PL162317B1 - Method for controlling transcription of a chemically controlled gene in plant material - Google Patents

Abstract

1 A method of regulating the transcription of a chemically regulatable gene in a plant material, characterized in that A / isolates a substantially pure, chemically regulatable DNA sequence from a chemically regulatable gene found in a natural system containing the gene, the way of activating RNA expression from a chemically regulated gene in this system, the isolation of this RN A, a differential screening of a genomic RN A bank generated from an RN A isolated from this activated system, as well as an RNA bank generated from RN A isolated from a control system which was not activated, isolating a genomic clone, a sub-clone of a chemically regulatable gene from that genomic clone and isolating a preset, chemically regulable DNA sequence, B / producing a chemically regulable chimeric DNA sequence containing the first, non-coding, regulate chemically component DNA sequences out isolated in step A /, having the ability to regulate the transcription of an associated DNA sequence in a growth line or plant tissue, where this regulation is dependent on a chemical regulator, and a second DNA component sequence that can be transcribed as RN A having the ability to regulate the expression of the phenotypic trait input by mechanical anti-sense, or that can be transcribed and translated in a plant or plant tissue to produce a polypeptide revealing a phenotypic trait, the first ligation route ..................... ... F ig 1 PL

Description

The present invention relates to a method for regulating the transcription of a chemically regulable gene in plant material.

Advances in recombinant DNA technology, in conjunction with the achievements in plant transformation and regeneration technology, made it possible to introduce new genetic material into plant cells, plants or plant tissues, and thus introduce new features, i.e. phenotypes, increasing the value of plants or plant tissues. Plants resistant to pathogens (European patent applications EP-A240332 and EP-A223452) or insects, produced in recent years by modified techniques of genetic engineering, [M. Vaeck et al., Nature 328, 33 (1987)] and obtaining herbicide tolerant plants [M. DeBlock et al., EMBO J. 6, 2513 (1987)] clearly demonstrate the potential for crop improvement. A wide variety of plants can be targeted for modification, from trees and shrubs to floral ornamental plants and crops. In fact, it turned out that "the crop may be a culture of plant tissue grown in a bioreactor, which is the source of a natural product.

In some cases, it would be beneficial to control the timing and / or the extent of expression of the genetic material introduced into plants, plant cells, or plant tissues. Ideally, the expression of the genetically introduced trait would be freely regulated by regulating the intermediate, which could be easily applied to crops, ornamental shrubs, bioreactors and the like. This can now be accomplished with a chemically regulable chimeric gene expression system. Such a system comprises a chemically regulated non-coding DNA sequence linked, for example, to a gene encoding a phenotypic trait such that expression of the trait is under the control of a regulator, meaning that expression of the regulated gene is determined by the presence or absence of a chemical regulator. This system is the first embodiment of the concept of chemically regulating the expression of a chimeric gene in plants or plant tissues. It allows obtaining transgenic plants and the control of the expression of the desired traits as a function of a chemical regulator.

A. Overview of plant transformation technologies.

Various methods are known for carrying out genetic transformation of plants and plant tissues (i.e., the permanent introduction of foreign DNA into plants). These include transformation by the genus Agrobacterium and transformation by direct gene transfer.

1. Agrobacterium mediated transformations.

Agrobacterium tumefaciens is the etiological factor of cancerous growths (crown gala), a disease of many dicotyledonous and gymnosperm plants, causing the formation of tumors or galli in the plant tissue at the site of infection. Agrobacterium that is normally banned

The plant at the site of injury carries a large extrachromosomal element called the Ti (tumor inducing) plasmid. Ti plasmids contain two regions responsible for tumor activity. One such region is T-DNA (foreign, transferred DNA), which is a DNA sequence that is permanently transferred into the genomic DNA of a plant. The second region required for tumor manifestation is the vir region (virulence region), involved in the transmission mechanism. Although the vir region is an absolute prerequisite for sustained transformation, the vir-DNA itself is not transferred into the infected plant. Transformation of plant cells by the use of Agrobacterium tumefaciens infection and subsequent transfer of T-DNA alone is well documented [e.g. Μ. V. Bevan and M. D. Chilton, Int. Rev. Genet. 16, 357 (1982)].

After several years of intensive research in many laboratories, the Agrobacterium system developed in such a way that it allowed for the routine transformation of many different plant tissues. Plant tissues transformed in this way include: tobacco, tomato, sunflower, cotton, rapeseed, soybean and poplar tissues. Although a large number of hosts are known to be transformed with the Ti plasmid using A. tumefaciens as the infecting agent, tobacco is the host of choice for ease of handling.

Agrobacterium rhizogenes is also used as a vector for plant transformation. This bacterium, which promotes rooting in many types of dicotyledons, carries a large extrachromosomal element called the Ri (root induction) plasmid, which functions in a manner analogous to the Ti plasmid in A. tumefaciens. The transformation with A. rhizogenes developed similarly to the transformation with A. tumefaciens. It has been used successfully to transform e.g. alfalfa, Solanum nigrum L. and poplars.

2. Direct gene transfers.

In order to transform plants or plant tissues without the use of Agrobacterium as an intermediary, a number of so-called direct gene transfer procedures have been developed. In the direct protoplast transformation technique, the entry of exogenous genetic material into the protoplast can be enhanced by the use of a chemical agent or an electric field. Such exogenous material can then be incorporated into the nuclear genome. In the early works, experiments were carried out on dicotyledonous tobacco. It turned out that the foreign DNA introduced into the plant passed to the daughter plants [e.g. J. Paszkowski et al., EMBO J. 3, 2717 (1984) and J. Potrykus et al., Mol. Gen. Genet. 199, 169 (1985)]. Protoplasts of monocotyledonous plants, e.g., Triticum monococcum, Lolium multiflorum (Italian ryegrass), maize and Mexican black sweetcorn, have also been transformed using this procedure.

Alternatively, the foreign DNA can be introduced into cells or protoplasts by micro injection. The plasmid DNA solution is injected directly into the cell using an appropriately pulled glass capillary. Alfalfa protoplasts were thus transformed with a series of plasmids [e.g. T. J. Reich et al., Bio / Technology, 4,1001 (1986)].

A relatively recent procedure for directly transforming genes is the bombardment of cells with DNA-containing microprojectiles. See T. M. Klein et al., Nature 327, 70 (1987). In this procedure, tungsten particles coated with exogenous DNA are accelerated towards the target of cells. In the described example (onion), at least transient expression was obtained.

B. Regeneration of transformed plant tissues.

As many methods are known for transforming plant tissues, there are also many methods for regenerating plants from plant tissues. The choice of a particular regeneration method depends on the starting plant tissue and on the type of plant to be regenerated. In recent years it has become possible to regenerate plant species from callus tissue derived from cuttings. Plants capable of regenerating from calluses include monocotyledons such as corn, rice, barley, wheat and rye, and dicotyledons such as sunflower, soybean, cotton, canola and tobacco.

Regeneration of plants from tissues transformed with A. tumefaciens bacteria is already carried out on many plant species, which include: sunflower, tomatoes, white clover, rapeseed, cotton, tobacco and poplar. Lucerne regeneration was also performed from tissue transformed with A. rhizogenes bacterium. A particularly useful technique is to regenerate plants from protoplasts

162 317 (D. A. Evans et al., Handbook of Plant Cell Culture, Vol. I, Ed. MacMillan Publ. Co 1983, p. 124). If a given plant species can be regenerated from protoplasts, then direct gene transformation procedures can be used. Such a transformation is not dependent on the use of A. ' tumefaciens. Plant regeneration from protoplasts is successfully carried out on rice, tobacco, rapeseed, potato, eggplant, cucumber, poplar and corn.

Advances in plant transformation and plant regeneration technologies increase the potential for plant improvement through genetic engineering. Thus, it has already been reported that genetically improved tobacco and tomato plants resist infection with e.g. tobacco mosaic virus (TMV) and are resistant to various classes of herbicides. Insect resistance has been genetically engineered into tobacco and tomato plants.

C. Cell cultures.

The possibilities for genetic engineering are not limited to arable crops, but also include ornamental plants, forage plants and trees. A less obvious goal of a plant biotechnology, encompassing both genetic engineering techniques and the application of tissue cultures, is to increase the production of plant-derived chemicals such as fragrances, dyes, natural sweeteners, industrial raw materials, antimicrobials and pharmaceuticals. In most cases, these compounds belong to a broad metabolic group, generally known as secondary products. Plants can produce such products for defense against pests, for attracting pollinators, or for combating infectious diseases.

Plant cell cultures that can be propagated in a bioreactor can be obtained from a wide variety of plant species. Most of the plants producing commercially important secondary products are of the usual species. The fact that it is possible to induce and select permanent genetic varieties derived from tissue cultures of plant somatic cells (somaclonal varieties) has been proven in experiments on many agricultural crops. The production of secondary compounds by the cultivation of plant tissues has many advantages. They include (1) the possibility of obtaining a product with higher purity, (2) the conversion of cheap precursors into expensive end products in a biotransformation process, (3) the possibility of producing new compounds by feeding substrate analogs to the culture.

D. Benefits of Controlled Gene Expression.

Whether the target of plant genetic manipulation is a crop, ornamental shrub, flower, tree, or a culture-culture to be grown in a bioreactor, the main benefit that should be achieved is the control of the expression of the chimeric gene such that that expression occurs in a bioreactor. at the right time, at the right time and only in certain situations in certain parts of the plant. For example, to obtain the desired phenotype, the chimeric gene may need to be expressed at 1 percent or more of the total protein. Such a case may be for fungal resistance resulting from chimeric gene expression and chitinase production, or for insect resistance due to increased expression of a proteinase inhibitor producing gene. In these cases, the energy expended in producing high levels of the foreign protein can harm the plant, while if the gene is expressed only when needed, e.g. if there is a danger of infection by a fungus or insect, energy losses and thus losses in productivity can be harmful. be limited.

Alternatively, if the expression of a chimeric gene encoding a phenotypic trait occurs at an inappropriate time in the plant's life cycle, this may adversely affect the plant. For example, if the product of the chimeric gene is a cleavage-inducing plant hormone, then early expression could result in the fruit being severed from the plant before the seed ripens, thus reducing yield. In this case, it would be much more advantageous to induce expression of this type of gene at a time when cutting off the pod is beneficial or less detrimental to the plant.

For tissues in a culture or in a bioreactor, the continuous production of a secondary product can lead to a reduction in the growth rate of the culture and consequently a reduction in product yield. It would be more advantageous to allow the culture to grow without expression of the secondary product and then induce the chimeric gene in a timely manner so as to obtain optimal expression of the desired product.

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Considering the above-mentioned aspects as well as others, it becomes apparent that it would be highly desirable to control the time, size and / or location of expression of a chimeric gene in plants or plant tissues. Controls that could be easily achieved in the field, in a greenhouse or in a bioreactor would be of particular commercial importance.

E. Known, regulated gene expression systems in plants.

It is known that some plant genes are induced by various internal and external factors such as plant hormones, heat shock, chemicals, pathogens, lack of light or oxygen. While few such systems are described in detail, observation of the best-characterized systems shows that increased mRNA accumulation leads to increased levels of a specific protein product.

As an example of a gene regulation by a plant hormone, mention can be made of the induction by abscisic acid (ABA) of a large amount of mRNA associated with late embryogenesis in cotton [G. A. Galau et al., Plant Mol. Biol. 7.155 (1986)]. Another example is the induction by gibberellic acid (GA3) of malate synthase transcripts in ricinus seeds and valeuronic alphaamylase isozymes in barley layers [D. Rodriguezi et al., Plant Mol. Biol. 9, 227 (1987), R. C. Nolan et al., Plant Mol. Biol, 8, 13 (1987)].

The regulation of the heat shock protein genes in soy has been thoroughly investigated. Keeping plants for several hours at 40 ° C causes de novo synthesis of certain so-called heat shock proteins [J. Key et al., Proc. Natl. Acad. Sci. USA, 78, 3526 (1981)]. Several such genes have been isolated and their regulation has been closely investigated. The expression of these genes is initially controlled at the level of transcription. The promoter of the hsp70 gene was fused with the neomycin phosphotransferase II (NptII) gene. It turned out that such a chimeric gene is induced by heat shock [A. Spena et al., EMPBO J. 4, 2736 (1985)], albeit at a lower level than endogenous heat shock genes.

Light-regulated genes are another class of genes induced in plants. These include the nuclear gene for the small subunit of ribulose-1,5-bisphosphate carboxylase (RUBISCO). [G. Morelli et al., Nature 315, 200 (1985) and L. Hererra-Estrella, Nature 310, 115 (1984)] that the 5 'flanking sequences of the RUBISCO pea gene may render the gene of interest capable of being induced by light if it is linked in a manner chimeric. This observation was valid for other light-induced genes as well, such as the chlorophyll-binding protein a / b gene.

The maize alcohol dehydrogenase (adh) genes have been extensively studied. The adhl-s gene was isolated from maize. It turned out that the 5'-flanking DNA part is capable of inducing the expression of the described chimeric gene (i.e. the chloramphenicol acetyltransferase gene, CAT), provided that the transiently transformed tissue is subjected to anaerobic conditions [E. Howard et al., Planta 170, 535, (1987)].

Recently, a group of compounds known as "protectants" have been developed to protect crops from potential damage by the use of herbicides. While the general mechanism of action of these compounds is not fully elucidated, it is assumed that one possible mechanism may be the regulation of naturally regulated genes. Recently it has been reported that higher levels of glutathione-S-transferase (SGT) are induced in 2-chloro-4-trifluoromethyl-5-methyl-thiazolecarboxylic acid benzyl ester treated maize, [R. C. Wiegand et al., Plant Mol. Biol., 7,235 (1986)]. Although an increase in GST mRNA levels has been found following treatment with the protective agent, the mechanism leading to such an increase has not been discussed.

Many plants reacting with hypersensitivity to various pathogens are stimulated to form a group of acid extractable low molecular weight proteins related to pathogenesis (PR) [L. C. Van Loon, Plant Mol. Biol. 4, 111 (1985)]. However, it is particularly interesting to observe that the same PR proteins accumulate at high levels in plants treated with chemical agents such as polyacrylic acid and acetylsalicylic acid [S. Gianinazzi et al., J. Gen. Virol. 23.1 (1974), R. F. White, Virology 99, 410 (1979)]. It turned out that there is a correlation between the presence of PR proteins and the induction of resistance against many different pathogens, both local and systemic. It has been observed to be intersspecific

162 317 tobacco hybrid resistant to tobacco mosaic virus (TMV) constitutively produces PR proteins [P. Ahli others, Plant Sci. Lett. 26, 173 (1982)]. Moreover, in vitro immunoprecipitation of translation products using mRNA from tobacco either infected with TMV or treated with a chemical indicates that the increased level of PR protein is due to RNA accumulation [B. J. C. Cornelissen et al., EMBO J., 5.37 (1986), J. P. Carr et al., Proc. Natl. Acad. Sci. USA 82, 7999 (1985)]. Thus, the induction of PR protein genes with chemicals or pathogens is a way to solve the problem of chemically regulating gene expression in plants and plant tissues.

The invention includes regulating the expression of a chimeric and / or heterologous gene in transformed plants.

The inventive method for regulating the transcription of a chemically regulable gene in a plant material consists in: a) isolating a substantially pure chemically regulatable DNA sequence from a chemically regulable gene present in a natural system containing the gene by activating RNA expression from a chemically regulated gene in this system, the isolation of this RNA, the differential screening of a genomic RNA bank generated from RNA isolated from this activated system, as well as an RNA bank generated from RNA isolated from a control system that was not activated, isolating a genomic clone, subcloning giving chemically regulate the gene from this genomic clone and isolate the desired chemically regulatable DNA sequence, B / produce a chemically regulatable chimeric DNA sequence containing the first, non-coding, chemically regulatable DNA component sequence isolated in step a / , having the ability to regulate the transcription of an associated DNA sequence in a plant or plant tissue, where this regulation is dependent on a chemical regulator, and a second component DNA sequence capable of being transcribed as RNA having the ability to regulate expression of the phenotypic trait by an anti-sense mechanism or to be transcribed and translation in a plant or plant tissue to produce a phenotypic polypeptide by ligating a first, non-coding, chemically regulatable component DNA sequence to a second component DNA sequence, c / transforming plant protoplasts, plant cells, plant tissues or whole plants transformed cells or plant tissues are selected with the help of the chimeric DNA sequence produced in step b /, using transformation methods involving Agrobacterium-mediated transformation, direct gene transfer, microimection or microprojectile bombardment, transformed cells or plant tissues are selected and / or regul a chemical agent on the plant tissue, plant or seed containing the chimeric DNA sequence of step b /.

Figure 1 shows the tobchrPR1013 restriction lambda map. Shown is a 49 kb recombinant phage genome with right and left lambda arms. Shown below the lambda phage genome map is an enlarged 19 kb long tobacco insert. The location of the PR-la gene is indicated by the grid and the direction of transcription is indicated by the arrow. P = Pst, H = Hind III, C = Body, R = EcoRI.

Figure 2 shows the construction of plasmid pBS-PR013Cla from lambda phage tobchrPR1013. The 19 kb fragment between the two Bodies sites is subcloned into the "bluescript plasmid." C = ClaI, LGT Agarose = Low Gelling Point Agarose.

Figure 3 shows the construction of the plasmid pBS-PR 1013Eco from lambda tobchrPR 1013. The 3.6 kb EcoRI fragment containing the PR-1A gene from lambda tobchrPr1013 is subcloned into the plasmid "bluescript. R = EcoRI, LGT Agarose = Low Gelling Point Agarose.

Figure 4 shows the construction of the pBS-PR1013Eco plasmid from pBS-PR1013Cla. The 3.6 kb EcoRI fragment containing the PR-Ia gene is subcloned into the EcoRI site of the plasmid "bluescript C = ClaI, R = EcoRI, LGT agarose = low gelling point agarose.

Figure 5 shows the construction of the pBS-PR 1013EcoAPst plasmid from pBS-PR 1013Eco. The 600 bp PstI fragment is removed from pBS-PR 1013Eco. P = PstI, R = EcoRI, X = XhoI, S - Sal, LTG agarose = low gelling point agarose.

Figure 6 shows the construction of pBS-PR1013EcoAPstAXho from pBS-PR1013EcoAPst. A 2 kb XhoI fragment is removed from plasmid pBS-PR1013EcoAPst. R = EcoRi, P = PstI, X = XhoI, S = SalI, LTG agarose = low gelling point agarose.

