WO2005026367A1

WO2005026367A1 - Overexpression of foreign genes in plants

Info

Publication number: WO2005026367A1
Application number: PCT/IN2004/000294
Authority: WO
Inventors: Vanga Siva Reddy; Huu Tam Nguyen; Sadhu Leelavathi
Original assignee: International Centre For Genetic Engineering And Biotechnology
Priority date: 2003-09-18
Filing date: 2004-09-17
Publication date: 2005-03-24

Abstract

A transcription systems for overexpression of foreign proteins in eukaryotic genomes of transgenic plants is disclosed. According to the invention, a modified RNA polymerase is expressed with a nuclear localization signal under the control of a plant tissue-specific promoter to direct the polymerase to the nucleus and to place the transgene under the control of a corresponding promoter and a terminator.

Description

OVEREXPRESSION OF FOREIGN GENES IN PLANTS

Field of the invention The present invention relates to transcription systems for overexpression of foreign proteins in higher eukaryotic genomes. More particularly, the present invention relates to. transcription systems for overexpression of foreign proteins in higher eukaryotic genomes, such as nuclear genomes of higher organisms. More particularly, • the present invention relates to T5 RNA polymerase, SP6 RNA polymerase or Bacteriophage T7 RNA polymerase based transcription systems for overexpression of foreign proteins in higher eukaryotic genomes, particularly nuclear genomes in plants. In particular, the present invention relates to bacteriophage T5 RNA polymerase, SP6 RNA polymerase or T7 RNA polymerase based transcription systems for use in overexpression of foreign proteins in a tissue specific and inducible manner. The present invention also relates to a method for overexpression of foreign proteins in plants using the novel Bacteriophage T5 RNA polymerase, SP6 RNA polymerase and T7 RNA polymerase based transcription systems. Background of the invention Genetic engineering offers enormous scope to utilize plants as protein production factories. However, commercialization of this important technology is hampered by generally observed low-level expression of recombinant proteins in a desired plant tissue. Plants are increasingly being used as "natural bioreactors" for large-scale production of foreign proteins ' for industrial application (Giddings, G. Transgenic plants as protein factories. Curr Opin Biotechnol. 12: 450-454 (2001)), an approach the success of which is highly dependent on the expression levels achieved for heterologous proteins in plants. Although, considerable increase in transgene expression has been achieved in the prior art through promoter optimization, protein targeting and codon optimization, lack of high level expression in a desired plant tissue is still a major limiting step in these approaches. An alternative approach to overproduce foreign proteins in plants is through chloroplast genetic engineering (Geert De Jaeger, Stanley Scheffer, Anni Jacobs, Mukund Zambre, Oliver Zobell, Alain Goossens, Ann Depicker & Geert Angenon.Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences. Nat. Biotechnol 20,1265 - 1268 (2002)). However, chloroplast transformation has been achieved routinely so far only in tobacco. Moreover, posttranslational modifications such as glycosylation of recombinant proteins is a limiting factor when expressed in chloroplasts that resemble prokaryotes. The bacteriophage T7 RNA polymerase (T7 RNAP) is the most commonly used transcription system to overproduce recombinant proteins in microbial systems. Although success has been achieved to establish T7-mediated expression in lower eukaryotic organism Trypanosoma brucei (Fujimoto H, Itoh K, Yamamoto M, Kyozuka J, Shimamoto K. Insect resistant rice generated by introduction of a modified delta- endotoxin gene of Bacillus thuringiensis. Bio/technology 11,1151-1155 (1993)), it has met with very little success in higher eukaryotic animal systems. Data from several reports (Maliga, P. Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21: 20-28 (2003)) establish that transcription of higher eukaryotic chromatin by the phage polymerase is not very processive. In plants, expression of GUS reporter gene integrated into chloroplast genome, a genome that resemble closely to prokaryotic genomes, was achieved through nuclear transformed and chloroplast-targeted T7 RNAP (Studier, F.W., Rosenberg, AH., Dunn, J.J., & Dubendorf J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185: 60-89 (1990)). However, it was not known if the T7 RNAP can transcribe a foreign gene integrated stably into higher plant nuclear genome, as plants and animals share similar chromatin organization. Development of a widely applicable regulated and tissue specific high level expression system for foreign genes in transgenic plants will have a profound impact on several currently ongoing plant biotechnology programs and on functional genomic studies. Though considerable increase in trangene expression was achieved through the use of strong viral and tissue specific promoters, protein targeting and codon optimization methods, lack of a generally applicable high level expression to a wide range of crop species in a desired tissue is still a major limiting step. An alternate approach for high level expression of recombinant protein could be to introduce transgene into chloroplast genome. However, lack of simple chloroplast transformation procedures for non-Solanaceae members and cereals and posttranslational modifications such as glycosylation may be limiting factors when expressed in chloroplasts. Moreover, majority of genes have their function outside chloroplasts and in the tissues that lack chloroplasts. The bacteriophage T7 RNA polymerase (T7 RNAP) based transcription is the used commonly most expression system to overproduce recombinant proteins in microbes (Studier, F. W., Rosenberg, AH., Dunn, J.J., & Dubendorjf, J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185: 60-89 (1990)). Although, T7 RNAP based expression (T7-system) has been achieved for transgene in lower eukaryotic organism Trypanosoma brucei recently (Wirtz, E., Hoek, M., & Cross, G.A. Regulated processive transcription of chromatin by T7 RNA polymerase in Trypanosoma brucei. Nucleic Acids Res. 26: 4626-4634 (1998)), it has met with very little success in higher eukaryotic animals. Data from several reports (Hartvig, L., & Christiansen, J. Intrinsic termination of T7 RNA polymerase mediated by either RNA or DNA. EMBO J. 15: 4767-4774 (1996)) suggest that transcription of higher eukaryotic chromatin by the phage polymerase is not very processive. In plants expression of uidA (GUS) reporter gene integrated into chloroplast genome which resembles prokaryotic genome structurally was achieved through nuclear transformed and chloroplast-targeted T7 RNAP (McBride, E. et al. Controlled expression ofplastid transgegenes in plants based on a nuclear DNA-encoded and plastid-targeted T7 RNA polymerase. Proc. Natl. Acad, Sci. 91: 7301-7305 (1994)). However, it was not known if the T7 RNAP can transcribe a foreign gene integrated randomly into higher plant nuclear genome, as plant genome is also organized into chromatin structure. The structure of naked DNA in microbial systems in which bacteriophage T7 RNA polymerase (T7 RNAP) has been successfully used to overproduce recombinant proteins is very similar to the chloroplast genome of higher plants. While, it does not automatically' predict that such transcription systems will effectively work to overproduce recombinant proteins in the chloroplast genomes of higher plants, given the unpredictable nature of biotechnological inventions in general, Monsanto, in its recent U.S. Patent teaches overexpression of recombinant proteins in the chloroplast genomes of higher plants. However, such transcription systems failed miserably when tried on animals which lack chloroplast genomes, thereby reaffirming the theory that bacteriophage T7 RNA polymerase (T7 RNAP) are successful in overproducing recombinant proteins only in prokaryotic or prokaryotic like genomes. Therefore, the prior art does not record any attempt to overproduce recombinant proteins outside prokaryotic or chloroplast genomes in higher plants. On the contrary, based on the teachings of prior art, it would be assumed by a person skilled in the art that T7 RNAP will not be effective to transcribe a foreign gene integrated stably into higher plant nuclear genome. This is because the prior art reports repeated failure in animal genomes and plants and animals share similar chromatin organization. The plastid genome is very small [(1.3-1.5) X 10⁵ kb] when compared to nuclear genomes of higher plants [1.1 - 4.3 X 10⁸ bp], and exists as a double-stranded circular DNA in multiple copies, resembling the genome of prokaryotic organisms in its structure. On the other hand, the nuclear genome is much more complex with a highly organized chromatin and a well-defined nucleus. Therefore, if T7 RNAP is reported to be ineffective to transcribe a foreign gene integrated stably into the chromatin genome of an animal, it would be expected to be equally ineffective to transcribe a foreign gene integrated into a higher plant nuclear genome, as plants and animals share similar chromatin organization. Therefore there is a tremendous need in the art for transcription systems for overexpression of foreign proteins in eukaryotic genomes of higher organisms, particularly, nuclear genomes of higher plants, especially since most of the important genes of an organism are located in the nuclear genome thereof. Objects of the invention Accordingly, it is one of the objects of the present invention to effectively transcribe a foreign gene integrated stably into eukaryotic genomes of higher organisms. It is another object of the present invention to provide a method for overexpression of foreign proteins in eukaryotic genomes of higher organisms. It is another object of the present invention to provide a method for overexpression of foreign proteins in eukaryotic genomes, particularly in higher plants. It is still another object of the present invention to provide transcription systems for overexpression of foreign proteins in eukaryotic genomes of higher organisms. It is still another object of the present invention to provide transcription systems for overexpression of foreign proteins in eukaryotic genomes of higher plants. It is yet another object of the present invention to provide Bacteriophage T5 RNA polymerase, SP6 RNA polymerase or T7 RNA polymerase based transcription systems for overexpression of foreign proteins in eukaryotic genomes, particularly nuclear genomes in plants. It is yet another object of the present invention to provide bacteriophage T5 RNA polymerase, SP6 RNA polymerase or T7 RNA polymerase based transcription to overexpress foreign proteins in a tissue specific and inducible manner. It is still another important object of the present invention to provide a method for overexpression of foreign proteins in non prokaryotic genomes, particularly nuclear genomes, such as in higher plants using the novel Bacteriophage T7 RNA polymerase based transcription systems. Summary of the invention The above and other objects of the present invention are achieved by the novel Bacteriophage RNA polymerase based transcription systems for overexpression of foreign genes in a higher transgenic organism comprising a foreign gene placed under the control of expression signals and a modified RNA polymerase to specifically transcribe said foreign gene, both said foreign said and said RNA polymerase being located either in cis or trans position with respect to each other in the nuclear genome of a higher organism, said RNA polymerase being selected from T5 RNA polymerase, SP6 RNA polymerase and bacteriophage. T7 RNA polymerase. The present invention will be described herein after with reference to T7 RNA polymerase. On the basis of the description provided herein, it will be apparent to a person skilled in the art that the invention will work equally with other polymerases such as T5 RNA polymerase and SP6 RNA polymerase. In an embodiment of the invention, said modified T7 RNA polymerase is a bacteriophage T7 RNA polymerase. In an embodiment of the invention, the transgenic organism is a plant. In another embodiment, said Bacteriophage T7 RNA polymerase based transcription system is in the form of a construct having a T7 promoter and terminator. The T7 RNA polymerase is preferably expressed with a nuclear localization signal (NLS) under the control of a plant tissue specific promoter to direct the polymerase to the nucleus and to place the transgene under the control of the T7 promoter and terminator in said construct. In an embodiment of the invention, the transgene is a uidA gene (GUS) and said higher transgenic organism is a monocotyledonous or a dicotyledonous plant. ' The present invention for the first time, demonstrates the use of the T7 RNAP to specifically transcribe a foreign gene integrated randomly into the nuclear genomes of plants. The present application, for the sake of convenience is described with reference to the uidA gene (GUS), a commonly used reporter gene in plants (Jefferson, R.A., Kavanagh, T.A., & Bevan, M.W. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901-3907 (1989)), integrated randomly into the nuclear genomes of tobacco (a dicot) and rice (a monocot), in a tissue-specific and inducible manner at high levels. However, a skilled reader of the present specification will be able to appreciate that the present invention can be successfully employed to overexpress any foreign gene in the nuclear genome of any tissue of any plant.

