidopsis sequence thaliana requirement of

ACGT-core of

motif A in ABRE

of the rice gene,

DRE and ABRE

are

interdependent in

the ABA- responsive

expression of the

rd29A. ACGTATERD1 ACGT +138, +167, ACGT sequence Arabidopsis

+226 (from -155 to - thaliana

+574, -138, - 152) required for

167, -226, - etiolation-induced

574 expression of erdl

(early responsive

to dehydration) in

Arabidopsis

ACGTOSGLUB 1 GTACGTG +136 ACGT motif" Oryza sativa found in GluB-1

gene in rice,

Required for

endosperm- specific

expression;

Conserved in the

5'-flanking region

of glutelin genes.

ACGTTBOX AACGTT + 166, -166 Plant bZIP Plant

Proteins gather at

ACGT elements.

AMYBOX1 TAACARA -305 Conserved Hordeum sequence found in vulgare, 5'-upstream Oryza sativa, region of alpha- Triticum amylase gene. aestivum

ARR1AT NGATT +315, -25, - "ARR1 -binding Arabidopsis

275 element" found in thaliana

Arabidopsis;

ARR1 is a

response

regulator;

N=G/A/C/T,

AGATT is found

in the promoter of

rice non- symbiotic

haemoglobin-2

gene.

ASFIMOTIFCAM TGACG +224, -569 TGACG motifs Cauliflower V are found in many mosaic virus, promoters and are Nicotiana involved in Tabacum, transcriptional Arabidopsis activation of thaliana several genes by auxin and/or

salicylic acid.

CACTFTPPCA1 YACT +68, 66, (CACT) is a key Flaveria

+90, -453, component of trinervia +108, -539, Meml (mesophyll

+310, -542, expression

+483, -552, module 1) found

+654 -648 in the Cis- regulatory

element in the

distal region of

the

phosphoenolpyru

vate carboxylase

(ppcAl) of the

C4 dicot F.

trinervia.

CANBNNAPA CNAACAC +500, +130 Core of "(CA)n Brassica element" in napus storage protein

genes in Brassica

napus,

Embryo and

endosperm- specific

transcription of

napA gene, seed

specificity;

activator and

repressor.

CARGCW8GAT CWWWWWWW + 205, + A variant of Arabidopsis

WG 328, -205, - CArG motif, with thaliana

328 a longer A/T-rich

core; Binding site

for AGL15.

CCAATBOX1 CCAAT + 341, -73 Common Eukaryotes,, sequence found in Glycine max the 5'-non-coding

regions of

eukaryotic genes.

CPBCSPOR TATTAG +62 The sequence Cucumis critical for sativus cytokinin- enhanced Protein

Binding in vitro,

found in -490 to - 340 of the promoter of the

cucumber POR

(NADPH- protochlorophylli

de reductase)

gene.

CURECORECR GTAC +67, +89, GTAC is the core Chlamydomo

+136, -67, of a CuRE nas -89, -136 (copper -response reinhardtii element) found in

Cyc6 and Cpxl

genes in

Chlamydomonas,

also involved in

oxygen-response

of these genes.

DOFCOREZM AAAG +300, -16, Core site required Zea mays

+379, -174, for binding of Dof

+523, -221, proteins in maize.

+533, -405,

-485, -603

DPBFCOREDCDC ACACNNG -550 A novel class of Daucus

3 bZIP transcription carota, factors, DPBF-1 Arabidopsis and 2 (Dc3 thaliana promoter-binding

factor- 1 and 2)

binding core

sequence.

EBOXBNNAPA CANNTG +550, -550 E-box of napA Brassica storage-protein napus gene of Brassica

napus, (also

known as R- response

element).

EECCRCAH1 GANTTNC +104 "EEC"; Chlamydomo

Consensus motif nas of the two reinhardtii enhancer

elements, EE-1

and EE-2, both

found in the

promoter region

of the Chlamydomonas

Cahl (encoding a

periplasmic

carbonic

anhydrase);

Binding site of

Myb transcription

factor LCR1.

ELRECOREPCRPl TTGACC +605, -617 EIRE (Elicitor Petroselinum

Responsive crispum); Element) core of Nicotiana parsley PR1 tabacum genes; consensus

sequence of

elements Wl and

W2 of parsley

PRl-1 and PR1-2

promoters; Box

Wl and W2 are

the binding site of

WRKY1 and

WRKY2,

respectively.

ERELEE4 AWTTCAAA -345 "ERE (ethylene Lycopersicon responsive esculentum, element)" of Dianthus tomato E4 and caryophyllus, carnation GST1 Lycopersicon genes. ERE chilense motifs mediate

ethylene -induced

activation of the

U3 promoter

region.