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Figure 7 shows the construction of plasmid pCIB270 from pBI 101.3 and pBSPR1013EcoAPstAXho. The Pstl-XhoI fragment, containing part of the PR-1α gene and the 5 'flanking sequence, is subcloned into pBI 101.3 previously digested with Sall-BamHI. The Xho and SalI sites are compatible with each other and both restriction sites are removed during ligation. The Pstl site is adapted to a BamHI site using a molecular adapter. X = XhoI, P = PstI, (X / S) = Xhol and Sal fusion site. The sites (X / S) will not be consumed by any enzyme. LB = left arm of T-DNA, RB = right arm of T-DNA. The direction of transcription from the inducible region of PR-1? In plasmid pCIB270 is shown by the arrow. The bars on the pCIB270 plasmid represent a 3 'percent position in the NOS gene. The shaded area in pCIB270 represents the beta-glucuronidase gene. The dotted box in pCIB270 represents the neomycin phosphotransferase II gene. The dashed box represents the NOS promoter.

Figure 8 shows the construction of the plasmids M13mpl8-PR1013EcoAPstAXho and M13mpl9-PR1013EcoAPstAXho. The PstI-Asp718 fragment, containing part of the PR-1? Coding sequence and the 5 'flanking sequence, is subcloned from pBS-PR1013EcoAPstAXho into M13mpl8 or 19 previously digested with the enzymes Asp718 and PstI. R = EcoRI, H = HindIII, P = Pstl, K = KpnI (Asp718 is the KpnI isoschizomer), LGT agarose = low gelling point agarose.

Figure 9 shows a schematic of the conversion of the ATG codon from PR-1? To an NcoI site. Single-stranded DNA from M13mpl8-PR1013EcoAPstAXho is marked with a solid line. The TCATGG sequence is converted to the CCATGG sequence by site-directed mutagenesis. K = KpnI, X = XhoI, B = BstEII, P = PstI.

Figure 10 shows the construction of the plasmid pCIB268. A BstEII-Pstl fragment derived from the replicating form M13mpl8-PR1013EcoAPstAXho.Nco is subcloned into pBSPR1013EcoAPstAXho previously digested with BstEII-Pstl to form pCIB268. X = XhoI, B - BstEii, N = NcoI, P = Pstl.

Figure 11 shows the construction of pCIB269 from pBS-GUS 1.2 and pCIB268. X = Xhol, N = NcoI, P = PstI. The square box in plasmid pCIB269 represents the GUS gene sequences and the shaded box represents sequences derived from the PR-1? Gene.

Figure 12 shows the construction of the pBS-GUS1.2 plasmid. The plasmid pBS-GUS1.2 was obtained by three-step ligation from fragments derived from pRAJ265, pBI221.1 and the plasmid "bluescript." S - Sali, R = EcoRI, N = Ncol.

Figure 13 shows the construction of plasmid pCIB271 from pCIB269 and pCIB200. X = Xhol, N = Ncol, R = EcoRI, S = Sali, (S / X) = fusion of Sal and Xhol sites, LGT agarose = low gelling point agarose.

Figure 14 shows the restriction map of the pCIB219 plasmid. This plasmid is constructed by adding an EcoRI / XhoI adaptive fragment to the XhoI / EcoRi fragment of pCIB269 containing the PR-1 and GUS genes and then ligating into SalI digested pCIB712.

Figure 15 shows the restriction map of the plasmid pCIB272. This plasmid is constructed by ligating the Asp718I / BamHI fragment from pCIB282 containing the GUS PR-1 gene (-833 to + 1 from PR-1 A) with the pCIB200 plasmid previously digested with Asp7181 / BamHI.

Figure 16 shows the restriction map of the plasmid pCIB273. This plasmid is contructed by ligating the Asp7181 / BamHI fragment from pCIB283 containing the GUS PR-1 gene (-603 to + 1 from PR-Ia) to the Asp7181 / BamHI digested plasmid pCIB200.

Figure 17 shows the restriction map of pCIB1004. This plasmid was constructed by ligating the XhoI / NcoI fragment from pCIB269 (containing the PR-Ia promoter) with the BT gene deleted from pCIB10 (35Bt / 607) as the NcoI / BamHI fragment and Sall / BamHI digested pCIB710.

Figure 18 shows the restriction map of pCIB200 / PR1-BT. This plasmid is constructed from pCIB1004 and pCIB200.

Figure 19 shows the restriction map of pCIB1207. A 5.8 kb XbaI fragment from a genomic lambda clone containing the Arabidopsis AHAS gene is cloned into an Xbal digested bluescript plasmid.

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Figure 20 shows the restriction map of pCIB 1216. A 3.3 kb NcoI / Xbal fragment from plasmid CIB1207 is cloned into pCIB269 previously cleaved with NcoI and Xbal to remove the GUS gene.

Figure 21 shows the restriction map of pCIB1233. A 4.2 kb Kpnl / XbaI fragment is isolated from plasmid pCIB 1216 and ligated to pCIB200 previously digested with the enzymes KpnI and XbaI.

Figure 22 shows the restriction map of pBSGluc39.1-GUS. The 1462 bp fragment from pBSGluc39.1 is cloned into BS-GUS1.2 previously digested with the enzymes NcoI and KpnI.

Figure 23 shows the restriction map of pCIB200 / Gluc39.1-GUS. The Kpnl / Xbal fragment containing the β-glucanase promoter and the GUS gene is isolated from the pBSGluc3 9.1 / GUS plasmid, and ligated with pCIB200 previously digested with KpnI and Xbal.

Figure 24 shows the restriction map of pCIB200 / GIuc39.1-BT. The ligated KpnI / NcoI fragment from pCIB 1004 containing the BT gene, the KpnI / NcoI fragment from pBSGluc39.1 / GUS and pCIB200 cut with the KpnI enzyme and treated with calf thymus alkaline phosphatase.

Figure 25 shows the restriction map of pBSGluc39.1 / AHAS constructed from the NcoI / Xbal fragment of pBSGluc.39.1 / GUS and the 3.3 kb NcoI / Xbal fragment of pCIB1207 containing the AHAS gene.

Figure 26 shows the restriction map of pCIB200 / Gluc39.1-AHAS. The Kpnl / XbaI fragment containing the promoter-glucanase was isolated from pBSGluc39.1 / AHAS and ligated to pCIB200 previously cleaved with the enzymes KpnI and Xbal.

Figure 27 shows the restriction map of pBSGLuc39.3 / GUS. The 1677 bp fragment from pBSGluc39.3 is cloned into pBS-GUS1.2 previously digested with NcoI and KpnI.

Figure 28 shows the restriction map of pCIB200 / Gluc39.3-GUS. From the pBSGluc3 9.3 / GUS plasmid, the Kpnl / Xbal fragment containing the j3-glucanase promoter and the GUS gene is isolated and ligated with pCIB200 digested with KpnI and Xbal

Figure 29 shows the restriction map of pCIB200 / Gluc39.3-BT. The ligated KpnI / NcoI fragment from pCIB 1004 containing the BT gene, the KpnI / NcoI fragment from pBSGluc39.3 / GUS and pCIB200 previously digested with KpnI and treated with calf thymus alkaline phosphatase.

Figure 30 shows the restriction map of pBSGluc39.3 / AHAS constructed from an NcoI / Xbal fragment, pBSGluc39.3 / GUS and a 3.3 kb NcoI / Xbal fragment from pCIB1207 containing the AHAS gene.

Figure 31 shows the restriction map of pCIB200 / Gluc39.3-AHAS. The Kpnl / Xbal fragment containing the promoter / 3-glucanase and the AHAS gene is isolated from pBSGluc39.3 / AHAS and ligated to pCIB200 digested with KpnI and Xbal.

Figure 32 shows the restriction map of pCIB 1208. A 5.8 kb XbaI fragment from the lambda genomic clone containing the mutant Arabidopsis AHAS gene is cloned into XbaI digested bluescript ¹¹ plasmid.

Figure 33 shows the restriction map of pCIB 1230. A 3.3 kb NcoI / Xbal fragment from plasmid pCIB 1208 is cloned into pCIB269 previously cleaved with NcoI and XbaI to remove the GUS gene.

Figure 34 shows the restriction map of pCIB1232. A 4.2 kb Kpnl / XbaI fragment is isolated from pCIB1230 and ligated to pCIB200 previously cut with KpnI and Xbal.

Figure 35 shows the restriction map of pBSGluc39.1 / AHAS-SuR constructed from the NcoI / Xba I fragment of the pBSGluc39.1 / GUS plasmid and the 3.3 kb NcoI / Xbal fragment derived from pCIB 1208 containing the AHAS gene.

Figure 36 shows the restriction map of pCIB200 / Gluc39.1-AHAS-SuR. A Kpnl / Xbal fragment containing the β-glucanase promoter and the AHAS gene is isolated from pBSGluc 39.1 / AHAS-SuR and ligated to KpnI and Xbal cut pCIB200.

Figure 37 shows the restriction map of pBSGluc39.3 / AHAS-SuR constructed from a NcoI / Xbal fragment derived from pBSGluc39.3 / GUS and a 3.3 kb NcoI / Xbal fragment derived from pCIB 1208 containing the AHAS gene.

Figure 38 shows the restriction map of pCIB200 / Gluc39.3 / AHAS-SuR. The Kpnl / XbaI fragment containing the / 3-glucanase promoter and the AHAS gene is isolated from the pBSGIuc39.3 / AHASSuR plasmid and ligated to pCIB200 previously digested with KpnI and Xbal.

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Figure 39 shows the genomic DNA sequence of the 2kb fragment between the Xho I and Bg 1II sites of the tobacco PR-1 gene. The arrow at nucleotide 903 indicates the transcription start site and the vertical arrows at nucleotides 207 and 313 represent the ends of two independent cDNA clones. The polypeptide sequence encoded by the coding portion of this gene is also disclosed.

Figure 40 shows the genomic DNA sequence of the tobacco PR-1 gene and the amino acid sequence of the polypeptide encoded by the coding region of that gene.

Figure 41 shows the cDNA sequence for a cucumber PR chitinase and the amino acid sequence of the polypeptide encoded by the coding region of that gene.

Figure 42 shows the cDNA sequence of a larger form of the tobacco PR-R gene and Figure 42a shows the sequence of Figure 42 and the amino acid sequence of the polypeptide encoded by the coding region of this gene.

Figure 43 shows the genomic DNA sequence of the tobacco basic β-1,3-glucanase gene contained in the pBSGluc39.1 clone.

Figure 44 shows the genomic DNA sequence of the tobacco basic / 3-1,3-glucanase gene contained in the pBSGluc39.3 clone.

Figure 45 shows the tobacco PR-Q cDNA sequence contained in plasmid pBSchtl5.

Figure 46 shows the DNA sequence of the isolated cDNA contained in the pBSGI6e plasmid. This cDNA encodes the acid form of β1,3-glucanase.

The present specification describes the isolation, cloning and identification of DNA sequences having the ability to regulate the transcription of an associated DNA sequence in plant tissues, where regulation is dependent on a chemical dredger. Such DNA sequences may be used to construct chimeric genes in which expression of the gene can be regulated by such regulators. The ability to regulate the expression of a chimeric gene in transgenic plants by a chemical method is applicable to obtaining the appropriate expression of the phenotypic trait with minimal adverse effect on plant growth and development. This type of regulation is important in the production of secondary products or other products cloned in plant tissue cultures or in bioreactors. Regulating a cloned sequence is also important for regulating the products of another gene by an anti-sense mechanism.

The expression of a given coding sequence at a particular, particular time can be regulated using a chemical regulator generally applied to plant tissue. Genes involved in the control of certain intermediate stages of development may also be regulated by linking a chemically regulated DNA sequence to an appropriate DNA coding sequence. In this way, by increasing or decreasing the level of the chemical regulator, it is possible to stop the growth of a plant at a certain stage or to accelerate it to a certain rate.

Chemically regulable DNA sequences can be used to increase the expression of foreign genes, e.g., impart resistance or tolerance to herbicides (e.g., atrazine tolerance in soybean), render insect resistance (e.g., Bacillus thuringiensis crystal protein in cotton), or require selective expression (for example, female or male infertility). Chemically regulable DNA sequences may also find use to increase the transcription of such DNA sequences which will control the expression of the second DNA sequence by an anti-sense mechanism.

Accordingly, the invention relates to a method of chemically regulating the expression of DNA coding sequences associated with chemically regulable DNA sequences, e.g., to regulate a phenotypic trait.

For a clear and unambiguous understanding of the description of the invention and the claims, definitions of the terms used are provided below.

An anti-sense mechanism. A gene regulatory mechanism based on controlling the rate of translation of mRNA into protein due to the presence in the cell of an RNA molecule complementary to at least a portion of the mRNA translated within the cell.

Related DNA sequence: A DNA sequence whose cellular activity (1) either regulates the activity of another DNA sequence, (2) or is regulated by another DNA sequence. This definition especially includes (but is not limited to) sequences that are contiguous in a continuous strand of DNA

162 317 or are physically separate from each other. Physical separation includes, e.g., separation within the same DNA strand, localization in different DNS, or staggered discrete sequences (e.g., alternating between regulating and coding sequences) on a single strand.

Chemically regulated DNA sequence: A DNA sequence that has the ability to regulate the transcription of an associated DNA sequence, where regulation is dependent on a chemical regulator. These sequences may be of natural or synthetic origin.

Chemically regulated gene: A gene that contains at least one non-coding chemically regulated DNA sequence and at least one associated DNA sequence. These genes may be of natural, synthetic or partially natural (synthetic) origin.

Chemical regulator: (for chemically regulatable DNA sequence). Elemental or molecular particles that control (initiate, terminate, increase or decrease), by direct or indirect action, the activity of a chemically regulable DNA sequence in a system where the chemical regulator is not normally present in an active form, in sufficient quantity to regulate transcribing a transcribed DNA sequence associated with a chemically regulated DNA sequence to the desired extent and at the desired time. This terminology includes situations where, when transcription is desired, the regulator is either absent or negligible, or where there is an amount of regulator but up or down regulation is required to effect transcription.

Thus, when the system containing a chemically regulable DNA sequence is a plant, e.g. a transgenic plant, then the chemical regulator is a substance not naturally occurring in the plant in an amount sufficient to chemically regulate and therefore transcribe the related gene to the desired extent and time.

The term "direct action" means that the action of a chemical regulator is the result of a direct interaction between the chemical regulator and the DNA sequence. The term "indirect action" means that the action of the regulator is the result of a direct interaction between that chemical regulator and some other endogenous or exogenous factor in the system. The ultimate result of this direct interaction is the activation or suppression of DNA sequence activity. The term "active form" means that the chemical regulator is in the form required to obtain control.

Chimeric sequence or chimeric gene: A DNA sequence that includes at least two heterologous portions, that is, portions derived from naturally occurring DNA sequences that are not related in their states of natural occurrence, or that contain at least one portion of synthetic origin not found in nature.

DNA coding sequence: A DNA sequence from which a cellular polypeptide is generated by its transcription and translation.

Gene: A secreted region of the chromosome responsible for a specific cell product.

Non-coding DNA sequence: A DNA sequence that is not transcribed or translated to produce a cellular polypeptide when it is associated with a given DNA sequence. Thus, a sequence that is a non-coding sequence in association with one coding sequence may become a coding sequence if it is associated with another coding or non-coding sequence.

Phenotypic trait: A noticeable property that results from the expression of a gene.

Plant tissue: Any plant tissue in a plant or culture. The term includes, but is not limited to, plants, plant cells, plant organs, seeds, protoplasts, calluses, cell cultures, and any groups of plant cells organized into structural or functional units. The use of this term, either alone or together with a particular type of plant tissue mentioned above or covered by this definition, is not intended to exclude another type of plant tissue.

PR or pathogenesis-related proteins: Proteins produced in plants that are responsive to pathogen hypersensitivity. The term includes the acidic and basic forms of the proteins, tobacco PR-1?, PR-1b, PR-1c, PR-1 ', PR-2, PR-N, PR-O, PR-P, PR-Q, PR-S , PR-R, chitinase, equivalent to PR-Plub PR-Q and beta-1,3-glucanase (glucan-endo-1,3 / 3-glucosidase, EC3.2.1.39), which

162 317 is equivalent to PR-2, PR-N or PR-O as well as pathogen-induced cucumber chitinase, but not limited to these proteins. The hypersensitivity reaction is characterized by tissue necrosis at the site immediately surrounding the site of pathogen infection and subsequent localization of the pathogen, which differs from a sensitivity reaction in which the pathogen spreads throughout the plant. The pathogens are viruses or viroids, e.g. e.g., Pseudomonas siringae or Pseudomonas tabaci, or masks, e.g. Myzus Persicae. This is not an exhaustive list by any means.

Regulation: Increasing (inducing) or reducing (repressing) the level of gene expression or the level of transcription of a DNA sequence. This definition does not include any specific mechanism.

Substantially pure DNA sequence: A DNA sequence isolated in substantially pure form from a natural or non-natural source. Such a sequence may be found in a natural system such as a bacterium, virus or plant, or an animal cell, or may be obtained synthetically or as a cDNA sequence. Substantially pure DNA sequences are generally isolated as a vector containing the specific DNA. The term "substantially pure" means that non-desired sequences are present in very low amounts, e.g., less than 5%, less than 1%, or preferably, less than 0.1%. Substantially pure DNA sequences and vectors containing them can and usually are maintained in a solution, e.g., an aqueous solution containing buffers, or in a culture medium.

Substantial sequence homology: The term "substantial sequence homology" means close structural links between nucleotide or amino acid sequences. For example, substantially homologous DNA sequences may have a homology of 80%, preferably 90%, even 95%, and substantially homologous amino acid sequences may generally be 50% or greater. Furthermore, linkages in which one or more nucleotide or amino acid sequences are omitted or sequences with additional nucleotides or amino acids are inserted are considered homologous.

A. Chemically regulated DNA sequences.

Non-coding DNA sequences have been described which, when influenced by a chemical regulator, are capable of regulating the transcription of an associated DNA sequence in a plant or in plant tissue. They are especially non-coding DNA sequences having the ability to regulate the transcription of an associated DNA sequence in a plant or in plant tissue, in which regulation is dependent on a chemical regulator. Preferably, the DNA sequences exist in substantially pure form for the gene or genome in which they are derived from a natural source, or for a mixture of DNA if present in a synthetic mixture.