Detailed description of the invention According to the present invention, a widely applicable bacteriophage T7 RNA polymerase directed tissue specific overexpression of foreign genes in transgenic plants was developed. This was achieved through the transformation of a modified T7 RNA polymerase placed under a tissue specific plant promoter that specifically recognized the transgene (uidA) placed under T7 expression sequences and integrated randomly into tobacco and rice genomes; Results from the use of six different promoters with different tissue specificities indicated that recombinant protein can be expressed at several fold high (3 - 10 times) as compared to transgene expressed directly under these tissue specific promoters. Another important feature of T7 system in plants was found to be the low variations in the transgene expression among independently transformed plants. In addition, using T7 system, transgene expression can be tightly regulated through chemically inducible mechanisms, extending the application of this powerful tool to various programs in plant biotechnology and to genomic studies. Brief description of the accompanying drawings Figure 1 & 1 (A). The sequence of chimeric GUS gene showing T7 promoter, ribosome binding site (rbs), partial GUS sequence (underlined), and T7 terminator (13). (Sequence ID 1). Forward arrow indicates the transcription start site and reverse arrow indicate the GUS internal primer used in primer extension. Figure 1. (B). Gene constructs' used for tissue-specific expression of GUS gene. Each promoter was designed with two constructs, one is control gus gene directly and the other is controlling GUS gene through T7-RNA polymerase system (T7 RNA polymerase with T7-promoter and T7-terminator). LB, left border; RB, right border; pro, promoter; ter, terminator; pA, poly A; Hyg, hygromycine resistant gene. Figure 1. (C). Northern blot analysis to detect the presence of GUS transcripts in Nt-441-1 (1), Nt-450-2 (2) and Nt-1301-1 (3) using uidA probe. Re-hybridization of the same blot was carried out with ribosomal 16S (16SrRNA) probe to show equal loading of RNA (lower panel). Figure 1. (D). Mapping of the 5' ends of the uidA transcripts by primer extension. ATGC represent partial nucleotide sequence of pITB450 generated by GUS internal primer. Lane 1 shows the extension product using total RNA from wild type