GADOWNAT ACGTGTC +226 Sequence present Arabidopsis in 24 genes in the thaliana G A -down

regulated dl

cluster (106

genes) found in

Arabidopsis seed

germination; This

motif is similar to

ABRE.

GARE10SREP1 TAACAGA -305 "Gibberellin- Oryza sativa responsive

element (GARE)" found in the

promoter region

of a cystein

proteinase (REP-

1) gene in rice.

GTl CONSENSUS GRWAAW +76, +390, Consensus GT-1 Pisum

+469, -17, - binding site in sativum,

242, -264 many light- Avena sativa, regulated genes, Oryza sativa, e.g., RBCS from Nicotiana many species, tabacum,

PHYA from oat Arabidopsis and rice, spinach thaliana,

RCA and PETA, Spinacia and bean CHS 15; oleracea,

R=A/G; W=A/T bean

.GT-1 can

stabilize the

TFIIA-TBP-DNA

(TATA box)

complex; Binding

of GT-1 -like

factors to the PR- la promoter

influences the

level of SA- inducible gene

expression.

GTGANTG10 GTGA +142 "GTGA motif" Triticum found in the aestivum, promoter of the CaMV, tobacco late Oryza sativa pollen gene glO

which shows

homology to

pectate lyase and

is the putative

homologue of the

tomato gene

lat56; Located

between -96 and

-93.

HEXMOTIFTAH3 ACGTCA (-) 224 "Hexamer motif" Triticum

H4 found in promoter aestivum, of wheat histone CaMV, genes H3 and H4; Oryza sativa CaMV35S; NOS;

Binding with

HBP-1A and

HBP-1B; Binding

site of wheat

nuclear protein

HBP-1 (histone

DNA binding

protein-1); HBP-1

has a leucine

zipper motif;

"hexamer motif"

in type 1 element

may play

important roles in

regulation of

replication- dependent but not

of replication- independent

expression of the

wheat histone H3

gene; Rice OBF1- homodimer- binding site.

IB OX GATAAG +449 Conserved Lycopersicon sequence esculentum, upstream of light- Arabidopsis regulated genes; thaliana Sequence found

in the promoter

region of rbcS of

tomato and

Arabidopsis;

Binding site of

LeMYB l,

(transcriptional

activator) that is a

member of a

novel class of

myb-like proteins.

IBOXCORE GATAA +390, +449, Conserved Monocots

+469, -243 sequence and dicots.

upstream of light- regulated genes.

INRNTPSADB YTCANTYY +177 Initiator elements Nicotiana found in the tabacum tobacco psaDb

gene promoter

without TATA

boxes; Light- responsive

transcription of

psaDb depends on

Inr, but not TATA

box.

LECPLEACS2 TAAAATAT +392 Core element in Lycopersicon

LeCp (tomato Cys esculentum protease) binding

c«-element (from

-715 to -675) in

LeAcs2 gene.

MYB1AT WAACCA +324 MYB recognition Arabidopsis site found in the thaliana promoters of the

dehydration- responsive gene

rd22 and many

other genes in

Arabidopsis.

MYBCORE CNGTTR +306 Binding site for Arabidopsis all animal MYB thaliana, and at least two animal, plant MYB Petunia proteins hybrida ATMYB1 and

ATMYB2, both;

ATMYB2 is

involved in

regulation of

genes that are

responsive to

water stress in

Arabidopsis; A

petunia MYB

protein

(MYB.Ph3) is

involved in

regulation of

flavonoid

biosynthesis.

MYBST1 GGATA -643, -650 Core motif of Solanum

MybStl (a potato tuberosum MYB homolog)

binding site.

MYCCONSENSUS CANNTG +550, -550 MYC recognition Arabidopsis AT site found in the thaliana promoters of the

dehydration- responsive gene

rd22 and many

other genes in

Arabidopsis;

Binding site of

ATMYC2

(previously

known as

rd22BPl); (E- box; CANNTG),

(MYCATRD22);

N=A/T/G/C;

MYC recognition

sequence in CBF3

promoter;

Binding site of

ICE1 (inducer of

CBF expression

1) that regulates

the transcription

of CBF/DREB 1

genes in the cold

in Arabidopsis.

NAPINMOTIFBN TACACAT -420 Sequence found Brassica in 5' upstream napus region (-6, -95, - 188) of napin

gene in Brassica

napus; Interact

with a protein

present in crude

nuclear extracts

from developing

seeds.

NODCONIGM and AAAGAT +300, -14, - One of two Glycine max OSE1ROOTNODU 601 putative nodulin

LE consensus

sequences.

NODCON2GM and CTCTT +564 One of two Glycine max OSE2ROOTNODU putative nodulin

LE consensus sequences.

NRRBNEXTA TAGTGGAT - 651 "NRR (negative Brassica regulatory napus region)" in

promoter region

of Brassica napus

extA gene;

Removal of this

region leads to

expression in all

tissues within the

stem internode,

petiole and root.