Preferably, the non-coding chemically regulable DNA sequences are those sequences which, in association with a DNA coding sequence, regulate the expression of that coding sequence in plant tissue, the degree of the regulation being dependent on a chemical regulator. Such sequences may be de novo synthesized or may be derived (e.g., may be isolated or cloned) from a naturally occurring and chemically regulated gene. The presence of chemically regulable DNA sequences is not limited to plant tissues. Such chemically regulable sequences can be derived from a variety of natural sources, such as bacterial, viral and animal sources. For example, they can be isolated from the 5 'flanking region or from the 3' flanking region of a naturally occurring chemically regulable gene. Alternatively, the non-coding DNA sequence can be chemically synthesized or enzymatically obtained as cDNA identical to the isolated sequence or having substantial homology to the sequence. By cloning a chemically regulated non-coding DNA sequence, the sequence can be separated from other adjacent sequences in the naturally occurring gene. In this way, an essentially pure DNA sequence can be obtained.

According to the invention, regulation occurs under the influence of chemical regulators which are either inductive or repressive regulators. Examples of repressed genes

162 317 ie genes inhibited by a repressing chemical are, for example, TrpR or AroH genes. The addition of a tryptophan repressor or tryptophan alone inhibits the expression of these genes. There are many other genes regulated by this type of end product repression, and in any case the end product acts as a chemical regulator that can be used to inhibit gene expression. The regulable sections of such genes can be part of chimeric constructs that can be chemically regulated, thereby regulating the transcription of related DNA sequences in a plant or plant tissue.

Examples of chemically inducible genes, i.e. those induced by an inducing chemical regulator, are PR protein genes, especially tobacco PR protein genes, e.g. PR-1a, PR-1b, PR-1c, PR-, PR-Q, PR -R and PR-S, cucumber chitinase gene and tobacco alkaline and acid / 3-1,3-glucanase genes. In particular, substantially pure DNA sequences that are chemically controllable non-coding DNA sequences or DNA sequences substantially homologous to such sequences that are part of a naturally occurring chemically regulated gene, e.g. naturally occurring chemically regulable genes in plants and plant tissues of monocotyledonous, dicotyledonous or gymnospermous plants. Preferably, said DNA sequence has the ability to regulate the transcription of a related DNA sequence in a plant or in a plant tissue, wherein said sequence related to it is a coding sequence. Transcription of this DNA sequence can be regulated by a repressive or inducing chemical regulator. Particularly preferred DNA sequences are those in which the chemically regulated gene is a PR protein gene, e.g. from dicots such as tobacco or cucumber. Most preferred are DNA sequences in which the chemically regulated gene is the tobacco PR-1α, PR-1b, PR-1c, PR-T, PR-Q or PR-R gene, the cucumber chitinase gene, or the alkaline or acidic gene / 3 -1,3-glucanases, especially the tobacco PR-1α and PR-gen gene, but also the cucumber chitinase gene and the tobacco alkaline and acidic / 3-1,3-glucanase genes. Primarily considered herein are DNA sequences in which the chemically regulated gene is the PR-1α gene or the tobacco basic / 3-1,3-glucanase gene.

The chemically inducible DNA sequence of the preferred PR and related genes appeared to be present in the coding sequences of an adjacent 5 'region flanking the coding sequences. For example, in a sequence isolated from a fragment of the 6500 bp tobacco PR-la gene containing part of the coding region, a region of about 900-1200 bp was found naturally adjacent to the transcription initiation site and chemically inducible. This feature remains in the fragment containing approximately 500-700 bp adjacent to the transcription start site. Thus, DNA sequences located in the 5 'flanking region of the PR or related genes, e.g. 1200 base pairs adjacent to the transcription start site are most preferred.

A non-coding DNA sequence having the ability to regulate the transcription of an associated DNA sequence in a plant or plant tissue, this regulation being dependent on a chemical regulator, and DNA sequences substantially homologous to this non-coding sequence are prepared such that the DNA is isolated from a gene found in in its essentially pure form, or it is synthesized by chemical or enzymatic means.

In particular, a substantially pure, chemically regulated DNA sequence from a chemically regulated gene in a naturally occurring system containing that gene is made such that (a) the system activates RNA expression from a chemically regulated gene, ( b) this RNA is isolated, (c) a genomic bank of RNA generated from RNA isolated from such an activated system is screened, as well as RNA generated from RNA isolated from a control system that has not been activated, (d) a genomic clone is isolated, ( e) the chemically regulable gene is subcloned from the genomic clone; and (f) the desired chemically regulable DNA sequence is isolated.

If the natural system is a plant, a chemically regulated DNA sequence is also obtained (a) the plant activates the synthesis of polyA + RNA from a chemically regulable gene, (b) this polyA + RNA is isolated, (c) a cDNA bank from this polyA + RNA is made, (d) this cDNA bank is screened for cDNA generated from RNA derived from control plants, not activated, (e) cDNA clones are isolated that give rise to

162 317 be chemically regulated, (f) isolating a genomic clone from the genomic bank of this plant using the cDNA clone from step (e) as a probe, (g) subcloning a chemically regulated gene from that genomic clone, and (h) isolating the desired chemically regulable DNA sequence.

A method is preferred in which the chemically regulated gene is a chemically inducible gene, e.g. a PR protein gene, thus the genes PR-1a, PR-1b, PR-1c, PR-, PR-Q or PR- Tobacco R, cucumber chitinase gene, or tobacco alkaline or acid / 3-1,3-glucanase gene. Furthermore, a method in which the PR protein gene is activated in step (a) by a chemical inducer or pathogen is preferred.

B. Chemically adjustable DNA sequences with portions of the naturally occurring coding sequences.

In addition to the fully non-coding, chemically regulable DNA sequences described in Part A, non-coding DNA sequences of naturally occurring, chemically regulable DNA sequences have also been obtained in association with some but not all of the coding sequence with which the regulatable sequence is associated in a naturally occurring gene. More particularly, they are, preferably in substantially pure form, DNA sequences which include a first DNA component sequence which is a non-coding, chemically regulable DNA sequence of a naturally occurring chemically regulable gene (or a sequence substantially homologous to that sequence). wherein the first component sequence is capable of regulating the transcription of an associated DNA sequence in a plant or plant tissue, where regulation is mediated by a chemical regulator, and the second DNA component sequence being part (but not the entire sequence) of the DNA sequence being transcribed or displaying substantial homology to that part with which the first component sequence is associated in a naturally occurring chemically regulable gene. The naturally occurring chemically regulated gene may be a gene of plant origin, for example found in monocotyledonous or dicotyledonous plants, and may be regulated by a repression or induction chemical regulator. The second component DNA sequence is generally a coding sequence. A preferred second DNA sequence is that encoding the signal peptide of any protein produced by the expression of a naturally occurring chemically regulable gene.

In particular, DNA sequences are described, comprising a first non-coding, chemically regulatable DNA sequence and a second coding DNA sequence from a PR protein gene, e.g. from a PR protein gene of dicotyledons, preferably from tobacco or cucumber, such as PR-1a, PR-1b. , PR-1c, PR-, PR-Q or PR-P, the chitinase gene, or from the alkaline or acid / 3-1,3-glucanase gene. Preferred is a DNA sequence from a PR protein gene in which transcription is regulated by an inducing chemical regulator. In particular, DNA sequences are described wherein the first non-coding sequence is located in the flanking region of one of the aforementioned PR protein genes, e.g., is located at about 2000 base pairs adjacent to the transcription start site of the PR protein gene. Most preferred is a DNA sequence comprising the above-mentioned first non-coding sequence and the second signal peptide coding sequence mentioned above. '

The substantially pure DNA sequences shown in Figures -9, 40, 43 and 44 and substantially pure DNA sequences substantially homologous to these sequences are most preferred. These sequences are examples of chemically controllable DNA sequences containing a first non-coding and a second coding DNA sequence. They are derived from the PR-1α and Pr-1 for the tobacco PR protein and from the two forms of basic tobacco β-1,3-glucanase, respectively. Figure 39 shows the sequence of a representative tobacco PR-Ia gene. Nucleotides 1 to 1150 are a chemically inducible 5 'non-coding flanking DNA sequence and part of the PR-1α coding sequence that is present in tobacco plants. In this case, the chemically inducible component is adjacent to the coding sequence. The nucleotides encoding the first thirty amino acids constitute the PR-1? Protein signal peptide encoding sequence. The coding sequence can be separated from the non-coding sequence to provide a non-coding, chemically inducible DNA sequence free of any coding sequence described above in section A. Such separation can be accomplished by known techniques such as restriction enzyme digestion.

162 317

A method of producing a DNA sequence comprising a first component DNA sequence that is a non-coding, chemically-regulatable DNA sequence of a naturally occurring chemically regulated gene (or a sequence substantially homologous to that sequence), the first component sequence having the ability to regulate associated transcription therewith. DNA sequence in a plant or in plant tissue, where regulation is dependent on a chemical regulator and a second DNA component sequence being part of (or having substantially homology to that sequence) the transcribed DNA sequence (but not all such sequence) to which the first is related a component sequence in a naturally occurring chemically regulable gene consists in isolating DNA in substantially pure form from the naturally occurring gene or synthesizing this DNA chemically or enzymatically. Preferably, the methods described in Part A above and substantially pure DNA sequences obtained by these methods are provided.

C. Chimeric genes containing chemically regulated DNA sequences.

A chimeric DNA sequence (chimeric gene) contains at least one chemically regulable DNA sequence. Two types of such chimeric sequences serve as examples. A simpler or two-part chimeric DNA sequence comprises a chemically regulable DNA sequence and a transcribed DNA sequence. Such a chimeric gene is capable of being expressed in plant tissue under appropriate chemical regulation conditions. In particular, the chemically regulable chimeric DNA sequence comprises a first, non-coding, chemically regulable, DNA component sequence capable of regulating the transcription of an associated DNA sequence in a plant or plant tissue, this regulation being dependent on a chemical regulator and a second , a component DNA sequence that is transcribed in a plant or plant tissue. The component DNA sequences may be derived from natural sources or may be produced synthetically.

Transcription of the second DNA sequence may result in the generation of RNA capable of regulating expression of the phenotypic trait by an anti-sense mechanism. Alternatively, the second DNA sequence in the chimeric DNA sequence can be transcribed and translated in plant tissue, yielding in part A above and substantially pure DNA sequences obtained by these methods.

C. Chimeric genes containing chemically regulated DNA sequences.

Included within the scope of the invention is a chimeric DNA sequence (chimeric gene) comprising at least one chemically regulable DNA sequence. Two types of such chimeric sequences serve as examples. A simpler or two-part chimeric DNA sequence comprises a chemically regulable DNA sequence and a transcribed DNA sequence. Such a chimeric gene is capable of being expressed in plant tissue under appropriate chemical regulation conditions. Particularly within the scope of the invention is a chemically regulable chimeric DNA sequence comprising a first non-coding chemically regulable DNA component sequence capable of regulating the transcription of an associated DNA sequence in a plant or plant tissue, this regulation being dependent on the regulator chemical and a second, component DNA sequence transcribed in the plant or in plant tissue. The component DNA sequences may be derived from natural sources or may be produced synthetically.

Transcription of the second DNA sequence may result in the generation of RNA capable of regulating expression of the phenotypic trait by an anti-sense mechanism. Alternatively, the second DNA sequence in the chimeric DNA sequence can be transcribed and translated in plant tissue, resulting in the polypeptide and consequently a phenotypic trait. The chimeric gene is constructed such that the second DNA sequence is suitably associated with a chemically regulated DNA sequence, generally in an adjacent orientation, which ensures transcription. Linking is carried out by known techniques.

A chimeric DNA sequence comprising a first non-coding chemically regulated DNA component and a second transcribable DNA component is preferred, wherein the first non-coding DNA sequence is one of the preferred DNA sequences described above in section A. First DNA component sequence it can be regulated by a repressive or inducing chemical regulator. A particular sequence is a chimeric DNA sequence in which the first component DNA sequence has substantial homology

162 317 to a chemically regulated DNA sequence in a chemically regulated gene naturally occurring in the plant.

A chimeric DNA sequence is preferred in which the first component DNA sequence is a chemically controllable DNA sequence (or a sequence substantially homologous to that sequence) contained in a naturally occurring, chemically regulated gene from a plant and the second DNA component sequence is the sequence. coding subject to transcription and translation to produce a polypeptide resulting in a phenotypic trait. Preferably, this second DNA component sequence is adjacent to the first DNA coding sequence. Particularly preferred is a chimeric DNA sequence in which the first component DNA sequence is (or has substantial homology thereto) a chemically induced DNA sequence in a gene for a PR protein, e.g. a PR protein from dicotyledons such as tobacco or cucumber. Examples are listed in Part A.

A second exemplary type of chimeric DNA sequence comprises three component DNA sequences derived from two or more sources. In the simplest embodiment, such a chimeric DNA sequence comprises a two-part DNA sequence as described in section B above and a third DNA sequence derived from at least one different source. More specifically, this type of chimeric DNA sequence comprises a first DNA component sequence that is a non-coding, chemically controllable DNA sequence (or a sequence substantially homologous to that sequence) of a naturally occurring chemically controllable gene, which first component DNA sequence is capable of regulating the transcription of an associated DNA sequence in a plant or plant tissue, where regulation is dependent on a chemical regulator, a second DNA component sequence (or a sequence essentially homologous to that sequence) being part of (but not all of) the DNA sequence being transcribed; to which is associated the first component sequence in a chemically regulated gene occurring in a natural state, and a third, component DNA sequence transcribed in a plant or plant tissue. Preferably, the chemically controllable naturally occurring gene is a plant gene.

The second and third DNA component sequences are generally coding sequences. The third component sequence may be from more than one natural or synthetic source. The first two DNA component sequences will typically be from a natural source. If the source is a plant, it is a monocotyledonous, dicotyledonous or gymnosperm plant. In a preferred embodiment, the second DNA sequence comprises a nucleotide sequence encoding a signal peptide from a chemically regulable gene in which the first two DNA sequences are present. If a chemically regulable DNA sequence is associated with a part of the coding sequence of the gene from which it is derived, the third DNA sequence must exist not only in the correct orientation but also in reading phase compatible with the second DNA sequence. This orientation can be achieved by known techniques.

Chimeric DNA sequences in which the first and second component sequences are the preferred sequences described in section B above are preferred. For example, in a preferred chimeric DNA sequence, the first component DNA sequence is a chemically inducible DNA sequence in a PR protein gene from a dicotyledonous plant, e.g., tobacco or cucumber, or a sequence substantially homologous to the above sequence. Examples of PR protein genes are given above.

The chimeric genes described above include many possible constructs. The chemically regulatable mecoding sequence may be linked to a gene that controls fruit flowering or ripening, a gene that affects herbicide tolerance or resistance, or many types of pests such as fungi, viruses, bacteria, insects, roundworms, or arachnids, a gene that controls enzyme production or secondary metabolites, male or female sterility gene, growth inhibition gene, odor, nutritional quality gene, etc. Thanks to the described chimeric genes, these traits can be enhanced by the farmer or the gardener. They no longer have to be dependent solely on natural factors. Of particular interest is the control of the phenotypic trait in the production of metabolites in tissue culture or in a bioreactor.

162 317

A preferred chimeric DNA sequence is a two- or three-component sequence in which the component coding sequence encodes a phenotypic trait, e.g., herbicide, fungus, virus, bacterial, insect, roundworm or arachnid tolerant or resistant trait, secondary metabolite production, male sterility or female, producing an enzyme or other compound of interest. A two- or three-component chimeric DNA sequence is particularly preferred. in which the component coding sequence codes for herbicide tolerance or resistance, e.g., for a wild-type or herbicide-resistant acetohydroxy acid synthetase (AHAS), or in which the coding sequence encodes for insect resistance, e.g. for Bacillus thuringiensis (BT) endotoxin resistance ).

If a chimeric sequence is to be used to designate chemical regulators then the phenotypic trait is a determinable marker. Suitable markers include, but are not limited to, luciferase (LUX), chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (NPT), nopaline synthase (NOS), octopine synthase (OCS), / 3-1,3 glucuronidase (GUS) , acetohydroxy acid synthase (AHAS) and Bacillus thuringiensis endotoxin (BT). Preferred markers are / 3-1,3-glucuronidase (GUS), acetohydroxy acid synthase (AHAS) and Bacillus thuringiensis endotoxin (BT). A representative example of such a chimeric DNA sequence detailed in the examples is a two-part chimeric DNA sequence containing a 5 'flanking, non-coding sequence from the PR-la gene.

Although the coding sequence of the GUS gene is mentioned as an example of the marker, any of the above-mentioned marker may be used therein. The analogous chimeric three-part sequence contains part of the coding sequence of the PR-1? Gene. Constructions of this type are particularly useful because a chemical induction effect, e.g. enzyme / 3-glucuronidase activity, is easily detectable in plant cells or extracts of these cells. Other particular solutions, for example, those containing a non-coding sequence of one of the tobacco species / 3-1,3-glucanase, and those containing a sequence coding for a wild-type or herbicide-resistant acetohydroxy acid synthetase, or for Bacillus thuringiensis endotoxin, are described in the Examples .

A preferred chimeric DNA sequence is a two- or three-component DNA sequence in which the first component sequence is the flanking region of the tobacco PR-Ia 5 gene having greater than 300 bp, e.g. 300-2000 bp, preferably 600-1000 bp adjacent to a transcription state site. A chimeric DNA sequence comprising three components is also preferred, wherein the second component DNA sequence encodes a signal peptide, for example the latter sequence encodes a signal peptide from a PR protein gene, preferably the PR-1? Gene or a peptide substantially homologous to said peptide.

A method of producing a chemically controllable chimeric DNA sequence comprising a first, non-coding, chemically controllable DNA component sequence having the ability to regulate the transcription of an associated DNA sequence in a plant or plant tissue, this regulation being dependent on a chemical regulator and a second a DNA component sequence transcribed in a plant or in plant tissue, such that these DNA component sequences are ligated. A method of producing a chimeric DNA sequence comprising a first DNA component sequence that is a non-coding, chemically controllable DNA sequence (or a sequence substantially homologous to that sequence) from a chemically controllable naturally occurring gene, the first DNA component sequence being capable of regulating the transcription of an associated DNA sequence in a plant or in plant tissue, where regulation is dependent on a chemical regulator, a second DNA component sequence (or a sequence with substantial homology to that sequence) being part of (but not all of) the DNA sequence that is transcribed, with which first component sequence is associated with a chemically regulated naturally occurring gene, and a third component DNA sequence which is transcribable in a plant or plant tissue, is that the component DNA sequences are ligated simultaneously or sequentially.