(not shown) and Nt.450-2 plant. (E). Histochemical staining with X-gluc indicating the tissues specificity for GUS expression. Top panel: The Nt.441-1, Nt.450-2 and Nt.1301-1 plants obtained by transforming pITB441, pITB450 and pCAMBIA1301 constructs, respectively. Lower panel show representative stem and root sections corresponding to the same plants shown in the top panel. Note that while GUS staining was observed in all tissues of Nt-1301-1 plant, it was restricted to green chloroplast containing tissues and totally absent in roots of NT-441-1 and Nt-450-2 plants. Figure 2. In situ analysis of GUS expression. Figure 2. (A) Comparison of GUS activity in leaf, stem and roots of Nt.1301-1, Nt.441-1 and Nt.450-2 plants. Transgenic tobacco roots fromNt.541-1 Figure 2. (B), Nt.550-2 Figure 2. (C) and Nt.228-2 Figure 2. (D). (RH, root hail; RE, root elongation zone; RC, root cap). Figure 2. (E) Epidermis peeled from the Nt.550-2 plant. Note strong expression of US in guard cells (inset). Tobacco roots from Nt.741-1 Figure 2, (F) and (Nt.750-2 Figure 2. (G). (VT, vascular tissue). Tobacco leaves from Nt.750-2 Figure 2. (H) and Nt.450-2 Figure 2. (ϊ). (J and K). Comparison of GUS expression under rbcS, kinl, cor6.6, pail, palΔ and CaMV 35S promoters directly and through T7-system among independently transformed plants. Figure 3: (A). Gene constructs used for tetracycline inducible expression of GUS under T7 RNAP transcription; The pBin-tetR contained tetracycline repressor gene (tetK) under CaMV35S promoter (35S-pro). The pITB228 contained T7 RNAP under a modified tripleX 35S promoter (12) and GUS under T7 promoter (T7-pro) and terminator. pNOS, nopaline synthetase ' promoter; NPTII, neomycinphospho- transferase; Hyg, hygromycine resistant gene; LB, left border; RB, right border. Figure 3: (B). Northern blot analysis showing the expression of GUS and T7 RNAP upon induction with tetracycline. Blots were probed either with uidA (left panel) or uidA and T7 RNAP together (right panel). UN, uninduced; IN, induced. Re- hybridization of the same blot was carried out with ribosomal 16S (lόSrRNA) probe to show equal loading of RNA (lower panel). Figure 3: (C). Levels of GUS expression in the induced and uninduced leaf samples after 48 hours. Figure 3: (ϊ)). Kinetics of Tc-induced GUS expression. Figure 4: (A). Histochemical staining to detect the GUS expression in rice leaves. Leaves from wild type (Wt), transformed with pCAMBIAl301 (Os.1301-1) and pITB228 (Os.228-2). Figure 4: (B). Roots from Os.228-2. Figure 4: (C). Northern blot to detect the presence of GUS transcripts in Os.1301-1 and Os.228-2 plants. Same membrane was hybridized with ribosomal 16S rRNA to show equal loading of RNA (lower panel). Figure 4: (D). Quantification of GUS activity in leaves of transgenic rice plants among independently transformed plants. The present invention will now be described in greater detail with reference to the following examples, which are included merely to illustrate and demonstrate the invention. These examples should not be construed to limit the scope of the invention in any way. It will be apparent to a skilled reader of the present specification that the present invention can be successfully employed to overexpress any foreign gene in the nuclear genome of any tissue of any plant. Example 1 Tissue specific high level expression To achieve tissue specific high level expression for a transgene, the general object of the present invention was to express a modified T7 RNAP with a nuclear localization signal (NLS) (Dunn, J.J., Krippl, B., Bernstein, K.E., Westphal, H, & Studier, F. W. Targeting bacteriophage T7 RNA polymerase to the mammalian cell nucleus. Gene 68: 259-266 (1988)), to target the T7 RNAP to nucleus, under a plant tissue specific gene promoter and express the transgene under T7 promoter and terminator in the same construct. ^■ To test this, gene constructs containing six differentially expressed gene promoters with various tissue specificities were used to and transform tobacco and rice (Fig. 1A and B). The pITB450, pITB550, pITB650, pITB750 and pITB850 constructs contained uidA placed under T7 promoter and terminator sequences (Fig. 1 A) and the modified T7 RNAP with NLS was placed under the control of the small subunit of ribulose-bisphosphate carboxylase (rZ>cS:3A) (Kuhlemeier, C. et al Localization and conditional redundancy of regulatory elements in rbcS-3A, a pea gene encoding the small- subunit of ribulose-bisphosphate carboxylase. Proc Nail Acad Sci USA. 85: 4662-4666 (1988)), stress inducible kinl, cor6.6 (Wang H., and Cutler J. Promoters from kinl and cor6.6, two Arabidosis thaliana low-temperature-and ABA-inducible genes, direct strong β-glucuronidase expression in guard cells, pollen and young developing seed. Plant Molecular Biology 28: 619-634 (1995)); (Wang H, Datla R, Georges F, Loewen M., and Cutler A. Promoters from kinl and cor6.6, two homologous Arabidopsis thaliana genes: transcriptional regulation and gene expression induced by low temperature, ABA, osmoticum and dehydration. Plant Molecular Biology 28: 605-617 (1996)), phynylalanine ammonia-lyase (pall) md pall A (Ohl S., Hedrick S.A., Chory J., and Lam C.J. Function Properties of a Phenyllalanine Ammonia-Lyase Promoter from Arabidopsis. The plant Cell 2: 837-848 (1990)), promoters, respectively Fig. IB). For a direct comparison, the uidA was also placed directly under kinl, cor6.6,pall mάpallA promoters, in pITB541, pITb641, pITB741 and pITB841 constructs, respectively. In addition, pCAMBIA1301 vector containing uidA under the control of a strong cauliflower mosaic virus (CaMV) 35S promoter (Benfey, P.N., and Chua, N.H. The cauliflower mosaic virus 35S promoter: Combinatorial regulation of transcription in plants. Science 250: 959-966 (1990)) that express constitutively in most tissue types was also transformed into tobacco and rice plants for comparison. Tobacco transgenic plants were produced for each of the construct following Agrobacterium mediated transformation (Horsch, R.B. et al A simple and general method for transferring genes into plants: Science 227: 1229-1231 (1985)). Southern hybridization or polymerase chain reaction (PCR) was used to confirm the transformation. Northern blot and real time PCR (data not shown) techniques were used to