POLASIG1 AATAAA + 296, + PolyA signal Pisum

429, +529, - found in legA sativum, 60 gene of pea, rice Oryza sativa, alpha-amylase; - Arabidopsis 10 to -30 in the thaliana. case of animal

genes. Near

upstream

elements (NUE)

in Arabidopsis.

POLASIG2 AATTAAA +149 Poly A signal Oryza sativa, found in rice animal alpha-amylase; - 10 to -30 in the

case of animal

genes.

POLASIG3 AATAAT +579 Consensus Zea mays sequence for plant

polyadenylation

signal.

POLLEN 1 LELAT5 AGAAA +521, -171, - One of two co- Lycopersicon

2 663 dependent esculentum regulatory

elements

responsible for

pollen specific

activation of

tomato lat52

gene; Found at - 72 to -68 region;

Also found in the

promoter of

tomato endo-beta- mannanase gene.

PRECONSCRHSP7 SCGAYNRNNNN + 99 Consensus Chlamydomo OA NNNNNNNNNN sequence of PRE nas

NHD (plastid response reinhardtii element) in the

promoters of

HSP70A in

Chlamydomonas ;

Involved in

induction of

HSP70A gene by

both MgProto and

light.

QELEMENTZMZ AGGTCA + 616 "Q (quantitative) - Zea mays M13 element" in maize

ZM13 gene

(pollen-specific

maize gene.)

promoter; Found

at -107 to -102;

Involved in

expression

enhancing

activity.

RAV1AAT CAACA -184, -336

REALPHALGLHC AACCAA -407 "REalpha" found Lemna gibba B21 in Lemna gibba

Lhcb21 gene

promoter;

Located at -134 to

-129; Binding site

of proteins of

whole-cell

extracts; The

DNA binding

activity is high in

etiolated plants

but much lower in

green plants;

Required for

phytochrome

regulation.

ROOTMOTIFTAP ATATT -395, -582 Motif found both Agrobacteriu 0X1 in promoters of m rhizogenes rolD

SIFBOXSORPSIL ATGGTA -19 "S IF box" Spinacia 21 conserved both in oleracea spinach, RPS1

and RPL21 genes

encoding the

plastid ribosomal

protein SI and

L21, respectively;

Negative element;

Might play a role

in downregulating

RPS1 and

RPL21 promoter

activity.

SEF4MOTIFGM7S RTTTTTR -47 Consensus Glycine max sequence found in

5 'upstream region

(-199) of beta- conglycinin (7S

globulin) gene;

"Binding with

SEF4 (soybean

embryo factor 4.

TAAAGSTKST1 TAAAG -16 Target site for Solanum trans-acting tuberosum StDof 1 protein

controlling guard

cell-specific gene

expression; KST1

gene encodes a

K+ influx channel

of guard cells.

TATABOXOSPAL TATTTAA +206 Binding site for Oryza sativa

OsTBP2, found in

the promoter of

rice pal gene

encoding

phenylalanine

ammonia-lyase;

OsTFIIB

stimulated the

DNA binding

and bending

activities of

OsTBP2 and

synergistically

enhanced

OsTBP2- mediated

transcription from

the pal promoter.

TATAPVTRNALE TTTATATA +457 Frequently Phaseolus U observed vulgaris, Zea upstream of plant mays tRNA genes;

Found in maize

glycolytic

glyceraldehyde-3 - phospate

dehydrogenase 4

(GapC4) gene

promoter;

Binding site of

TATA binding

protein (TBP).

TATCCACHVAL2 TATCCAC + 650 "TATCCAC box" Hordeum 1 is a part of the vulgare conserved xacting response

complex (GARC)

that most often

contain three

sequence motifs,

the TAACAAA

box , GA- responsive

element (GARE);

the pyrimidine

box, CCTTTT ;

and the

TATCCAC box,

which are

necessary for a

full GA response

TATCCAOSAMY TATCCA +643, + 650 "TATCCA" Oryza sativa element found in

alpha-amylase

promoters of rice

at positions 90 to

150bp upstream

of the

transcription start

sites; Binding

sites of OsMYBSl,

OsMYBS2 and

OsMYBS3 which

mediate sugar and

hormone

regulation of

alpha-amylase

gene expression.

TATCCAYMOTIF TATCCAY +650 TATCCAY Oryza sativa OSRAMY3D motif and G motif

are responsible

for sugar

repression.