D. Vectors.

The vectors are prepared by standard techniques and contain either the chemically controllable DNA sequences described in Part A or B, or the chimeric DNA sequences described in

162 317 in part C above. Vectors are recombinant DNA sequences that can be used to isolate and multiply said DNA sequences and to transform suitable hosts with these sequences. Preferred vectors for the purposes of isolation and multiplication are plasmids which can be grown in a suitable host microorganism, e.g. E. coli. Vectors which are favorable for transformation are those which are used to transform plant cells or Agrobacteria. More particularly, if plant cells other than protoplasts are transformed, then a vector derived from a Ti plasmid is a preferred vector. Any of the vectors mentioned can be used for the direct introduction of DNA into the protoplasts. Suitable vectors to be used as starting materials are known. Vectors suitable for transforming plant tissues and protoplasts are described in the following publications: A deFramond et al., Bio / Technology, 1. 263 (1983), G. An i mm, EMBO J 4, 277 (1985), 1. Potrykus et al., Supra, JS Rothstein et al., Gene 53, 153 (1987). In addition, many other vectors have been described that can be used as starting materials in the methods described herein.

Vectors that contain only a chemically regulated DNA sequence described in part A or B can be used as intermediates to prepare a vector containing the chimeric DNA sequence described in part C above. Insertion of an appropriate sequence, which is transcribable into such an intermediate vector, results in a vector containing the chimeric DNA sequence. This sequence can then be used to transform a desired plant tissue, protoplast, or other host. Alternatively, a chimeric DNA sequence can be prepared and then inserted into an appropriate vector, which in turn can be used to transform the desired plant tissue or other host.

The construction and multiplication of the vectors can be carried out in a suitable host, e.g., E. coli. Suitable strains of E. coli include: HB101, JM83, DH1, DH5a, LE392, and the like. Vectors may be used as such in direct gene transfer or in the microinjection technique. In some cases, it is preferable to linearize the vector prior to use. Alternatively, the vectors can be transformed into an Agrobacterium host. Such transformation is carried out using known techniques, which include: pairing two parents [R. Simon et al., Bio / Technology, 174 (1983)], three-parent mating [G. Ditta et al., Proc. Natl. Acad. Sci. USA, 77, 7347 (1980)] or transformation [M. Holsters et al., Mol. Gen. Genet. 163, 181 (1978)]. Suitable strains include, but are not limited to, A. tumefaciens LBA4404, CIB542, and C58Z7O7.

Preferred vectors include those containing the preferred DNA sequences described in sections A, B, and C above. A vector that is further preferred is one which is functional in plant cells or in Agrobacterium. The vectors described in the examples are particularly preferred.

E. Plant tissues, plants and seeds.

The obtained plant tissues, plants or seeds contain the chimeric DNA sequences described above. Plant tissues, plants or seeds containing those chimeric DNA sequences mentioned to be preferred are preferred.

Plant tissue cells are transformed with the vectors described above using known techniques, including the techniques given in the above publications and the techniques detailed in the examples below. These techniques include direct infection or co-culturing of plants or plant tissues with Agrobacterium. A very convenient technique is the transformation of leaf discs described by R. B. Horsch et al., Science 225, 1229 (1985). Alternatively, the vector is transformed directly, e.g., by electroporation, by microimection, or by transforming protoplasts in the presence of polyethylene glycol (PEG), calcium chloride, or in an electric field, as further described below.

Transformed cells may be derived from monocotyledonous or dicotyledonous plants and may contain one or more chemically regulated chimeric genes. Thus, genes encoding resistance or tolerance to herbicides and a wide variety of pests, including insects, viruses, bacteria, fungi and other pests, genes encoding sterility, size, flowering and fruit ripening are introduced into plant tissue, and these cells or protoplasts regenerate to produce plants in which these traits can be

162 317 Controlled by manipulation of chemical regulator. Alternatively, the cells can be expanded in tissue culture or in a bioreactor, producing enzymes or secondary metabolites. If enzyme determination is required, then the coding portion of the chimeric gene may contain e.g. the previously indicated LUX, CAT, NPT, NOS, OCS, GUS, AHAS or BT gene. Such chimeric genes containing a detectable chemical sequence from the PR gene are advantageous for the ease of use of the regulator and the easy detection of the enzyme product.

After transformation, selection or sknmg of transformed plant cells or tissues is performed by known techniques. The transformed cell or plant tissue contains the chimeric DNA sequence described above. Such a cell is regenerated using known techniques, including those described in the publications cited above and those described in the examples below, for both monocotyledonous and dicotyledonous plants. Crops that can be regenerated by these techniques include, but are not limited to, corn, sunflower, canola, clover, tobacco, carrot, cotton, alfalfa, rice, potatoes, eggplant, and cucumber. The regenerated plants are then screened by known methods to obtain transformed only plants. To obtain improved plant and seed lines, the progeny of regenerated plants are continuously screened and selected for the constant presence of integrated DNA sequence. This DNA sequence can be transferred to other genetic lines in a variety of ways, including classical breeding, protoplast fusion, nuclear transfer, and chromosome transfer.

F. Benefits and Uses.

The invention provides a number of advantages and uses resulting from readily controlled, regulatable expression in plants and plant tissues of genes containing chemically regulable DNA sequences. It becomes possible to regulate genes acting according to the antisense mechanism or genes whose expression is revealed by the formation of a phenotypic trait. Of particular interest is the ability to control the timing and rate of gene expression, and the ease of this control, either uniformly throughout the plant or localized parts of the plant.

Such control may be performed in the usual manner by applying a chemical regulator on a plant tissue, or on a plant or part of that plant, in such a manner and amount as to achieve regulation of a chimeric gene whose expression is desired in plant cells, plant tissues or plants. If, for example, expression of a trait is to occur only in leaves, then by spraying or dusting the leaves at the optimal time for expression to occur in the leaves and before migrating to other parts of the plant, the target can be easily and effectively achieved.

Alternatively, uniform expression throughout the aerial part of the plant can be achieved by applying a regulator to the whole plant (i.e., the stem and both sides of the leaves). For root expression, a possible method of regulation is application to seed, to the soil around the seed or to the roots. Expression in a bioreactor is achieved quite easily, e.g. by adding a chemical regulator to the medium in contact with the cells.

Being able to control the time, degree and / or expression of a gene encoding a new phenotypic trait in transgenic plants offers many advantages. If, for example, a herbicide or other pesticide tolerant trait is introduced into a plant at the root of the detoxification mechanism, this trait can be maximized by timely dosing of a chemical regulator. Such a regulator may be applied before, during or after application of the herbicide or other pesticide, whichever method provides the optimal tolerance.

It is now also becoming possible to regulate the production of compounds, biosynthesis which is controlled by endogenous or foreign genes. After chemical induction, these products can be produced at the desired time. The processes induced may be multi-step biosynthesis or single-step metabolite conversion.

Another advantage of the invention is that it is possible to regulate the developmental processes in plants over time by using a chemical regulator. For example, plant development can be synchronized (germination, shoots, budding, flower formation, fruit ripening, drying out, detachment of fruit). Moreover, normal development of the plant can be prevented. This can be achieved by introducing e.g. a toxin gene that will interfere with the desired stage of development, a DNA sequence that will block the stage of development on

162 317 by an anti-sense mechanism, or gene, the expression of which will block the transition to a new stage of development and which will be subject to chemical repression, preventing development from proceeding. A useful application is the introduction of male or female sterility during controlled hybridization of crops.

As additional advantages resulting from the use of the invention, new methods for determining, preferably methods for determining enzymes, can be mentioned. It has now become quite simple to identify new chemical regulators. These methods use transformed plants or plant cells containing a chimeric DNA sequence as defined above. The chemical is applied to the transformed host and the transcription of the transcribed DNA sequence is measured against the control. Transcription is generally detected as a translation product or as a result of the action of such a product. Such assays can be performed in many different ways, but are generally performed using whole regenerated plants (applying a chemical to the plant) or cell or tissue cultures (applying a chemical to cells or tissues), and then providing a substrate for the enzyme. The product of the enzymatic activity is detected by standard techniques, e.g. spectrophotometric.

The method for identifying a chemical regulator consists in transforming the host with a chimeric DNA sequence described above in section C or with a vector containing such a chimeric DNA sequence, administering the putative chemical regulator to the transformed host, and measuring expression of the phenotypic trait. A method in which the transformed host is plant tissue or plant cells is preferred. In addition, a method is preferred in which the chimeric DNA sequence is the one mentioned above as being preferred, e.g. where the first component DNA sequence in said chimeric DNA is a chemically inducible sequence (or a sequence of substantial homology) that is chemically inducible. a PR protein gene sequence and wherein the phenotypic feature encoded by the chimeric DNA is a detectable enzyme marker.

The invention enables the development of new methods for identifying other chemically regulable DNA sequences. A vector is prepared containing a putative chemically controllable DNA sequence, e.g., from a selectable host marker and a selectable or assayable marker. Selectable markers include antibiotic resistance genes and herbicide resistance genes. Representative antibiotic resistance genes are the genes for hygromycin, kanamycin, chloramphenicol, neomycin, puromycin, kinkomycin, G418 and methotrexate. A particularly suitable hygromycin resistance gene is the aminoglycoside phosphotransferase IV gene. Suitable plant transformation vectors containing the hygromycin resistance gene are described by S. J. Rothstein et al., Gene 53, 153 (1987). Examples of herbicide resistance genes are those encoding resistance to e.g. sulfonylureas, glyphosate, phosphinotricin and atrazine.

Suitable markers that can be labeled are genes for enzymes, antigens, immuglobins, etc., as well as antibiotic resistance genes. Enzyme markers include LUX, CAT, NPT, NOS, OCS, GUS, AHAS, and BT. A particularly suitable enzyme is) 3-1,3-glucuronidase (GUS) due to the ease of determination of this enzyme. The insertion of the DNA sequence encoding the measurable marker into the vector is made by known techniques.

A putative controllable DNA sequence is usually incorporated adjacent to the gene of a markable marker such that expression of that marker is under the control of the putative DNA sequence. This vector is then used to transform plant tissue or other suitable host. Selection of transformed plant tissue or other hosts is based on a selectable plant marker, generally an antibiotic resistance marker. A chemical regulator is then administered to that plant tissue or other host, and the transformed tissue is selected or regenerated. Induction or repression of such a marker upon application of a chemical regulator indicates the presence of the putative DNA sequence as a sequence having the ability to regulate the transcription of an adjacent DNA sequence, the regulation of which is dependent on a chemical regulator. The assay can be carried out on whole plants, regenerated or transformed plants,

For example, by applying a chemical regulator to leaves or other plant tissue and measuring the expression of the marker to be labeled.

Alternatively, the assay is performed on transformed callus tissue or on another host in cell culture, using a putative chemical regulator on the callus or other culture, and measuring the expression of an assayable marker.

The method of identifying a chemically regulable DNA sequence consists of (a) transforming the host with a putative chemically regulable DNA sequence or a vector containing that sequence, a second DNA sequence being a selectable gene marker in the host and a third DNA sequence encoding a phenotypic trait, (b) applying a chemical regulator to said transformed host; and (c) measuring the expression or selecting for changes in expression of a phenotypic trait encoded by a third DNA sequence.

A method in which the third DNA sequence is a host selectable marker or a marker for a determinable gene and a method in which the second or third DNA sequence is a herbicide resistance gene marker or an antibiotic, e.g. hygromycin, kanamycin, chloramphenicol, is preferred. bleomycin, puromycin, lincomycin and methotrexate, especially the aminoglycoside phosphotransferase IV gene which confers hygromycin resistance.

A method in which a putative chemically controllable DNA sequence is related to and preferably adjacent to the third sequence is also preferred. Furthermore, a method in which the transformed host is plant tissue or plant cells is preferable.

Also preferred is a method in which the third DNA sequence encodes a marker for a measurable enzyme such as luciferase (LUX), chloramphenicol acetyltransferase (CAT), chloramphenicol phosphotransferase (NPT), nopaline synthetase (NOS), octopine synthetase (OCS), / 3-1 , 3-glucuronidase (GUS), acetohydroxy acid synthetase (AHAS) and Bacillus thuringiensis endotoxin (BT).

A method of selecting transformed plant material was also developed. Plant material exposed to an exogenous DNA sequence containing a chemically controllable DNA sequence for which a chemical regulator is known and a second DNA sequence for the phenotypic trait is treated with the regulator and expression of the phenotypic trait is observed. The observation of this feature confirms that the transformation of the putative transformants did indeed take place. Typical phenotypic features suitable for this method are the selectable host markers and the assignable markers described above.

A method of selecting transformed plant cells or tissues that have been transformed with DNA containing a first component DNA sequence that is chemically regulated by a known chemical regulator and an associated second DNA sequence encoding a phenotypic marker is to (a) treat putative transformants with this known chemical regulator, and ( b) selecting transformants for expression of the phenotypic trait encoded by the second DNA sequence. A method in which the phenotypic trait is resistance to an antibiotic, e.g. to hygromycin, kanamycin, chloramphenicol, bleomycin, puromycin, lincomycin, G418 and methotrexate, or a marker of an assayable enzyme e.g. luciferase (LUX), acetaminophenikolase CAT), neomycin phosphotransferase (NPT), nopaline synthetase (NOS), octopine synthetase (OCS), β-1.3-glucuronidase (GUS), acetohydroxy acid synthesis (AHAS) and Bacillus thuringiensis endotoxin (BT).

G. Signal Peptides.

A signal peptide or signal sequence is a special N-terminal amino acid sequence in a protein that enters the endoplasmic reticulum. Such a sequence in eukaryotic cells is generally about 15-40 amino acid residues, many of which are hydrophobic. This sequence is cleaved from the mature protein.

The chemically regulated gene signal peptide is useful in the construction of the three-piece chimeric constructs described above. Such chimeric constructs contain a first chemically controllable DNA sequence, a second DNA sequence encoding a signal peptide in proteins, and a third sequence encoding a phenotypic trait. An advantageous phenotypic trait is one that is easy to detect, e.g. by an assay procedure. Incorporation into

The construction of the chimeric DNA sequence encoding the signal peptide allows the expression product of the third DNA sequence to leave the endoplasmic reticulum of the host cell and be directed to a target site.

Thus, a particularly useful peptide is a tobacco PR-Ia protein and a protein with an amino acid sequence substantially homologous to that peptide. This peptide has the following amino acid sequence:

MetGlyPheValLeuPheSerGlnLeuProSerPheLeuLeuValSerThrLeuLeuLeuPheLeuValIleSerHisSerCysArgAla.

Also useful is a DNA sequence encoding this peptide (see Figure 39) and DNA sequences substantially homologous to this sequence. Also useful are the above-mentioned (I) DNA sequences containing a chemically controllable DNA sequence in conjunction with a signal peptide coding sequence, and (2) three-piece DNA signal sequences containing a chemically regulated component, a signal peptide coding section and a DNA sequence subject to transcription, preferably with translation.

H. New PR genes.

New isolated and identified genomic DNA sequences of PR and cDNA were obtained from the tobacco genes labeled PR-1a, PR-Γ and PR-R, the pathogen-induced cucumber chitinase gene, from the tobacco PR-Q gene, basic / 3-1.3 -glucanases 39.1 and 39.3 from tobacco, tobacco acid / 3-1,3-glucanase, and sequences substantially homologous to these genes. The DNA sequences of these genes are shown in Figures 39-46. The new genes from the sequences shown in Figures 39, 40, 43 and 46 (PR-Ia, PR-Γ, and / 3-1,3-glucanases 39.1 and 39.3, respectively) are also a particular example of non-coding chemically regulable sequences DNA obtained by the method of the invention. The new sequences shown in Figures 4I and 42, 45 and 46 encode cucumber chitinase PR, tobacco PR-R proteins, respectively. Tobacco PR-Q and the acid form / 3-1,3-glucanase of tobacco. Details on gene isolation and characterization are given in the examples.

I. Chemical regulators.

A chemical regulator is defined as a substance that regulates the expression of a gene through a chemically regulable DNA sequence under appropriate conditions as given in the definition of the term

A substance, in ionic or neutral form, with or without the addition of solvating or complexing molecules or anions, will typically be exogenous to a system containing a chemically controllable gene at a time when regulation is desired. The use of exogenous chemical regulators is advantageous for the ease and convenience of controlling the amount of regulator in the system. However, endogenous regulators may also be used, that is, chemicals whose activity or level in the system can be artificially controlled by other components in the system or acting on the system.

Chemical regulators include chemicals known as inducers of PR proteins in plants or their closely related derivatives. Examples are benzoic, salicylic and polyacrylic acid and substituted derivatives thereof, having substituents such as lower alkyl, lower alkoxy, lower alkylthio or halogen. When applied to plant tissue, usually leaves and whole plants, they increase mRNA levels and PR proteins are produced in the plant tissue.

An additional group of regulators for chemically regulated sequences of DNA and chimeric genes are compounds with the structure of benzo-1,2,3-thiadiazole, e.g. benzo-1,2,3-thiadiazolecarboxylic acid, benzo-1,2,3-thiadiazolothiocarboxylic acid, cyanobenzo -1,2,3-thiadiazole, benzo-1,2,3-thiadiazole carboxylic acid amide, and benzo-1,2,3-thiadiazole carboxylic acid hydrazide and derivatives thereof.