confirm the transcription of uidA (Fig. 1C). As can be seen from Fig. 1C, in Nt.450- 2 plant, the uidA transcription under T7-system was 2 - 3 times higher when compared to uidA transcription directly Under rbcS3A promoter and the transcript levels were comparable to uidA transcripts under strong CaMV 35 S promoter in Nt.1301 - 1. As the transcript initiation from the T7 promoter by T7 RNAP was highly specific, primer extension analysis was carried out to authenticate the transcription of uidA by T7 RNAP. It can be seen from figure ID, GUS transcripts initiated from the nucleotide 'G', specific for T7 promoter in Nt.450-2. These results clearly demonstrate that the T7 RNAP recognizes its promoter in the randomly integrated plant genome and transcribed uidA accurately, akin to its transcription in E. coli. The histochemical analysis revealed that the GUS expression under rbcS:3A promoter was Hmited to green tissues with maximum activity localized in leaves followed by stem and absent in roots (Fig. IE). Most significantly, similar tissue specific expression pattern was observed among the transgenics that were transformed with ρITB450 construct (Fig. IE). On the other hand, GUS under CaMV 35S promoter expressed in all tissues tested. It can be noted that the intensity of blue colour was more in the Nt.450-2 leaves when compared to the Nt.441-1 leaves indicating that the expression level could, be high in the Nt.450-2 plant. Further analysis with the cross sectioned stem and root tissues (insets) reveled that while GUS expressed in all cell types within the stem in the' Nt.1301-1 plant'-ithe activity was localized to cortex (green tissue within the stem) in both Nt. 441-1 and Nt. 450-2 plants and was absent in the cambium-ring^ vascular tissue (xylem and phloem) and in the middle pith tissue. Within the roots, as expected, GUS activity was present in Nt.1301-1 and absent in Nt441-1 and Nt.450-2 plants. Quantification of GUS activity in various tissues further confirmed that the GUS expression under rbcS:3A promoter was highly tissue specific with the highest activity in leaves and lowest in^' roots (Fig. 2A). On the other hand, GUS in all the tissues investigated under 35S promoter expressed and the activity was 3 times high in leaves and 15 times high in roots when compared to GUS expressed under r£cS:3A promoter in leaves and roots, respectively. The pattern of GUS expression was similar in both Nt.441-1 and Nt.450-r2 plants^' with the highest activity in leaf followed by stem and roots. Significantly, expression' of GUS was 3 - 4 fold high under rbcS T1- expression when compared to the uidA expressed directly under rbcS:3A promoter, a level comparable to GUS expressed under the strong viral CaMV 35S promoter (Fig. 2A). Example 2 To further confirm the high level tissue specific GUS expression observed under rέcS.3A promoter using T7-system, ^: four additional promoters (kinl, cor6.6, pall and pall A) from Arabidopsis thaliana that were shown to express at different levels in different tissues were exarnined. While kin and cor<5.6 genes express at high level in the stem, roots and in reproductive tissues, their expression in leaf is relatively low. Within the leaf, the expression was more prominent in guard cells. In the present study, similar expression pattern was observed in trasgenic plants transformed with pITB641 and pITB541 (Fig. 2B) constructs where GUS was expressed directly under cor6.6 and kinl promoters respectively. Within the roots, the GUS expression varied considerably in different regions, with more activity localized in the root cap and was absent in the root elongation zone and in root hairs (Fig. 2B). Similar expression pattern was observed under T7-system for both kinl (Fig. 2C) and cor6.6 (data not shown) promoters. As expected, the intensity of blue colour in root and root cap region was high in Nt.550-2 when compared to GUS expression observed in Nt.541-3. On the other hand, expression of GUS under 35S promoter (pITB228) was uniformly high in all zones of root (Fig. 2D). Within the leaf, as expected, GUS expressed more prominently in the guard cells under cor6.6 (data not shown) and kinl (Fig. 2E) promoters. Analysis of phenylalanine ammonia-lyase (pall) gene promoter from Arabidopsis revealed that the pall promoter is highly tissue specific with maximum activity in the vascular tissue of roots and leaves. In the present invention, a full length (pall, +1 to -832) and a truncated (pallA, +1 to -540) promoters that have same tissue specificity but differ in their strength were used to test the GUS expression under T7- system. For comparison, uidA vf&s also expressed directly under both /1 wad pall A promoters. Under pall promoter, expression of GUS was high in vascular tissue of roots (Fig. 2F) and leaves. Similar expression pattern was observed for GUS under pall promoter using T7-system (Fig. 2G). Again, the GUS activity was high in Nt.750-1 when compared to Nt.741-1. Similar results were obtained for pallA promoter. Strong GUS activity was observed in the vascular tissue of leaf (Fig. 2H) and roots under T7- system. In contrast, the GUS expression under rbcS:3A promoter was restricted to green mesophyll cells and was absent in the vascular tissue (Fig, 21). This was expected for rόeS.3A promoter as the rbcS expression is linked to presence of chloroplasts whereas the vascular tissue is devoid of them. A detailed quantification of GUS activity in the leaf tissue of ten randomly chosen transgenic plants revealed that the GUS activity was significantly high under T7-system when compared the GUS expressed directly under any promoter investigated. On an average, there was 3 - 10 fold increase in the expression under T7- system (Fig. 2F-G). A remarkable feature of T7-expression system in plants was found to be uniform levels of transgene expression among independently transformed plants, as opposed to large variations found under direct expression of plant promoters. It establishes that very low expression of T7 RNAP is just sufficient to transcribe the transgene at maximum level. This feature will be particularly useful in plants such as legumes, cereals and tree species where it is most difficult to transform and regenerate large number of transgrnic plants required to identify high-expressing plant(s).