TGACGTVMAMY TGACGT + 224 Required for high Vigna mungo level expression

of alpha-Amylase

in the cotyledons

of the

germinated seeds

WBBOXPCWRKY TTTGACY + 604, -497 "WB box"; Ipomoea 1 WRKY proteins batatas, bind specifically Triticum to the DNA aestivum, sequence Hordeum motif vulgare,

(T)(T)TGAC(C/T Avenafatua, ), which is known Petroselium as the W box crispum,

Arabidopsis thaliana

WBOXATNPR1 TTGAC +223, +605, "W-box" found in Arabidopsis

-498, -570, - promoter of thaliana 618 Arabidopsis

thaliana NPR1

gene;

WBOXHVISOl TGACT SUSIBA2 bind to Hordeum

+143, -476, - W-box element in vulgare 497 barley isol

(encoding

isoamylasel)

promoter

WBOXNTERF3 TGACY +143, +606, "W box" found in Nicotiana

-476, -497, - the promoter tabacum 617 region of a

transcriptional

repressor ERF3

gene in tobacco; WRKY710S TGAC +143, +224, "A core of Oryza sativa,

+606, -477, - TGAC-containing Petroselinum

498, -570, - W-box" of, e.g., crispum

618 Amy32b

promoter;

Binding site of

rice WRKY71, a

transcriptional

repressor of the

gibberellin

signaling

pathway.

WUSATAg +556, -327 Target sequence Oryza sativa of WUS in the

intron of

AGAMOUS gene

in Arabidopsis

(+) represents for SEQ ID NO: l and (-) represents for SEQ ID NO:2

[00134] Bidirectional promoter: The transcript levels of At3gl7140 and At3gl7150 genes were assessed in WT and GUS-650 line by semi-quantitative RT-PCR. Homozygous and hemizygous GUS-650 mutant plants were identified with PCR based genotyping in the T ₂ generation. With the primer combination of IF- IR and IF- NRBGUS , hemizygous plants gave 350 bp and 400 bp amplicons, respectively, whereas plants homozygous for T-DNA insertion gave only 400 bp amplicon (data not shown).

[00135] Transcripts of both At3gl7140 and At3gl7150 genes were found in inflorescence of WT transcripts of AT3gl7150 were not detected in the GUS-650 homozygous plants (Fig 7), but uidA transcripts were found in both hemizygous and homozygous GUS-650 plants. From the results obtained it is inferred that identified intergenic sequence functions as a bi-directional promoter and simultaneously transcribes both At3gl7140 and AT3gl7150 genes in WT.

[00136] Gene expression in the native context could be regulated by sequences besides the immediate upstream cis sequences, such as introns, 3' UTRs etc. Therefore, it was essential to determine if the 672 bp sequence alone is sufficient to drive gene expression, uidA expression cassettes were prepared in pORE-R2 vector. The 672 bp fragment was amplified and cloned in forward or reverse orientation to drive the uidA reporter gene (Fig. 8). Figure 8A and 8C depict the two vector constructs generated to confirm that 672bp intergenic fragment functions as a bi-directional promoter. Arabidopsis plants transformed with these constructs were examined for GUS expression. GUS expression was detected in plants with either type of construct and was similar to that observed in the GUS-650 line. Further, GUS expression was confined to the female gametophyte in both orientations (Fig. 8B and 8D). More or less identical GUS expression pattern was observed throughout the developmental stages of female gametophyte in the transgenic plants generated using the promoter in either of the orientation. GUS expression was first detectable at the FG3 stage at the micropylar end and gradually extended towards the chalazal end as the embryo sac development progressed (Fig. 8Ba-d and 8Da-d). At maturity, intense GUS expression was observed in the gametes and after fertilization in the embryo and the endosperm (Fig. 8Be-f and 8De-f) exactly mimicking the GUS-650 mutant line. Further, transcript levels of uidA gene in homozygous T ₂ transgenic plants carrying 672 nt promoter: :uidA constructs in either orientations were assessed through RT-PCR. All lines showed more or less similar levels of uidA transcripts (Fig. 9a-b). These data confirm that the 668 bp intergenic sequence perfectly functions as an embryo sac specific bidirectional promoter.

[00137] Presence of multiple copies of positive regulatory elements such as CAAT box, GATA box, ACGT, CArG motif might contribute to the strong promoter activity of the 672 bp fragment. Further, presence of strong negative regulatory elements such as AACA that are known to inhibit gene expression in floral or vegetative parts might explain the ovule and seed specific nature of this promoter. Skn-motif reported in promoters that upregulate carotenogenic gene expression during flower development in Gentiana lutea was also found in this sequence. Embryo sac specific expression could result from the interactions between various positive and negative elements in this 672 bp fragment. [00138] This type of strong tissue specific promoter could find many important biotechnological applications. For instance, it could be used to drive a pair of antibody fragments to ensure the two types of molecules are produced in equimolar quantity in the same cell/tissue. Being specific to ovules and developing seeds, this promoter could be employed in improving nutritional quality of seeds through engineering of metabolic pathways. Further, ability of this promoter to drive gene expression in the embryo sac during early stages of differentiation could be deployed to manipulate reproductive pathways in plants.