Suitable derivatives are especially compounds in which the benzo-1,2,3-thiazole moiety is unsubstituted or substituted with small substituents customary in aromatic ring systems of compounds used in agrochemistry, such as lower alkyl, lower alkoxy, lower haloalkyl, lower haloalkoxy, lower alkylthio, cyano, nitro or halogen. This type of derivatives also includes benzo-1,2,3-thiadiazoles in which the carboxy-functional group is 162,317l. The thiocarboxylic acid, carboxylic acid amide, or carboxylic acid hydrazide is unsubstituted or substituted with aliphatic, arylaliphatic or aromatic groups. Examples of such groups are alkyl (especially lower alkyl), alkoxy (especially lower alkyl), lower alkoxyalkyl, alkoxyalkoxyalkyl, cycloalkyl, cycloalkylalkyl, phenylalkyl (especially benzyl), naphthylalkyl, phenoxyalkyl, alkenyl and alkynyl part of which is unsubstituted or substituted with hydroxyl, halogen, cyano or nitro, and the aromatic part of the substituent is unsubstituted or substituted with small substituents commonly found in aromatic ring systems of compounds used in agrochemistry, such as lower alkyl, lower alkoxy, lower haloalkyl, a lower haloalkoxy group. lower alkylthio group, cyano group, nitro group or halogen atom.

Regulators based on the benzo-1,2,3-thiadiazole structure encompass all molecular systems with the property of releasing a molecule actually acting as a regulator.

A preferred group of regulators based on the benzo-1,2,3-thiadiazole structure include benzo-1,2,3-thiadiazole carboxylic acid and alkyl esters of benzo-1,2,3-thiadiazole carboxylic acid. in which the alkyl group contains 1-6 carbon atoms, and substituted derivatives of these compounds. Suitable substituents include a lower alkyl group, a lower alkoxy group. lower alkylthio group and halogen. Preferred inducers of chimeric DNA sequences containing chemically controllable DNA sequences isolated from PR protein genes are especially benzo-1,2,3-thiadiazole-7-carboxylic acid and its alkyl esters, e.g. benzo-1,2,3-acid methyl ester. thiadiazole-7 carboxylic acid. Methods of making the above-mentioned chemical regulators and their use as biocides are disclosed in British Patent No. 1,176,799 and in P. Kirby et al., J. Chem. Soc. C 2250 (1970).

Benzo-1,2,3-thiadiazole derivatives which can also be used as regulators are described in US patent application US Am. No. 234 241 of August 18, 1988.

Preferred compounds with the structure of benzo-1,2,3-thiadiazole are, for example, benzo-1,2,3-thiadiazolecarboxylic acid-7, benzo-1,2,3-thiadiazole-carboxylic acid-7 methyl ester, benzo-1-acid η-propyl ester Benzo-1,2,3-thiadiazole-carboxylic acid, 2,3-thiadiazolecarboxylic acid, benzyl ester-7,1-tertiary-butylhydrazide-7, benzo-1,2,3-thiadiazolecarboxylic acid, etc.

A second group of regulators for chemically controllable DNA sequences are compounds based on a pyridine carboxylic acid structure, e.g., an isonicotinic acid structure, and preferably a haloisonicotinic acid structure. Dichloroisonicotinic acids and their derivatives, e.g. their lower alkyl esters, are preferred. Suitable regulators of this class of compounds are, for example, 2,6-dichloroisonicotinic acid and its lower alkyl esters, especially the methyl ester.

As already described above, a method of regulating the transcription of a chemically regulated gene by applying such a chemical regulator to plant tissue, plant or seed containing a chemically regulated DNA sequence described in section A, B or C above has been developed. A method in which said plant tissue, plant or seed comprises a chemically controllable DNA sequence mentioned above as preferred is preferred.

Chemical regulators can be used in pure form, in solution or in suspension, in the form of powders or dusting powders, or in any other convenient form for use in agriculture or in bioreactor processes. Such formulations may contain solid or liquid carriers, that is, a material with which a regulator is attached for easier application to plants, tissues, cells, tissue cultures or the like, or to improve storage, handling or transportation conditions. Examples of suitable carriers are silicates, clays, carbon, sulfur, resins, alcohols, ketones, aromatic hydrocarbons, etc. When preparing a frequently used wettable powder or an aqueous emulsion, one or more surfactants, ionic or non-ionic, may be added to the formulation of the regulator. such as wetting, emulsifying, or dispersing agents.

Regubitors may also be applied to plants in combination with another plant beneficial agent, which benefit may or may not be related to a trait controlled by a chimeric gene under the control of the regulator. The regulator can, for example, be mixed with fertilizer

162 317 and applied immediately before expression of the transgenic trait independent of fertilization. The regulator may also be used in conjunction with a herbicide to mitigate the adverse effect of the herbicide at a time when the effect would be at its maximum.

In the form of liquid formulations, the regulator can be applied as a spray to leaves, stems or branches of plants, to seeds prior to sowing, or to soil or other plant growth medium. Regulators can also be used in cultures grown in bioreactors to achieve regulation by simply adding a formulation of the regulator to the reaction medium or by gradual dosing over a period of time.

The controller is used in the amount and for the time needed to obtain the correct regulation. A preferred regulator is one that has no or only minimal phytotoxic or other adverse effects on the plant, plant tissue or plant cells to which it is applied.

Of the DNA sequences described in parts A and B and the chimeric DNA sequences described in part C, especially those whose transcription is regulated by a chemical regulator mentioned above, especially benzoic, salicylic, acetylsalicylic and polyacrylic acid and substituted derivatives of these compounds or benzo1 acid, are preferred. 2,3-thiadiazole carboxylic acid, benzo-1,2,3-thiadiazolecarboxylic acid, 1,2,3-cyanobenzo-thiadiazole, benzo-1,2,3-thiadiazole carboxylic acid amide and benzo-1,2,3-thiadiazole carboxylic acid hydrazide and their derivatives or with dichloroisonicotinic acid or its derivatives. Most preferred are those DNA sequences whose transcription is regulated by the aforementioned preferred chemical regulators, e.g. benzo-1,2,3-thiadiazole-carboxylic acid-7, benzo-1,2,3-thiadiazole-7-carboxylic acid methyl ester, n-propyl ester benzo-1,2,3-thiadiazolecarboxylic acid-7, benzo-1,2,3-thiadiazole-carboxylic acid benzyl ester-7, tertiary butylhydrazide benzo-1,2,3-thiadiazole-carboxylic acid-7. 2,6-dichloro-isonicotinic acid or 2,6-dichloro-isonicotinic acid methyl ester, in particular renzo-1,2,3-tladyl-7-carboxylic acid methyl ester.

J. Deposits with ATCC.

The following bacteria, plasmids and culture suspensions have been deposited in the American Type Culture Collection:

E. Coli HB101 / pTJS75-Km, ATCC No. 67628, deposited February 12, 1988, plasmid pBS-PR1013Cla, ATCC No. 40426, deposited February 12, 1988, plasmid pBS-PR1019, ATCC No. 40427, deposited February 12, 1988. , Zea mays 6-2717-GC 'culture suspension, ATCC No. 40326, deposited on May 20, 1987, plasmid pBS-Glu39.1, ATCC No. 40526, deposited on December 29, 1988, plasmid pBS-Glu39.3, ATCC No. 40527, deposited December 29, 1988, plasmid pBScucchi / Chitinase, ATCC No. 40528, deposited on December 29, 1988, plasmid pBSGL6e, ATCC No. 40535, deposited January 18, 1989.

K. Description of the experimental part.

Chimeric genes contain chemically regulated DNA sequences from any source, natural or synthetic, in combination with one or more transcribed DNA sequences. In general, at least a portion of the transcribed DNA sequence will be translated to result in a phenotypic trait, although anti-sense chimeric genes may also be useful. While the following description and many examples refer to chemically controllable DNA sequences naturally occurring in plant genomes, sequences that occur in other natural systems, e.g., bacterial, viral, and animal systems, are equally useful as well. techniques for isolating such sequences are generally known. This also applies to sequences derived from synthetic or other natural systems.

The chemically regulable DNA sequence is isolated from the source in its natural state or from a synthetic source and, if necessary, characterized in a known manner. If a DNA sequence is isolated from a naturally occurring gene, the gene is activated by an appropriate regulator, chemical or other known for that gene. The RNA resulting from such activation is isolated and used to construct a cDNA bank. This bank is used for differential screening using labeled with a radioactive element

CDNA generated from both (1) RNA isolated from an activated system and (2) RNA isolated from a second, unactivated regulator. Clones containing chemically regulable cDNA are isolated and preferably sequenced at this stage. These cDNA clones are then used to identify and isolate the genomic clone from the genomic bank, isolating the genomic clone containing a chemically controllable DNA sequence. Preferably, this gene is sequenced and a functional map of a chemically regulable DNA sequence is constructed by subcloning fragments of this gene, ligation with the gene to be described and evaluating their activity in transgenic plant material or in transient plant expression systems.

Using standard techniques, a lambda genomic bank is constructed for the tobacco PR protein genes, and clones containing chemically controllable DNA sequences are identified and purified. One of these clones is characteristic. Restriction fragments containing a chemically controllable DNA sequence are identified. For the PR-1? Gene, these fragments include a 20,000 bp Body fragment, a 6,500 bp Hind III fragment, and a 3,600 bp EcoRI fragment. The HundIII fragment contains about 500 coding base pairs and about 600 non-coding base pairs in the 5 'flanking region adjacent to the transition start site. Some of these fragments are cloned into plasmids and used as DNA sources for further subcloning and full characterization, i.e., restriction mapping, DNA sequencing, and identification of a transient gene start site. For the PR-1α gene, an EcoRI fragment of 3600 bp is directly cloned from the lambda genomic clone and then subcloned into the final 1200 bp clone flanked by Xhol and Pstl sites. This clone contains a chemically inducible DNA sequence from the PR-1? Gene and a portion of the contiguous region for that gene. This section contains the coding sequence for the signal peptide from the gene for the PR-1? Protein.

Genomic clones encoding the basic form of β1,3-glucanase are similarly isolated. The two clones 39.1 and 39.3 are characterized and restriction fragments containing chemically regulated DNA sequences are identified.

Using the vectors described herein, these clones can be used to prepare chimeric genes containing the three parts described above. These chimeric DNA sequences contain a chemically regulable DNA sequence, a portion of the transcribed DNA, and a third DNA sequence from a foreign source. Alternatively, these clones may be further manipulated, e.g., by site-directed mutagenesis, to remove all or most of the coding fragment from the parent gene prior to annealing to a coding sequence from a foreign source, to construct the two-piece chimeric gene described above.

In a preferred embodiment, the non-chemically regulated portion of the chimeric gene constitutes a gene for an easily observable and detectable phenotypic trait. The following examples describe the genes for β1,3-glucuronidase, wild and herbicide resistant acetohydroxy acid synthetase and Bacillus thuringiensis endotoxin. In addition, many other genes can be listed for identifiable traits. In a further preferred embodiment, the coding component sequence in the chimeric gene encodes herbicide tolerance or resistance, or insect resistance. This solution is described on the example of the genes mentioned for the acetohydroxy acid synthetase and for the Bacillus thuringiensis endotoxin.

Chimeric genes and vectors containing these genes are introduced into plant cells by a variety of techniques, resulting in the growth of transformed cells, tissues and plants, and cell cultures in bioreactors. Several techniques applicable to both monocotyledonous and dicotyledonous plants are described in detail in the following examples. Some of these methods have been the subject of other patent applications, particularly US patent applications US Am. No. 056506 of 29 May 1987 and No. 165 665 of 8 March 1988.

The transformed plant material is then treated with a chemical regulator and expression of the gene encoding the phenotypic trait is observed and / or measured. This type of system is an easy screening tool to identify the regulatory activity of a specific chemical compound. Therefore, an improved method for the determination of enzymatic activity / 51 ronidase (GUS) was developed, allowing the screening of a large number of samples with high precision

162 317 in no time. This determination is based on the reaction of plant tissue extracts in samples below 0.5 ml containing 1.5-3 mM 4-methyl-umberliferyl-glucuronide at a temperature of about 37 ° C for 1-5 hours and determining the concentration of the released fluorescent indicator.

Quantification of constant RNA levels is an essential step in gene expression analysis. For this purpose, a method of primer extension was developed. According to this method, a gene-specific oligonucleotide is labeled with a radioactive element rendering it highly specific radioactivity, this oligonucleotide is hybridized with an RNA sample, and the hybrid is extended with reverse transcriptase. The elongation products are separated on denaturing acrylamide gels and visualized by autoradiography. The advantages of this method over the previous assay methods are the ease of preparation of the probe, the simplicity of the assay procedure and the shorter time needed to perform the assay. This primer extension method is as sensitive and quantitative as the SI endonuclease mapping method.

The primer extension method used in the specific reaction conditions described in the examples below was optimized for determining the level of PR-1 mRNA in the total RNA extracted from TMV-infected tobacco leaves. In these leaves, 1% of the total MRNA is produced by PR-1 mRNA. These results were obtained based on the quantitative analysis of the data obtained using the primer extension method and the analysis of the frequency of cDNA clones in a cDNA bank derived from this RNA. Improvements were achieved by the method described herein over the previously described methods by labeling the oligonucleotide for highly specific activity, reducing the molar amount of probe used for the assay (generally 0.01-0.05 pmol), optimizing the hybridization and elongation time, and optimizing triphosphate concentrations nucleotides. Thus, the method of determining the amount of specific RNA in an RNA solution consists in (a) labeling an RNA-specific oligonucleotide primer of 12-18 nucleotides in length, rendering it highly specific radioactivity, (b) hybridizing this RNA with a labeled oligonucleotide at a concentration of 0.1-20 mM for 2 minutes to 24 hours at about 37 ° C, (c) reverse transcriptase elongation of this oligonucleotide primer in the presence of nucleotide triphosphates at a concentration of 0.003-1 mM for 1-120 minutes, and (d) isolation and visualization of elongation products by the autoradiographic method.

L. Examples.

The following examples illustrate the invention.

Enzymatic reactions are performed according to the procedures recommended by the manufacturers, unless otherwise indicated. Chimeric genes and vectors are constructed using commonly known techniques. Suitable techniques are described in the following publications: T. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, New York (1982), Methods in Enzymology, volumes 68, 100, 101 and 118 (1979, 1983, 1983 and 1986) and DNA Cloning DM Glover, IRL Press, Oxford (1985). The compositions of the media are described in J. H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, New York (1972), as well as in the publications reported previously.

Shortcuts

ATP adenosine triphosphate bp pas; nsad

CETAB hexadecyltrimesylammonium bromide

2,4-D 2,4-dichloroenossyacetic acid

DTT dttlOtΓettol dicamba 3,6-douchloro-2-methoxybenzene acid

EDTA esylenediamine-N, N, N ', N'-tetraacetic acid kb yysieni pasa zaad

MES 1 ^ w ^ ^ s 2- (N-moffoiline ^ ranosulfonic

MU 4-mesyloumbeilfesyl glucuronide

PEG giiko 1 polyethylene picloram 4-amlno-3,5, ί ^ trichloropicic acid

Sodium SDS

TFA tI · eHuoroacetic acid

TMV in ^ us mosaics and zto ^ nui

Tris-HCl hydrochloride ttoj (hzdtoeszmetzlo) metzlorminz

162 317

Media.

SH-O medium: medium according to R. U. Schenk et al., Can. J. Bot., 50 (1972) 199-204, without hormones. The SH medium can be liquid or solidified with 0.8% agar or 0.5% GelRite. The medium is heat sterilized in an autoclave at about 110-121 ° C for 15-20 minutes.

SH-30 medium: SH-O medium containing 30μΜ of dicamba.

SH-45 medium: SH-O medium containing 45 μΜ dicamba.

RY-2 medium · medium according to Y. Yamada et al., Plant Cell Reports, 5, 85-88 (1986).

OMS medium: medium according to T. Murashige and F. Skoog, Physiologia Plantarum 9, 473 (1968). The medium can be solidified by adding 0.8% agar or agarose or 0.5% GelRite.

Table 1 shows the composition of the media used. Macronutrients ^{a /} . micronutrients ^{a /} and FeEDTA are added according to the literature: KM medium according to KN Kao et al., Planta 126, 105-110 (1965), N6 medium according to Chu et al. Scientia Sernica 18, 659 11975).

Table 1

Media KM-8p ° ' ^c ' N6 Organic compounds and vitamins (mg / liter) biotin 0.01 - pirvdoksvna-HCl 1.00 0.5 thiamine-HCl 10.00 a.m. 0.1 nicotinic acid amide 1.00 mkotvnowv acid 0.10 0.5 Folic acid 0.40 - D-Ca-pentothenate 1.00 - p-aminobenzoic acid 0.02 - choline chloride 1 00 - riboflavma 0.20 - vitamin B12 0.02 - glycine 0.10 2.0 Sugars 1 alcohols derived from sugar (g / l) saccharose 0.25 30.0 glucose 68.40 - mannitol 0.25 - sorbit 0.25 - cellobiosis 0.25 - fructose 0.25 - mannose 0.25 - rhamnosis 0.25 - rvboza 0.25 - xylose 0.25 - myoinositide 0.10 - Final pH 5.8 5.6 Sterilization filtering ainoklauouanie

Explanation of references a / Macronutrients are prepared as a tenfold concentrated stock solution, micronutrients as a thousandfold concentrated stock solution b / A solution of citric, fumaric and malic acids (each with a final concentration of 40 mg / liter) and sodium pyruvate (with a final concentration of 20 mg / liter) prepared as 100% concentrated stock solutions, with the pH adjusted to 6.5 with NH OH, and added to this medium

162 317

Ro / ivorv adenine (final concentration 0 I mg / L) and guamna. Thymidine, Uracil, Hypoxanthine and Cytosine (each with a final concentration of 0.03 mg / liter) are prepared as 1,000-fold stock solutions. adjusted to pH 6.5 with NH4OH, 1 is added to this medium. d) The following amino acids are added to the nutrient hopper using a tenfold concentrated stock solution (pH 6.5 adjusted with NH4OH). reaching the stated final concentrations of glutamine (5.6 mg / liter). alanine, glutamic acid (0.6 mg / liter each) cysteine (0.2 mg / liter). asparagine, aspartic acid. cystine. histidine. isoleucine. leucine. lysine, methionine. phenylalanine. serine proline. threonine. tryptophan. tyrosine 1 valine (0.1 mg / liter each) e / A stock solution of vitamins is prepared in a standard 100% concentrated manner. Materials.

Agarose: Preparation and purification of agarose is described by Guiseley and Renn. The Agarose Monograpg. Marina Colloids Division FMC Corp .. 1975. Agarose is one of the components of agar. Commercial agar consists of a mixture of neutral agarose and ionic agaropectins with a large number of side groups. Typically, a number of side chains remain intact and determine the physicochemical properties of the agarose. such as gel formation and melting point. A preferred solidifying agent is low melt agarose. especially SeaPlaque agarose.