Example 3 To test the wider application of T7-expression system in plants, the expression of GUS in rice, a monocot plant, with worldwide significance as a major source of staple food was examined. Biolistic mediated transformation (Cao J., Wang Y-C, Klein TM., Sanford JC, Wu R. Transformation of rice and maize using the particle gun method. Pages 21-33 in Plant gene transfer. Lam C.J. and Beachy RN, eds, Wiley- Liss, New York (1990)) was followed to introduce ρCAMBIA1301 and pITB228 constructs into rice genome. A large number of putative transgenic plants, regenerated on hygromycin selection, were screened for the expression of GUS. Similar to tobacco, GUS activity under CaMV 35S promoter using T7-system was high in leaves and roots of transgenic plants when compared to GUS expressed directly under CaMV 35S promoter (Fig. 4A-B). To verify that the high expression of GUS in Os.228-1 is due to increase in transcription under T7-system, Northern blot analysis was carried out to verify the transcript levels. When compared to Os.1301-1, a high GUS expressing plant, the uidA transcripts were 3 times more in the Os.228-1 plant (Fig. 4C), suggesting that the increased level of GUS expression could be due to increase in uidA transcription. Quantitative analysis also reveled that the GUS expression was 3 - 5 times high in plants that were independently transformed with pITB228 construct when compared to GUS expression in plants that were transformed with pCAMBIA1301 (Fig. 4D). Also, as opposed to large variations observed in the GUS expression among the independently transformed plants with pCAMBIA1301, variations were minimum among the pITB228 transformed plants, similar to the observations made in tobacco. Example 4