Casein Hydrolyzate: Casein Hydrolyzate-Enzyme Hydrolyzate From Cow Milk. Type 1. Sigma Co. P. O. Box 14508. St. Louis. MO 63178. USA

Cellulase RS: Cellulase RS. Yakult Honsha Co .. Ltd .. 1. 1. 19 Higashi-Shinbashi. Minato-ku. Tokyo. 105 Japan.

GelRite: GelRite Gellan Gum. Scott Laboratories Inc .. Fiskerville. R. J. 02823. USA.

GeneScreen Plus: Catalog No. NEF 976. NEW Research Products. 549 Albany St. .. Boston. MA 02118. USA.

IBI Random primer kit: "Pnme Time" set of random primers. International Biotechnologies Inc .. P. O. Box 1565. New Haven. CT 07606. USA.

Nalgene Filter: Nalge Co .. Division of Sybron Corp. Rochester. N.Y. 14602. USA.

Pectolyase Y-23: Seishin Pharmaceutical Co. Ltd .. 4-13 Koamicho. Nihonbashi. Tokyo. Japan.

Parafilm: Parafilm laboratory foil - American Can Co. Greenwich. CT 06830. USA.

SSC: 1.54 mM NaCl. 0.154 mM sodium citrate.

Spin column: ready. a packed Sephadex G25 column. Catalog No. 100402. Boehringer Mannheim Biochemicals. Piscatway. NJ. USA.

TAE buffer and TBE buffer: Tris-acetate buffer and Tris-borate buffer, respectively. commonly used electrophoresis buffers. see Maniatis et al. j. w.

1. General techniques.

This group of examples describes the general techniques used in the following specific examples.

Example I. Ligation in agarose.

After restriction digestion of plasmid DNA and electrophoretic separation of the fragments on a low-melting gel in TAE buffer. bands containing the appropriate fragments are carefully cut and heated to 65 ° C to melt the agarose. 2-5 µl of this agarose is added to 15 µl of water. and then the solution is left for 10 minutes at 65 ° C. This solution is cooled to 37 ° C and left for 5 minutes for the temperature to equilibrate. 2 µl of tenfold concentrated ligase buffer (200 mM Tris. PH 8.0, 100 mM MgCl 2, 100 mM DTT. 10 mM ATP) is added along with 1 µl of T4 DNA ligase (New England BioLabs). this solution is then allowed to solidify and incubated overnight at 15 ° C.

Example II. Transformation from agarose.

Agarose containing the appropriate DNA is melted by incubating for 10 minutes at 65 ° C. 10 µl of this solution is added to 30 µl of TE buffer (10 mM Tris pH 7.5.1 mM EDTA). stirred and left at room temperature. Frozen competent cells (E. coli strain DH5a) are placed on wet ice to thaw. The diluted DNA solution is added to 200Al cells and left on ice for 20 minutes. DNA containing cells are then heat shocked at 41 ° C for 90 seconds. These cells are then left at room temperature for 10 minutes. 0.8 ml of SOC medium [D. Hanahan

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J. Mol. Biol. 166, 557-580 (1983)] and the culture is incubated for one hour at 37 ° C. 100 µl of the culture is plated on LB medium plates (Miller, as above) containing ampicillin (L-amp) at a concentration of H ≤ 0 µg / ml. The plates are then incubated overnight at 37 ° C. Positive colonies are selected and plated onto a second plate with the same medium, then the plates are incubated overnight at 37 ° C.

Example III. The Sozthern technique.

^ g of tobacco DNA is digested with various restriction enzymes under the conditions recommended by the supplier. DNA is phenol extracted, ethanol precipitated and then resuspended in gel loading buffer (15% ficoll, 0.025% bromophenol blue, 10 mM EDTA, pH 8). The samples are loaded and electrophoresed on a 0.5% agarose gel at 5V / cm, until the dye bromophenol blue reaches the end of the gel. DNA is transferred to Gene-Screen Plus (DuPont) using the basic transfer procedure as described by the supplier. Pre-hybridization, hybridization and washing are performed according to the manufacturer's recommendations. Hybridization is detected by autoradiography.

Example IV. Molecular adapters.

A typical molecular adapter for converting a PstI restriction site into BamHI is sequence

5'-GGGATCCCTGCA-3 '.

This molecule is synthesized. in an Applied Biosystems Synthesizer using the [5-cyanoethyl phosphoramidite method and purification by reverse phase HPLC. About 2 µg of this oligonucleotide is phosphorylated according to Maniatis et al., Pp. 125. The oligonucleotide solution is heated to 65 ° C in a water bath and then allowed to cool to room temperature over approximately 30 minutes. An approximately tenfold molar excess of this annealed adapter is added to digested DNA with tenfold concentrated ligase buffer, T4 DNA ligase and an appropriate amount of water.

A typical reaction mixture includes: DNA to be combined with the 1-2 µ adapter] (~ 1 pmol); 1 μ \ (—Opmol) adapter; 10-fold concentrated ligase buffer 1P1; 1 µl T4 DNA ligase; water 5-6 / 1.

This solution is incubated at 12-15 ° C for 30 minutes and heated at 65 ° C for 30 minutes to inactivate the ligase. The salt concentration and volume are adjusted to provide the appropriate conditions for restriction digest and the DNA connected to the adapter is digested to expose the "sticky end". Non-embedded adapters are removed by agarose gel electrophoresis or by sequential isopropanol precipitation.

Example 5 Extended Primer Mapping.

A. Synthesis and labeling of 5 'primers for primer extension process.

The following primer oligomers are synthesized in an Applied Biosystems Synthesizer using the jJ-cyanoethyl phosphoramidite method

PR-1: 5'-ATAGTCTTGTTGAGAGTT-3 '

GUS: 5'-TCACGGGTTGGGGTTTCTAC-3 '

AHAS: 5'-AGGAGATGGTTTGGTGGA-3 '

BT: 5'-ATACGTTCTACTATCATAGT-3

Oligonucleotides are purified by reversed-phase high pressure liquid chromatography (HPLC). 5pmoli of each phosphorylated oligonucleotide (T. Maniatis et al, supra p. 125) using 200 // Ci ³² P ATP (6000Ci / mmol, 10μΟ / μ0. After 30 min incubation at 37 ° C the mixture was diluted to a volume of 100 µl, extracted with phenol / chloroform mixture and then precipitated three times with 50 µg carrier RNA. The final pellet is resuspended in reverse transcriptase buffer (50 mM Tris-HCl pH 7.5.40 mM KCl, 3 mM MgCk) at concentration 2nM. The determined specific activity of the labeled oligonucleotide is about 3 X ⁶ Cvcpm I0 / pmol.

B. Production of total RNA.

Total RNA is prepared essentially as described by L. M. Lagrimini et al., Proc. Natl. Acad. Sci. USA 84, 7542 (1987). The tissue is ground under liquid nitrogen with a pestle in a mortar. The grinded tissue is added to the grinding buffer (Lagrimini et al. U. V.) Using 2.5 ml per gram of tissue. An equal volume of phenol is then added and the emulsion is homogenized with a homogenizer

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Brinkman polytron. Half the volume of chloroform is added and the emulsion is gently stirred for 15 minutes. The phases are separated by centrifugation and the aqueous phase is removed. RNA is precipitated by adding sodium acetate to a concentration of 0.3 M and 2.5 volumes of ethanol. The precipitate is separated by centrifugation and resuspended in 2 ml of sterile water. Lithium chloride is added to a final concentration of 3 M and left overnight at 4 ° C. The solution is swirled and the resulting precipitate is washed with ice-cooled 80% ethanol. The pellet is then dried and resuspended in 500 µl of sterile water. The total RNA concentration in this preparation is determined spectrophotometrically. Alternatively, RNA is extracted from the callus as described above with the difference that the callus tissue is cut into about 3 mm cubes and inserted into a pre-cooled mortar and rubbed with a pestle in liquid nitrogen prior to the Brinkman polytron homogenization step.

C. Extending the primer.

50 µg of total RNA is lyophilized in a 500 µl Eppendorf tube. RNA freezes again at 30μ! of the radiolabeled probe solution and heated to 70 ° C for 10 minutes. The tube is cooled slowly to 37 ° C and incubated overnight. 2 μ1 of a tenfold concentrated buffer for reverse transcriptase (500 mM Tris-HCl, pH 7.5, 400 mM KCl, 30 mM MgCl ₂ ), 1 μ1 of bovine serum albumin at a concentration of 5 mg / 5 are introduced into a test tube placed in a water bath at 37 ° C. ml, 5μ1 100mM dithiothreitol, 5μ1 tenfold concentrated dNTP (10mM aqueous solution of each dNTP), 3μ1 water, 2μ1 RNazine (80 units) and 2μ1 reverse transcriptase (400 units) and the reaction is incubated for 30 minutes at 37 ° C. 5 µl of 3M sodium acetate (pH 5) and 150 µl of absolute ethanol are added to stop the reaction. The tube is left at -20 ° C for 30 minutes, the precipitate is separated by centrifugation, then washed with 80% ethanol and allowed to air dry. The pellet is resuspended in 10-20 μ / l of the electrophoresis dye buffer (90% formic acid amide, 0.05% bromophenol blue, 0.05% xylene cyanol, 1 mM EDTA) and the elongation products are separated on a 6% gel for sequencing (T. Maniatis et al., ibid.). The elongation products are visualized by autoradiography.

Example VI. SI nuclease mapping.

The plasmid pBS-PR 1013Cla is digested with SfaNI restrictase, dephosphorylated with calf greens phosphatase and phosphorylated with ³² P-ATP. After phenol extraction and ethanol precipitation, the DNA is digested with BstEII restrictionase and the 300 bp fragment corresponding to the sequence from nucleotide 750 to 1035 in Figure 39 is isolated after low gel temperature agarose electrophoresis. The probe is resuspended in hybridization buffer containing formic acid amide [AJ Berk et al., Cell 12, 721 (1977)] at a concentration of approximately 2 nM. The specific activity of the probe is approximately 5X10 Cvcpm / pmol.

Lyophilized total RNA (50 µg) is dissolved in 30 µl of the probe solution and the tubes are heated to 65 ° C for 10 minutes and then allowed to hybridize at 48 ° C overnight. The treatment with S11 nuclease gel electrophoresis is performed essentially as described, using the S11 nuclease at a concentration of 400 units / ml and an incubation temperature of 30 ° C. The correct concentration of SI nuclease is determined in preliminary experiments.

Example VII. Transcription start site mapping.

The transcription start site of the PR-1? Gene is determined by a combination of SI nuclease mapping and primer extension analysis. The autoradiogram from the primer extension experiment is examined using either RNA isolated from TMV infected leaves or the mpl9 restriction fragment mpl9 is subcloned from Xhol-Pstl as a template and the 17 base oligonucleotide complementary to positions 1025-1042 of the PR-la sequence as specific primer. The primer itself is labeled at 5 'phosphate, therefore the size of the extension product will be identical to the size of the band in the sequential electrophoresis lane. The appearance of two strong bands corresponding to positions 902 and 903 and a weak band corresponding to position 901 of the genomic clone suggest starting transcription at either of these positions. Primer extension analysis alone cannot be used to identify the 5 'end of the mRNA. For example, due to the splicing process, the mRNA may contain a 5 'end from a different (5' direction) position.

High resolution S1 nuclease mapping in combination with primer extension is used to define the 5 'end. The SfaNI fragment is tagged at the 5 'end

162 317 and digested with BstEII restrictionase to give a specific probe extending from position 759 to L040. This probe is used to map the 5-end of PR-1? Transcripts in RNA isolated from TMV-infected tobacco leaves. There is a major band 137 ± 2 bases long corresponding to positions 901-905 of the genomic clone. High-resolution experiments with SI nuclease electrophoresis with the molecular weight standards from the probe sequencing reaction show three bands corresponding to positions 901, 902, 903. Regarding transcription initiation, one possible interpretation is of these results is that RNA polymerase starts transcription at either base 901, 902, or 903 with less or less equal probability. However, since eukaryotic transcription usually starts at site A, the most likely explanation for the seemingly multiple 5 'ends is that PR-la mRNA starts at position 903 (position A) and PR-Ib and -lc mRNA each start at position 903 (position A). one of the other positions on their corresponding genes.

2. Identification and characterization of proteins.

The PR proteins described in these examples are isolated, purified, and sequenced, in some cases for the first time, and in others according to procedures described in the literature to finally confirm the identity of their chemically inducible genes.

Example VIII. Purification of PR-1a and PR-1b proteins.

Plants of Nicotiana tabaccum cv. Xanthi are grown in a greenhouse and infected when they are 8 weeks old by gently rubbing the leaves with a suspension of the common (UI) TMV strain (0.5 µg / ml). The leaves are harvested 7 days after infection, the intracellular fluid (ICF) is washed from the leaves and harvested according to a procedure described in the literature. 250 ml of ICF is concentrated to 50 ml by lyophilization and applied to an Ultragel ACA54 gel column equilibrated with Tris-HCl pH 8.0 and 1 mM EDTA. The eluate is analyzed by electrophoresis on 10% polyacrylamide gels. PR-1 protein containing fractions are pooled, lyophilized, resuspended in 3 ml of water and then dialyzed overnight against water. This preparation is further purified by anion exchange HPLC on a TSK-DEAE 5 PN column by gradient elution with 0-0.6 M NaCl in 50 mM Tris-HCl, pH 8.0, 1 mM EDTA. Fractions are analyzed by polyacrylamide gel electrophoresis (PAGE). First, the PR-1b is eluted from the column at 0.18 M NaCl, and the PR-Ia is eluted at 0.28 M NaCl. Fractions containing each of these proteins are pooled, dialyzed against water and lyophilized. The purity of the PR-1a and PR-1b protein preparations is confirmed by reversed-phase HPLC using an Aquapora phenyl column (2.1 X 100 mm, Brownlee) and gradient elution with linearly increasing mixtures (5-80%) of the acetonitrile mixture / isopropanol (1: 1) in 0.1% TFA.

Example IX. Protein sequence determination.

Purified, lyophilized PR-Ia proteins are dissolved in 6M guanidine hydrochloride containing 1M Tris-HCl, pH 8.6, 10mM EDTA. Dithiothreitol is added to a concentration of 20 mM i

4-vinylpyridine to 50 mM final concentration. The sample is then incubated for 1.5 hours under nitrogen. The pyridylethylated material is desalted by HPLC on an Aquapore phenyl column (2.1 X 10 cm, Brownlee), gradient elution with linear ascending mixtures (5-80%) of acetonitrile / isopropanol (1: 1) at 0.10 1% trifluoroacetic acid (TFA).

Automatic Edman degradations are performed in an Applied Biosystems 470A gas phase protein sequencer. Phenylthiodantoin (PTH) amino acids are identified on an Applied Biosystems 120A PTH analyzer.

Cyanogen bromide digestion is performed m situ according to R. J. Simpson et al., Biochem. Intl. 8, 787 (1984). Digestion with PR-1 pyroglutamiman aminopeptidase (Boehringer Mannheim) is performed according to G. Allen, Plant Sci. Lett. 26, 173 (1982).

PR-la is digested with Lys-C endopeptidase (Eoehringer Mannheim) in 0.1 M Tris-HCl, pH 8.5, for 24 hours at room temperature, with an enzyme to substrate ratio of 1:10. The peptides are isolated by HPLC on an Aguapore C-8 column (1x22cm, Browlee) by gradient elution with linearly increasing mixtures (0-60%) of acetonitrile / isopropanol (1: 1 ratio) in 0.1% TFA.

Digestion of the N-terminal peptide resulting from digestion with Lys-C endopeptidase, trypsin (Cooper) is carried out in 0.1 M ammonium bicarbonate, pH 8.2, containing 0.1 M chloride

162 317 calcium, for 5 hours at 37 ° C, with an enzyme to substrate ratio of 1: 100. Duplicate peptides are separated by HPLC using the same conditions as for peptides resulting from digestion with Lys-C endopeptidase.

Example X. Sequence of PR-Ia peptides.

The DNA sequences of three cDNA clones were first correlated with the PR-la, -lb, and -lc proteins by B. J. C. Cornelissen, et al., Based on a comparison of the published protein sequences of three peptides obtained from PR-la [J. Lucas et al., EMBO J. 4, 2745 (1985)] and the protein structure deduced from cDNA clones. However, the cDNA clone designated PR-1a is truncated at the 5 'end and can only be compared to two of the three peptides with one residue replaced. The encoded amino acid sequence deduced from the cDNA obtained and analyzed above has not only reportedly had an altered tryptophan residue compared to the published PR-1? CDNA sequence (U. M. Pfizner et al., J. V.), But also changed at three other amino acid positions. These abnormal sites question the identification of this clone as a PR-la. Therefore, the identity of the tobchrPR 1013 clone as the PR-1? Gene is verified by determining large parts of the primary structure of the purified PR-1? Protein by amino acid sequencing.

The amino acid sequence of the PR-la protein is compared to the amino acid sequences deduced from the cDNA clones for PR-la, -lb, and -lc. The cDNA sequence used in the analysis is derived from the cDNA clones isolated above and the sequences given by U. M. Pfitzner et al., Which are consistent at every position. More than 80% of the mature protein equation is determined and this sequence corresponds to the deduced protein sequence for tobchrPR 1013. By comparing the deduced amino acid sequence obtained from the putative PR-Ib and PR-lc cDNA clones (BJCCornelissen et al., As above), 7 and 6 changes are found, respectively . This evidence clearly demonstrates that tobchrPR1013 is indeed a clone of the PR-1? Gene.

3. Production of clones.

Examples in this group describe clones produced by gene isolation and identification of chemically controllable sequences and chimeric genes containing the sequences.

Example XI. Manufacture of tobchrPR1013.

The kernels are isolated from the leaves of Nicotiana tabaccum by freezing 35 g of tissue in liquid nitrogen and grinding to a fine powder with a pestle and mortar. The powder is added to 250 ml of grinding buffer (0.3 M sucrose, 50 mM Tris, pH 8.5, 5 mM magnesium chloride, 5 mM sodium bisulfite, 0.5% NP40) and stirred under ice cooling for 10 minutes. The mixture is filtered through 6 layers of gauze and the liquid is transferred to centrifuge tubes and centrifuged at 700 X g for 10 minutes. The pellets are resuspended two more times in the grinding buffer and vortexed. After the final centrifugation, the pellets are resuspended in the grinding buffer and this suspension is applied to the surface of 20 ml of cold sucrose layer containing 10 mM Tris, pH 8.0, 1.5 mM magnesium chloride, 140 mM sodium chloride, 24% sucrose and 1% NP40. These tubes are centrifuged at 17,000 Xg for 10 minutes. The sediments at this stage mainly contain starch kernels and granules. High molecular weight DNA is isolated from the nuclei essentially according to T. Maniatis et al. The DNA is partially digested with Mbol and ligated into the BamHI site of the EMBL3 vector. After testing 300,000 plates of labeled PR-Ib cDNA, 50 positive clones were isolated and characterized. Clone DNA from positive plates is purified and characterized using restriction analysis. As a result of this analysis, these clones were classified into eight different groups. One of the clones was identified as tobchrPR1013. A partial restriction map of tobchrPR1013 is shown in Figure 1. DNA isolation and manipulation is performed essentially as described by T. Maniatis et al., I.

Example XII. Preparation of pBS-PR1013CLa.

The CLaI restriction fragment from the tobchrPR 1013 clone is subcloned into the Bluescript plasmid (Stratagene Cloning Systems), resulting in the plasmid pBS-PR1013Cla (see Figure 2). A 2 kb Xhol-Bgl II restriction fragment from pBS-PR 1013Cla is sequenced. As found, positions 913 to 1711 (Fig. 39) correspond exactly to the sequence of the PR-1α cDNA clone isolated by U. M. Pfitzner et al., I. Based on sequence data and the restriction map of pBS-PR 1013Cla, digestion of tobacco genomic DNA

162 317 with EcoRI, Hind III, Xhol + Bgl II, or HindUI + PStl restrictionases should result in 3.2 kb, 6.7 kb, 2.0 kb or 6.4 kb fragments, respectively, that contain the PR gene -1 a. To test this assumption, 3pg of tobacco chromosomal DNA was digested with appropriate enzymes and electrophoresed on a 7% agarose gel. The DNA is transferred to a nylon membrane and probed with the labeled XhoI-BstEII restriction fragment from the PR-la gene. For controls, pBS-PR 1013Cla plasmid DNA is digested and electrophoresed. As predicted, digestion with EcoRI, HindUl, Xhol + B ^ Hl and HindUl + Pstl restrictionases resulted in fragments of the expected molecular weights that migrated with control bands. Therefore, the DNA contained in the tobchrPR1013 clone represents contiguous stretches of DNA from the tobacco genome.

Example XIII. Generation of the plasmid pBS-PR1013Eco.

A. The plasmid pBS-PR 1013Eco is constructed by subcloning a 3.6 kb EcoRI restriction fragment containing the PR-Ia gene from the tobchrPR 1013 (Example IX) at one EcoRI restriction site of the bluescript plasmid. The bluescript plasmid (Cat # 21203) is obtained from Strategene Cloning Systems, La Jolla, California. The structure of the pBS-PR 1013Eco plasmid is confirmed by both restriction mapping and DNA sequencing. This plasmid construction method is shown in Fig. 3.

B. Alternatively, plasmid pBS-PR1013Cla is digested with EcoRI restrictionase and the 3.6 kb fragment containing the PR-1 gene is isolated. pBluescript is digested with EcoRI, mixed, and ligated with the previously isolated 3.6 kb EcoRI restriction fragment. This plasmid construction is shown in Figure 4.

Example XIV. Preparation of the plasmid pBS-PR 1013Eco APst.

The plasmid pBS-PR 1013Eco was digested with PstI restrictionase and the DNA fragments were separated on a 2.5% low gelling agarose gel. A large fragment containing the bluescript vector and a 3.0 kb long tobacco DNA fragment is cut and heated to 65 ° C to melt the agarose. Then 2 µl is added to 15 µ water and the DNA is ligated into agarose as described in Example I. After incubation overnight, the DNA-containing agarose is melted by incubating at 65 ° C for 10 minutes and then transforming with agarose as essentially described in example II. An aliquot of the bacterial culture from each of the six putative positive constructs is inoculated with 5 ml of LB medium (Miller, uv) containing K 2 O / µg per ml of ampicillin and this culture is incubated overnight at 37 ° C. Cells are harvested by centrifugation in a clinical centrifuge at full rotation speed for 10 minutes. The supernatant is removed and the cells are resuspended in 100 µl of a solution with the following composition: 50 mM glucose, 25 mM Tris, pH 7.5, 10 mM EDTA and the suspension is transferred to 1.5 ml Eppendorf centrifuge tubes. Then 25 µl of a freshly prepared 10 mg / ml lysotime solution is added and the suspension is left on ice for 10 minutes. Then, 200 µl of a solution with the following composition: 0.2 N sodium hydroxide, 1% sodium dodecyl sulfate are added and the suspension is stirred and left on ice for 2 minutes.

Then 200 µl of 2M sodium acetate (pH 4.8) are added and the solution is left for 15 minutes on ice. The resulting solution is centrifuged at full hole speed in an Eppendorf centrifuge, the supernatant is removed to a new tube and extracted with 400 µl of phenol / chloroform (1: 1) mixture. The aqueous phase is removed to a new tube and extracted with 400 µl of chloroform. The aqueous phase from this extraction is removed to the next tube and DNA precipitated by adding two volumes of ethanol and storing at -20 ° C for 30 minutes. The precipitated DNA is collected by centrifugation for 15 minutes in an Eppendorf microcentrifuge at full speed of rotation. The ethanol is removed and the precipitates washed with 80% ethanol. The ethanol is removed again and the precipitates are allowed to air dry. DNA is resuspended in 100 µl TE buffer. The structures are verified by digestion with Pstl restrictionase. The parent plasmid gives a band of 6 kb and approximately 650 bp, the smaller of which does not persist in the new constructs. One of these plasmids was selected as the pBS-PR 1013Eco APst plasmid. The structure of this subclone is verified by sequence analysis. The construction of this plasmid is shown in Figure 5.

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Example XV. Preparation of the plasmid pBS-PR 1013EcoAPstAXho.

The initial restriction map of the pBS-PR 1013Eco plasmid is already established. The Xho I restriction site is located approximately 1200 bp from the Pstl site downstream. The plasmid pBS-PR 1013EcoA Pst was digested with XhoI restrictionase and the fragments were separated on a 0.9% low gelling agarose gel. As a result of electrophoresis, fragments with a length of 3.9 kb and 1.7 kb are visible. The band containing the 3.9 kb fragment is carefully cut from the gel and ligated in agarose essentially as described above. The agarose is then melted and the DNA transformed into a competent E. coli HB101 strain as described above. One permeable positive colony is inoculated into LB-amp medium and DNA is isolated essentially as described above. The structure of this novel pBS-PR 1013EcoA PstA Xho subclone is verified by restriction analysis and DNA sequencing. Fig. 6 shows the construction of this plasmid.

Example XVI. Generation of the plasmid pCIB270.

The plasmid pBHO1.3 (Cat # 6024. J) was obtained from Clonetech Labs, Palo Alto, California. This plasmid was digested first with BamHI and then with SalL. The plasmid pBS-PR 1013EcoA PstA Xho was digested with PstI restrictionase. Pstl BamHI adapter with sequence

5'-GGGATCCCTGCA-3 'is prepared as described in Example IV, and ligated to the pBS-PR 1013EcoA PstA Xho plasmid digested with PstI restriction. The ligation product is digested first with BamHI and then with XhoI. The BamHI / XhoI fragment is isolated, mixed and ligated into the digested plasmid pBT101.3, and then transformed into the bacteria. The putative positive clone pCIB270 is isolated and verified by restriction analysis and DNA sequence analysis. The method of producing this plasmid is shown in Figure 7.

Example XVII. Production of M13mpl8- or mpl9-PR1018EcoA PstA Xho plasmids.

The plasmid pBS-PR 1013EcoA PstA Xho was digested with Pstl and Asp718 restrictionases. An Asp718 Pstl fragment of 1.1 kb in length is isolated after electrophoresis on a 1% agarose low gel temperature wTAE gel. The replication form (RF) of the M13mpI8 or M13mpl9 coli phage was prepared according to J. Messing, Meth. Enzymol. 101, 20 (1983). Alternatively, these vectors can be obtained from Bethesda Research Labs. Gaithersburg, Maryland. Both vectors were digested first with Asp718 and then with Pstl. After removing the polylinker fragment by electrophoresis on a 0.7% low-gelling TAE agarose gel, the resulting vector is mixed and ligated in agarose with the 1.1 kb fragment prepared as described above. The DNA is transformed into bacteria, then putative positive colonies are isolated and the structure verified by DNA RF analysis. The construction of the MI3mpl8-i mpl9-PR1013EcoA PstA Xho is shown in Fig. 8.

Example XVIII. Production of plasmids M13mpI8- or mpI9-PR1013EcoA PstA Xho. Nco and pCIB 268.

Single-stranded M13mpl8-PR1013Ecoo Psto Xho phage DNA is isolated according to Messing, as in. An 18 bp primer oligonucleotide with the 5'-AATCCCATGGCTATAGGA-3 'sequence is synthesized as described above. The primer is phosphorylated with T4 polynucleotide kinase (T. Maniatis et al., Ibid., P. 125) using unlabeled ATP. After incubation at 37 ° C for 60 minutes, the reaction mixture is warmed to 68 ° C for 15 minutes and then cooled by placing it on ice. This primer and primer are sequenced Μ13-40 (New England Biolabs # 1212) heated to obtain M13mpl8-PR1013EcoA PstA Xho single-stranded DNA after mixing in a 1: 1: 1 molar ratio by slow cooling after heating for 10 minutes at 68 ° C C. The heated mixture is placed on ice and 1/10 volume of tenfold concentrated elongation buffer (20 mM Tris, pH 7.5, 6 mM MgCl2, 1 mM DTT, mM dATP, 1 mM dCTP, 1 mM dGTP and 1 mM dTTp) is added. .

Then 10 units of the large fragment of DNA polymerase I (Klenow fragment, New England Biolabs No. 210) and 0.1 units of T4 DNA ligase (NEB No. 202) are added and the reaction is carried out for 14 hours at 15 ° C. After this time, additional amounts of each enzyme are added and the reaction is carried out for an additional two hours at 25 ° C before using the mixture to transform a competent E. coli strain IM101. A diagram of the general flow of this procedure is shown in Figure 9.

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To identify mutant phages, plates are transferred to Gene Screen Plus (Dupont) and processed according to the manufacturer's instructions. The DNA on the filters is pre-hybridized for 4 hours and hybridized overnight at 42 ° C in manufacturer's hybridization solution containing 0.9 M NaCl. The filters are then washed continuously with 0.9 M NaCl and monitored by autoradiography, increasing the washing temperature by 5 ° C at each step. Phage DNA identified as mutated by hybridization is sequenced to confirm the base change.

DNA of a replicating form of one such candidate (M13mpl8-PRI013EcoA PstA Xho. Nco) was isolated and digested with PstI and BstEII to generate a 394 bp fragment which is used to replace the corresponding non-mutated 394 bp fragment in the pBSA-PR1013 plasmid PstA Xho as illustrated in Figure 10. The result is plasmid pCIB268 having an NcoI restriction site at position 930 followed by 217 bp of the coding sequence of the PR-1a gene. The structure of this plasmid is confirmed by sequence analysis.

Example XIX. Generation of chimeric genes containing GUS and variable length PR promoter sequence.

A. Construction of the plasmid pCIB269.

The 930 bp fragment is isolated by electrophoresis on a 1.0% low gelling agarose gel in TAE of plasmid pCIB268 digested with Xhol and NcoI restrictionases. This fragment is then ligated with the plasmid pBS-GUS.1.2 (see example ΧΙΧΒ) digested with Xho I and NcoI restrictionases. Transformants are isolated and subjected to restriction analysis and DNA sequencing. One positive plasmid was selected and named pCIB269. The construction of this plasmid is shown in Figure 11

B. Construction of the pBS-GUS1.2 plasmid.

The plasmid pBS-GUS1.2 is generated by a three-part ligation of the SalL / SnabI restriction fragment derived from pRA265 [R. A. Jefferson et al., EMBO J. 6.3091-3907 (1987) and GUS User Manual, Clonetech Labs., Palo Alto, Ca] with a 1707 bp Snabl / EcoRI fragment with plasmid pBI221 (RA Jefferson et al., EMBO J. , ibid.) and the plasmid pBS digested with Sa111 EcoRI restrictionases, see Figure 12. Transformants are isolated and analyzed by restriction digest and DNA sequencing. One of the verified clones was named pBS-GUS1.2.

C. Construction of the plasmid pCIB200.

First, TJS75Kan is generated by digesting plasmid pTJS75 [Schmidhauser and Helinski, J. Bacteriol. 164,446-455 (1985)] with Narl restrictionase to excise the tetracycline resistance gene followed by insertion of the AccI restriction fragment derived from plasmid pUC4K [J. Messing and

J. Vierra, Gene 19, 259-268 (1982)] carrying the NptI gene. Plasmid pCIB 200 is then generated by ligating the XhoI linkers with an EcoRI restriction fragment derived from plasmid pCIB7 [containing left and right T-DNA border sequences, plant selectable nos / nptII chimeric gene and pUC polylinker, SJ Rothstein et al., Gene 53, 153-161 (1987)] and cloning of the XhoI digested fragment into SalI digested TJS75Kan.

D. Construction of plasmid pCIB271.

Plasmid pCIB269 (Example 19A) was digested with Xho I and EcoRI restrictionases, and after electrophoresis on a 10% low-gelling TAE agarose gel, a 3023 bp fragment carrying the PR-1? Gene promoter connected to the 3 'end of the nose was isolated. This fragment is ligated with a vector that can be expressed in a variety of pCIB200 cells digested with Sal and EcoRI restrictionases. Transformants are then isolated and restriction analysis performed. One of the verified clones was named pCIB271. The construction of this clone is shown in Figure 13.

E. Construction of plasmid pCIB272.

Plasmid pCIB268 (Example 18) was digested with Alul and NcoI restrictionases, and then, after electrophoresis on a 0.7% TAE agarose gel, an 833 bp fragment was isolated. This fragment is ligated to the 3'-glucuronidase gene with the 3'-nose end in pBS-GUS1.2 (Example XIX B). The promoter and GUS are then excised from this plasmid, named pCIB282, as an Asp7181 / BamHI restriction fragment, and ligated into the pCIB200 plasmid that has been digested with restrictionases

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Asp71811 BamHI before transforming DH5aE strain. coli. Transformants are isolated and restriction analysis performed. One of the verified clones was named pCIB272.

F. Construction of plasmid pCIB273.

Plasmid pCIB268 was digested with Haelil and NcoI restrictionases and then, after electrophoresis on a 0.7% TAE agarose gel, a 603 bp fragment was isolated. This fragment is placed upstream of the 3'-nose / 3-glucuronidase gene in pBS-GUS1.2. The promoter and GUS are then excised from this plasmid, named pCIB283, as an Asp7181 / BamHI restriction fragment, and ligated to the pCIB200 plasmid, which was digested with Asp7181 and BamHI restrictionases prior to transformation of E. coli DH5a strain. Transformants are isolated and restriction analysis performed. One of the verified clones was named pCIB273.

Example XX. Generation of a chimeric gene in a vector carrying the hygromycin resistance gene.

Plasmid pCIB269 was digested with Xhol and EcoRI restrictionases, and then, after electrophoresis on a 1.0% low-gelling agarose gel in TAE, a 3023 bp fragment carrying the promoter of the PR-Ia gene linked to the 3 'GUS gene was isolated. nose. This fragment is then transformed into an Xhol Sali restriction fragment by ligation with an adapter having the sequence:

5'-AATTCGTCGACG-3 '.

The XhoI SalI restriction fragment is ligated to the Sa11-digested plasmid pCIB712 (S. J. Rothstein et al., I. V.) To generate plasmid pCIB219, (see Figure 14).

Example XXI. Generation of the plasmid pCIB200 / PR 1-BT.

A. Ligation with the pCIB200 plasmid.

Plasmid pCIB1004 (see below) is digested with SphI restrictionase, phenol-extracted, ethanol precipitated and resuspended. An oligonucleotide having the sequence is synthesized

Y-CCGGTACCGGCATG-3 'purifies it, phosphorylates it, and ligates it with the SphI-digested plasmid pCIB1004. After ligation, it is ligase inactivated and DNA digested with KpnI restrictionase. This DNA was electrophoresed on a 0.8% low gelling agarose gel. Plasmid pCIB200 (Example 19C) is digested with KpnI restrictionase and treated with calf gut alkaline phosphatase, followed by electrophoresis as described above. DNA from the pCIB200 vector PR-1 fusion vector bands DNA 5'-end flanking sequences (BT is mixed and ligated to form the pCIB200 plasmid) PR-1-BT.

B. Generation of plasmid pCIB1004.

Plasmid pCIB10 (35Bt / 607) was digested with NcoI and BamHI restrictionases and electrophoresed on a 0.8% low gelling agarose gel. Plasmid pCIB269 (Example XIX A) was digested with NcoI and XhoI restrictionases and electrophoresed as described above. The plasmid pCIB710 (S. J. Rothstein et al., Ibid.) Was digested with Sal and BamHI restrictionases and electrophoresed as described above. Bands containing the SalI and BamHI restriction digested vector comprising the 3 'end of the CaMV 35S promoter cloned into pUC19, the BT gene and the 5' flanking region of PR-1 are excised, mixed and the DNA ligated to form plasmid pCIBI004.

C. Generation of plasmid and pCIB10 (35Bt / 607).

Plasmid pCIB10 (35Bt / 607), a partially deleted protoxin gene containing a sequence encoding approximately 607 amino acids, is produced from plasmid pCIB10 / 35Sbt, a plasmid containing the Bacillus thuringiensis endotoxin protoxin gene. E. coli strain MC 1061 containing the pCIB10 / 35Stb plasmid was deposited with the American Type Culture Collection, ATCC No. 67329, February 27, 1987. The partially deleted protoxin gene is constructed by inserting a BamHI restriction site (GGATCC) recognition site after nucleotide 1976 in the BZ gene sequence. This is done by cloning the BamHI restriction fragment containing the protoxy sequence derived from the pCIB10 35Stb plasmid into mpl8 and using standard oligonucleotide mutagenesis procedure. Following mutagenesis, a double-stranded replicative form of DNA is prepared from the M13 clone, which is then digested with BamHI. An approximately 1.9 kb fragment containing the partially deleted protoxin gene is inserted into the BamHI-cleaved plasmid pCIB 10 710. The resulting plasmid is selected for kanamycin. For a detailed description of the preparation, see U.S. Patent Application No. US No. 122,109, filed November 18, 1987.