Inducible expression of transgene To test the inducible expression of foreign genes under T7-system, a previously tested tetracycline inducible expression system in plants was used (Gatz, G, Frohberg, C. and Wendenburg, R Stringent repression and homogeneous de-repression by tetracycline of a modified CaMV 35S promoter in intact transgenic tobacco plants.

Plant J. 2: 397-404 (1992)). Foreign gene placed under a modified CaMV 35S promoter was shown to be expressed normally but repressed completely when the Tet repressor protein is co- expressed in the same plant (Gatz, C, Frohberg, C. and Wendenburg, R Stringent repression and homogeneous de-repression by tetracycline of a modified CaMV 35S promoter in intact transgenic tobacco plants. Plant J. 2: 397-404 (1992)). The foreign gene could easily be de-repressed by treating the plants/tissues with low concentrations of tetracycline. For this purpose, tobacco plants were first transformed with pBin-tetR (Fig. 3 A) to express Tet repressor protein constitutively under CaMV 35S promoter. The Nt.BintetR-1 plant that expressed high levels of tetR was re-transformed with pITB228 (Fig. 3A). For comparison, Nt.228-1 plant obtained in the previous experiments was used. Kinetics of de-repression was followed by taking leaf samples at defined time intervals from tetracycline treated (Tc-treated) and untreated plants and assayed them for the presence of uidA transcripts by- Northern blot analysis (Fig. 3C), real time polymerase chain reaction (data not shown) and for GUS activity (Fig. 3D). In the

Northern blot analysis, a single band corresponding to the expected size of uidA transcripts was detected only in the tetracycline treated sample (Fig. 2C). Reprobing the same membrane simultaneously with uidA and T7 RNAP probes revealed the presence of both uidA and T7 RNAP genes in the Tc-treated leaf. No transcripts could be detected in the untreated sample. Real time PCR experiment also confirmed the tetracycline inducible expression of GUS in the Nt.228+tetR-l plant (data not shown). The GUS activity was detected after 12 hours of Tc-ixeatment with maximum activity at 48 - 72 hours (Fig. 3E). The non-Tc-treated leaves showed very low activity (<1%) throughout the test period. In the Nt.228-1 plant, the GUS activity remained high with no significant differences between treated and un-treated samples. The GUS activity in the Nt.228-1 plant was comparable to the activity observed after 48 - 72 hours of Tc-treated Nt.228+tetR-l plant. No significant difference was observed between the Tc-treated and untreated samples from Nt.1301-1 plant. The co-expression of both T7 RNAP and uidA only after Tc-treatment coupled with the presence of GUS activity clearly demonstrate the highly regulated expression of GUS under T7-system. The results clearly demonstrate that T7 RNAP based transcription can be used for high level and tissue specific expression of foreign genes in higher plants and the transgene expression can be regulated through inducible mechanisms. These results have profound impact on biotechnological application of transgenic plants in agriculture, industry and in functional genomic studies. Experimental protocol Expression vectors The ρITB450 that contained two chimeric genes (rbcS:3A:Tl RNAP:35SpolyA and T7pro:«/^'dA:T7ter) was constructed following standard PCR and recombinant DNA techniques (Sambrook J, Fritsch EF, Maniatis: Molecular cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. - (1989)) The 2.7 kb Bglll-BamHI fragment of T7 RNAP coding region along with nuclear localization signal (NLS) from pAR3283 ° was cloned into BamHI site of plasmid pFF19²¹ to yield plasmid pFF19-T7. The BamHI site just after the carboxy terminal end of T7 RNAP was destroyed by restriction enzyme digestion and end filling by Klenow enzyme. The 800bp 35S promoter from pBI221 (Clontech) was cloned into Hindlll- Smal sites of pFF19-T7 replacing 35S promoter and 35S enhancer element to create pFF19-T7-35S. The 3.5 kb Hindlll-Ncol (blμnt ended) containing CaMV 35S:T7- RNAP:35S polyA cassette from pFF19-T7-35S was cloned into a plant transformation vector pCAMBIA1300 at Hindlll-Smal sites to create pITB239. In the next step, the PCR amplified uidA gene fragment from pFF19G (using primers forward (5'gattccatggTCCGTCCTGTAGAAACCCCA3' Seq LD 2) and reverse

(5'cgcggatccTCATTGTTTGCCf CCCTGCTG3'....Seq LD 3) was digested with Ncol- BamHI and cloned into pET14b (Novagen) in the same sites to create pET14b-GUS. The T7Pro: uidA:T7Ter gene cassette was PCR amplified from ρET14b-GUS (using primers forward (5'ggggtaccaagcttGGATCCGTCCGGCGTAGAGGATCGAGAT3' ....Seq ID 4) and reverse

(5'ggggtaccaagcttGGATCCATCCGGATATAGTTCCTCCTTTC 3 '....Seq LD 5) and cloned into Hindlll site of ρITB239 to yield ρITB250. Finally, the pITB450 was constructed from pITB250 by replacing CaMV 35S promoter with pea rZ>cS:3A promoter. The rbcS:3A promoter (Gene bank Ace. No. M21356) was PCR amplified from pea genomic DNA using primers forward

(5'ggtctagaggatccagatctGATCCAAAAGCTTGGACAGG 3 ' ....Seq ID 6) and reverse (5'ggtctagacccgggATTTTTCTCACTTCTGTATGAAT....Seq LD 7) and cloned into pITB250 at Sm l-Bglll sites to yield pITB450 (Fig. IB). The plasmid pITB441 was created from pITB450 by removing T7Pro:«/flfA:T7Ter cassette by digesting with Bglll-BamHI and religation. TheT7 RNAP:35SpolyA was replaced with w dA:35SρofyA from pFF19G using Ncol-Smal. The plasmid pITB550, pITB650 were created from pITB450 by replacing rέcS.3A promoter with kinl and corό. <5-promoters as Smal-Bglll fragments, respectively. The plasmid pITB541 and pITB641 were created from pITB441 by replacing rZ>cS.3A promoter with kinl and cor6.6 promoters as BamHI-Hindlll fragments, respectively. The plasmid pITB750 and pITB850 were created from pITB550 by replacing cor6. d-promoter with pall and pall A promoters as Smal-Bglll fragments, respectively. The plasmid pITB741 and pITB841 were created from pITB541 by replacing cor6. o^'-promoter with pall and pall A promoters, respectively. The pCAMBIA1301 vector containing GUS under CaMV 35S promoter was used for the comparison. The plasmid pBin-tetR was constructed by ligating CaMV 35S:te/R:OCter gene cassette as EcoRI - HindHI fragment into pBin-Hyg in the same sites. The pITB228 construct was created first by cloning the T7 RNAP from pITB250 was cloned as BamHI-Sall fragment into pBinHygTX in the same sites to yield pBin-Hyg-TX-T7. In the' second step, the T7Pro:wtdΑ:T7Ter gene cassette from pITB450 was cloned into HindHI site of plasmid pBin-Hyg-TX-T7 to create pITB228. Transformation The LBA4404 strain of Argrobacterium tumefaciens carrying either of the gene constructs was used to transform tobacco (Nicotianatabacum cv. Petit Havana) by leaf disc method. Particle delivery system (PDS lOOHe, BioRad) was used to transform rice. GUS analysis For the detection of GUS expression in various tissues, intact plantlets grown in in vitro or cross sectioned stems or roots were vacuum infiltrated with histochemical staining solution containing 1 mM X-Gluc (5-bromo-4chloro-3-indolyl-b-D-glucuronic acid cyclohexyammonium), 0.1 M NaH2PO4 (pH 7.0), 0.25 M ethylenediaminetetraacetic acid (EDTA), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and incubated at 37°C. After 1 - 12 hours, tissues were treated with 70% ethanol and the GUS activity was visualized under microscope. GUS activity was measured fluorometrically using ImM 4-methylumbelliferyl-β-D-Glucoronide (MUG) as substrate. Nucleic acid analysis Total genomic DNA isolated from transgenic and wild type plants was digested with relevant restriction endonucleases, resolved on 0.8% agarose gels and transferred on to nylon membrane. About 20 μg of total RNA isolated from leaf tissue was separated in denaturing formaldehyde agarose gel (1.5%) and blotted on nylon membranes. The membranes were UV crosslinked and then probed with 32P labeled GUS and T7 RNAP coding regions. Standard procedures were followed for nucleic acid hybridization. Transcription start site Primer extension was performed using preamplification kit (Invitrogen) to locate the 5' ends of uidA transcripts. Reaction was carried out with 10 μg of total RNA using the GUS internal primer (Fig. 1A). Primer was labeled with (gamma ³²P) ATP using T4 polynucleotide kinase (Promega). The size of the extension product was determined by comparison with the DNA sequence generated using the same primer and pITB450 DNA (Sequeήase II kit, USB). Induction of GUS expression

Tetracycline (Tc, l' mg/L) was used for the induction of GUS expression in detached leaves or in in vitro grown plants. Kinetics of induction was followed by real time PCR and by quantifying the GUS activity in the Tc-treated and untreated leaf samples at defined time periods.

Claims

Claims: 1. Novel RNA polymerase based transcription system for overexpression of foreign genes in a eukaryotic genome of a higher transgenic organism comprising a foreign gene placed under the control of suitable expression

' 5 signals and a corresponding modified RNA polymerase to specifically transcribe said foreign gene, both said foreign gene and said RNA polymerase being located either in cis or trans position with respect to each other in the eukaryotic genome of a higher organism, said RNApolymerase being selected from bacteriophage T5 RNA polymerase, SP6 RNA polymerase or T7 RNA

10 polymerase. 2. Novel T7 RNA polymerase based transcription system for overexpression of foreign genes in a eukaryotic genome of a higher transgenic organism comprising a foreign gene placed under the control of T7 expression signals and a modified T7 RNA polymerase to specifically transcribe said foreign gene,

15 both said foreign gene and said T7 RNA polymerase being located either in cis or trans position with respect to each other in the eukaryotic genome of a higher organism. 3. A transcription system as claimed in claim 2 wherein said T7 RNA polymerase is a bacteriophage T7 RNA polymerase.

20 4. A transcription system as claimed in claim 3 wherein said transgenic organism is a monocot or dicot plant. 5. A transcription system as claimed in claims 3 or 4 wherein said Bacteriophage T7 RNA polymerase based transcription system is in the form of a construct having a T7 promoter and terminator. 25 6. A transcription system as claimed in claim 5 wherein said T7 RNA polymerase is expressed with a nuclear localization signal (NLS) under the control of a plant tissue specific promoter to direct the polymerase to the nucleus and to place the transgene under the control of the T7 promoter and terminator in said construct; 30

7. A transcription system as claimed in any preceding claim wherein said transgene is a uidA gene (GUS).

8. A transcription system as claimed in claims 5 or 6 wherein said construct has a sequence as shown in Seq ID # 1 and in Figures 1 and 1A.

9. A transgenic plant whenever incorporating the transcription system as claimed in any preceding claim.

10. A method of overexpressing a foreign gene in a eukaryotic genome of a transgenic organism which comprises expressing a modified T7 RNA polymerase with a nuclear localization signal under the control of a plant tissue-specific promoter to direct the polymerase to the nucleus and to place the transgene under the control of a T7 promoter and a terminator.

11. A method as claimed in claim 10 wherein said T7 RNA polymerase and said transgene are located either in cis or in trans position with respect to each other.

12. A method as claimed in claim 10 or 11 wherein said modified T7 RNA polymerase is a bacteriophage T7 RNA polymerase.

13. A method as claimed in any one of claims 10 to 12 wherein said modified T7 RNA polymerase and said transgene are located in a single construct.

14. A method as claimed in claim 13 wherein said construct has a nucleotide sequence as shown in Sequence ID # 1 and in Figures 1 and 1 A.

15. Use of a modified T7 RNA polymerase in tissue-specific overexpression of foreign genes in transgenic plants.