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Example XXII. Generation of the plasmid pCIB1233 (pCIB200 PR1-AHAS).

A. Ligation with the pCIB200 plasmid.

Plasmid pCIB200 followed by pCIB1216 was digested with KpnI and XbaI restrictionases and electrophoresed on a 0.8% low gelling agarose gel. The band containing the approximately 4.3 kb DNA containing the 5'-flanking PR-1 flanking sequences fused to the AH AS coding sequence and the band containing the pCIB200 vector is then excised, mixed the DNA and ligated to form plasmid pCIB1233

B. Generation of plasmid pCIB1216.

Plasmid pCIB269 (Example 19A) followed by pCIB1207 was digested with XbaI NcoI restrictionases and electrophoresed on a 0.8% low gelling agarose gel. The band containing the bluescript vector with PR-1 5 'flanking sequences and the 3.3 kb DNA band including the AHAS coding sequence are then excised, mixed and ligated to form plasmid pCIB1216.

C. Generation of plasmid pCIB1207 containing the Arabidopsis AHAS gene.

Total DNA is isolated from 5 week old Arabidopsis thaliana plants, Colombia ecotype. 250 µg of this DNA was digested with 4,000 units of Xbai restrictionase for 4 hours. The digestions were fractionated by electrophoresis on a 0.8% agarose gel. A DNA fragment with a length in between

6.5 kb and 4.36 kb (based on length markers from HindIII-digested lambda phage DNA) are isolated from the gel using NA-45 DEAE membrane (Schleicher and Schuell, Keene, NH, USA) according to the manufacturer's recommended procedure . An ongC (longC) phage DNA arm preparation digested with Xbal restriction and treated with phosphatase was obtained from Stratagene Cloning Systems (La Jolla, CA, USA). 0.1 pg of the XbaI Arabidobsis restriction fragment inserted is ligated to the longC arm DNA according to the procedure recommended by Stratagene. 2.5 µl of the ligation product is packed into lambda phage heads using a Gigapack Plus kit (Stratagene Cloning Systems) according to the manufacturer's recommended procedure. The activity of the bank is assayed on E. coli strain VCS257 (Stratagene Cloning Systems).

50pg DNA of pE168-c4 plasmid (J. Polarna, Carlsberg Res. Comm. 49, 577-584) was digested with EcoRI restrictase and the digests were electrophoresed on a 0.8% agarose gel. A 2 kb EcoRI restriction fragment containing the yeast AHAS gene sequence is isolated from the gel using a NA45 DEAE membrane according to the manufacturer's recommended procedure. Radiolabeled probes for a 2 kb fragment are synthesized on the day of the hybridization reaction with the random primer mix using the Prime Time kit (International Biotechnologies Inc., New Haven, CT, USA) according to the manufacturer's recommended procedure.

250,000 recombinant phages are plated on the lawn of strain VCS257. The plaque-derived phages are then transferred to the membranes using Colony / PIaque Screen membranes (NEW Research Products, Du Pont, Boston, MA, USA) according to the manufacturer's recommended procedure. The DNA on the membranes is pre-hybridized in LS Hybridization Buffer, then transferred to 150 ml of fresh LS Hybridization Buffer containing 0.25 µg of Phosphorus ³² P-labeled yeast AHAS probe prepared as described above. The membranes are incubated for 16 hours at 42 ° C with gentle shaking, then washed twice for 15 minutes, at room temperature with twice with concentrated SSC, washed three times for 2 hours at 42 ° C with LS wash buffer (25% formic acid amide, five times concentrated SSC, 1% SDS) and washed twice for 30 minutes with washing buffer (0.1-fold concentrated SSC, 0.1% SDS). The membranes are removed and fluorographed using Cronex Lightening PlusScreens (Du Pont, Wilmington, DE, USA) and XAR-5 film (Kodak). Plaques from the plaque regions that give positive signals on the film film are selected and phages derived from them are replicated at low density - phages from 300 plaques per plate with a diameter of 15 cm. Replication is repeated until single hybridizing phage clones are isolated.

Small scale preparation of phage DNA from the plaque purified clone is performed according to the procedure using the LambdaSorb Phage Adsorbent kit (Promega, Madison, WI, USA). The phage DNA is cut with XbaI restrictionase and the inserted 5.8 kb fragment is cloned into the Xbai site of the pBS (-) plasmid (Stratagene Clonig Systems). The identity of the cloned fragment with the XbaI restriction fragment containing the Arabidopsis AHAS gene is verified by comparing its restriction map and DNA sequence with those publications for the gene by

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Haughn et al., Mol. Gen. 211, 266 (1988) and Mazura et al., Plant Physiol, 85, 1110 (1987). This plasmid was designated pCIB1207.

Example XXIII. Generation of the plasmid pCIB1232 (pCIB200 / PRl-AHAS-SuR).

A. Ligation with the pCIB200 plasmid.

Plasmids pCIB200 (Example 19C) and pCIB1230 were digested with KpnI and XbaI restrictionases and electrophoresed on a 0.8% low gelling agarose gel. The band containing approximately 4.3 kb DNA containing the 5 'flanking sequences fused to the AHAS-SuR coding sequence and the band containing the pCIB200 vector are then excised and the DNA mixed and ligated to form plasmid pCIB1232.

B. Generation of plasmid pCIB1230.

Plasmid pCIB1208 (see below) was digested with XbaI and NcoI restrictionases and the fragments were separated by electrophoresis on a 0.8% low-gelling wTAE agarose gel. Plasmid pCIB269 (Example XIX A) was digested with NcoI and XbaI restrictionases and electrophoresed as described above. The fragment of the plasmid pCIB269 containing the bluescript vector and the promoter of the PR-1 gene and the 3.3 kb fragment containing the mutant AHAS gene [identified by cross-homology and restriction fragment analysis with the gene described by Mazura et al., Plant Physiol. 85, 1110-1117 (1987)] is cut, melted agarose and ligated together to form a clone designated pCIB1230.

C. Generation of plasmid pCIB1208.

A genomic DNA bank is constructed as described in Example XXIIC, using DNA isolated from Arabidopsis plants selected for sulfonylurea herbicide resistance as described by Haughn and Somerville, Mol. Gen. Genet. 204, 430-434 (1986). Plaques containing genes encoding acetohydroxy acid synthase (AHAS) are identified by cross-hybridization with a yeast acetohydroxy acid synthase gene probe (see Example XXII C). The recombinant DNA is subcloned into Bluescript (Stratagene) creating a clone designated as pCIB1208. This plasmid contains a gene encoding a mutant acetohydroxy acid synthase that is resistant to inhibition by sulfonylurea herbicides.

Example XXIV. Isolation of a genomic clone encoding the basic form of / 3-1,3-glucanase (glucan endo-1,3 / 3-glucosidase) from Nicotiana tabaccum.

High molecular weight DNA is obtained from the leaves of Nicotiana tabacum cv. Havana 425 via the CETAB procedure [Murray and Thompson, Nucleic Acid Res. 8, 4321 (1980)]. 100pg of DNA is digested completely with SacI restrictionase. This DNA is separated by 0.8% agarose gel electrophoresis. The region of the gel corresponding to the molecular weight range of the fragments from 3 to 7 kb is cut into 6 sections of equal size and the DNA of these sections is electroeluted and precipitated as separate fractions. The DNA is resuspended and the amount from each fraction is electrophoresed on an agarose gel and analyzed by Southern blot. Fractions containing DNA that hybridizes to the cDNA / 3-1,3-glucanase probe [H. Shinshi et al., Proc. Natl. Acad. Sci. USA 85, 5541-5545 (1988)] is summed up and used to construct the bank.

LambdaOngC vector purchased from Stratagene Corp. digested with SacI. 1 µg of this DNA is mixed with 0.1 µg of SacI-restricted tobacco DNA and ligated with T4 ligase according to the manufacturer's instructions. The ligated DNA is then packaged into phage particles using the m vitro packaging procedures of Stratagene. The phages are seeded with bacteria as suggested by the descriptions for the lambda phage. Approximately 75,000 plaques were tested using a phosphorus 3 ² P labeled cDNA / 3-1,3-glucanase probe. 11 positive plaques were identified. The phages contained in these plaques are purified by successive screening and testing.

DNA is isolated from the purified clones and the clones analyzed by restriction digestion of their DNA using HindUI, EcoRI and SacI restrictionases. Two types of clones were found, one represented by clone 39.1 and the other represented by clone 39.3. 4.5 and 4.7 kb inserts in clones 39.1 and 39.3, respectively. is subcloned in SacI restractase digested bluescript plasmid and the pBSGluc39.1 (SacI restriction fragment obtained from lambda 39.1 clone) and pBSGluc39.3 (restriction fragment obtained from lambda 39.3 clone) subclones are isolated. The DNA sequences of the restriction fragments contained in the pBSGluc39.1 and pBSGluc39.3 subclones are determined by DNA sequencing. The sequences are shown in Figures 43 and 44 respectively.

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A coding sequence was found and a large intervening sequence was located near the 5 'end of the coding sequence.

Example XXV. Identification of the transcription start site of the gene / 5-1,3-glucanase.

A. Mapping of the elongated primer.

A synthetic DNA primer complementary to bases 2399 to 2416 is synthesized and used for primer extension experiments as described in Example 5. RNA for these experiments is isolated from tobacco infected with Phytophora parasitica var. nicotiana. Primer extension products are electrophoresed along with molecular weight standards in which the labeled primer is used in a DNA sequencing reaction with didesoxynucleotides with the pBSGluc39.1 subclone used as the reference. Extension products corresponding to positions 1433, 1446 and 1447 of the sequence / 3-1,3-glucanase 39.1, Figure 43, are identified after gel electrophoresis and autoradiography as described in Example V. Since there is a large intron between positions 1554 and 2345 of the pBSGluc39.1 sequence , the resulting molecular weight image may not correctly reflect the elongation products. Therefore, a second primer extension mapping experiment is performed using a primer complementary to positions 1530 to 1547. Using this primer, the three 5 'ends of the glucanase mRNA are mapped to site A at positions 1434, 1448 and 1449.

B. SI nuclease mapping.

A synthetic oligonucleotide complementary to positions 1415 to 1504 is synthesized for use as probes in SI nuclease mapping. The oligonucleotide is phosphorylated at the 5 'terminus using ^ P-ATP and T4 polynucleotide kinase according to the supplier's recommendations. After phenol extraction and ethanol precipitation, the tagged oligonucleotide is resuspended in hybridization buffer containing formic acid amide (see Example 6) at a concentration of approximately 2 nM. The RNA used in these experiments is isolated from tobacco infected with Phytophthora parasitica as described in the previous example. S1 nuclease mapping is performed on this RNA using a tagged oligonucleotide as described in Example VI. After gel electrophoresis, three bands were detected which correspond to positions 1434, 1448 and 1449 of the pBSGluc39.1 DNA sequence. '

C. Determining the start of transcription.

Both the primer extension mapping and SI nuclease mapping procedures locate the 5 'ends of the mRNA at positions 1434, 1448 and 1449. Therefore, these sites, all adenine residues, correspond to the transcription start site of the gene / 5-1,3-glucanase.

Example XXVI. Production of the plasmid pBSGluc39.1 / GUS. Oligonucleotides with sequences

A / 5'-GTTTATGTGAGGTAGCCATGGTGGGAAGACTTGTTGGG-3 '

B / 5'-GATCGCGGTACCGAGCTCCTCTAGGGGGCCAAGG-3 'is synthesized, purified and used in a polymerase catalyzed reaction (PRC) according to the supplier's directions (Perkin Elmer Cetus, DNA thermal cycler and Perkin Elmer Cetus, GeneAmp Reagent Kit) for amplification of a DNA fragment 1494 bp from plasmid pBSGluc39.1. The oligonucleotides are designed to add a KpnI restriction site immediately to the 5 'side of the SacI restriction site at the first base pair of the glucanase sequence and to generate a new NcoI restriction site at 1462 base pair. The DNA obtained after the PCR amplification procedure is phenol / chloroform extracted and ethanol precipitated. DNA is resuspended and digested with both NcoI and KpnI restrictionase and electrophoresed on a 0.8% low gelling agarose gel. The band containing the 1462 bp DNA is cut and used in the next ligation. The plasmid pBS-GUS 12 (Example XIX B) was digested with NcoI and KpnI restrictionases, electrophoresed on a 0.8% agarose gel and the band containing the vector excised. The band containing the pBS-GUS 12 vector and the band containing the 1462 bp DNA from the PCR reaction are ligated and used to transform E. coli. Positive colonies are selected and tested for a 1462 bp DNA insert. The plasmids containing the inserts are presumably clones of pBSGluc39.1 / GUS, however, due to the relatively high mutation frequency of this procedure (about 1/1000 bases), several clones were sequenced from the start to the end of the 1462 bp fragment. One of the clones having the expected sequence was selected as the pBSGluc39.1 / GUS clone.

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Example XXVII. Generation of the plasmid pSGluc39.3 / GUS.

Oligonucleotides with sequences.

A / 5'-GTTTATCTGAGGTAGCCATGGTGAGAAGACTTGTTGGA-3 '

B / 5'-GATCGCGGTACCGAGCTCCCTTGGGGGCAAG-3 'is synthesized, purified and used in a PCR catalyzed reaction to amplify a 1677 bp DNA fragment from plasmid pBSGIuc39.3. The oligonucleotides are designed to insert a new KpnI restriction site immediately adjacent to the SacI restriction site at position 39.3 first glucanase sequence and a new restriction site at position 164.5. DNA obtained after PCR amplification is extracted with fcnol / chloroform, precipitated with ethanol, resuspended, digested with both NcoI and KpnI, and electrophoresed on a 0.8% low-gelling agarose gel. The band containing the 1646 bp DNA is cut and used in the next ligation. Plasmid pBS-GUS1.2 is digested with NcoI I and KpnI restrictionases and subjected to electrophoresis as described above. The band containing the vector is cut, mixed with the band containing the 1646 bp DNA from the PCR reaction, and ligated to form the pBSGluc39.3 / GUS plasmid. Several permeable positive plasmids are isolated and sequenced previously. One of the plasmids with the expected sequence was selected as the pBSG! Uc39.3 / GUS plasmid.

Example XXVIII. Generation of the plasmids pCIB200 / Gluc39.1-GUS and pCIB200 / Gluc

39.3- GUS.

A. The plasmids pCIB200 (Example 19C) and pBSGluc39.1 / GUS (Example XXVI) were digested with KpnI and XbaI restrictionases and electrophoresed on a 0.8% low gelling agarose gel. The band containing the pCIB200 vector and the band containing the KpnI-XbaI restriction fragment including the 5 'flanking sequences of the glucanase gene are fused to the GUS gene, excised and DNA ligated to form the plasmid pCIB200 / Gluc39.1-GUS.

B. Similarly, plasmid pBSGluc39.3 / GUS (Example XXVII) is digested and ligated to the digested plasmid pCIB200 to generate plasmid pCIB2OO / Gluc39.3-GUS.

Example XIX. Generation of the plasmids pCIB200 / Gluc39.1-BT and pCIB200 / Gluc39.3-BT.

A. PCIB200 / Gluc39.1-BT.

The plasmids pBSGluc39.1 / GUS and pCIB1004 (example ΧΧΙ / Β) were digested with KpnI and NcoI and electrophoresed on a 0.8% low gelling agarose gel. Plasmid pCIB200 (Example XIXC) is digested with KpnI restrictionase, dephosphorylated using calf intestinal alkaline phosphatase and subjected to electrophoresis as described above. The band containing the pCIB200 vector, the band containing the 5'-flanking region of the glucanase gene, and the band containing the BT gene are excised, mixed together and ligated with DNA to form the plasmid pCIB200 / Gluc39.3-BT.

Similarly, the pBSGluc39.3 / GUS plasmid was digested and ligated with the digested plasmids pCIB 1004 and pCIB200 to generate the plasmid pCIB200 / Gluc39.3-BT.

Example XXX. Generation of pCIB200 / GIuc39.1-AHAS and pCIB2 (M) / Gluc plasmids

39.3- AHAS.

A. Ligation with the pCIB200 plasmid.

The plasmids pBSGluc39.1 / AHAS and pCIB200 (Example XIXC) were digested with KpnI and XbaI restrictionases and electrophoresed on a 0.8% low-gelling agarose gel. The band containing the glucanase / AHAS fusion and the band containing the expression vector pCIB200 are cut, mixed and DNA ligated to form the plasmid pCIB200 / Gluc39.1-AHAS.

Similarly, the pBSGluc39.3 / AHAS plasmid is digested and ligated to the digested pCIB200 plasmid to form the pCIB200 / Gluc39.3-AHAS plasmid.

B. Generation of the plasmids pBSGluc39.1 / AHAS and pBSGIuc39.3 / AHAS.

PBSGluc39 plasmids. 1 / GuS (Example XXV) and pCIB 1207 (Example XXIIC) were digested with NcoI I and Xbal restrictionases and electrophoresed on a 0.8% low gelling agarose gel. The band containing the 5 'flanking glucanase sequences and the vector, and the band containing the AHAS coding sequence are excised, mixed, and DNA ligated to form the pBSGluc39.1 / AHAS plasmid.

Similarly, plasmid pBSGluc39.3 / GUS (Example XXVII) is digested and ligated to the digested plasmid pCIB 1207 to generate plasmid pBSGluc39.3 / AHAS.

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Example XXXI. Preparation of the plasmids pCIB200 / Gluc39.1-AHAS-SuR and pCIB200 / Gluc39.3-AHAS-SuR.

A. Ligation with the pCIB200 plasmid.

The plasmids pBSGluc39.1 / AHAS-SuR and pCIB200 (Example XIXC) were digested with KpnI and XbaI restrictionases and electrophoresed on a 0.8% low-temperature agarose gel. The band containing the AS-SuR glucanase / AH fusion and the band containing the pCIB200 expression vector are excised, mixed and DNA ligated to form the pCIB200 / Gluc39.1-AHAS-SuR plasmid.

Similarly, the pBSGluc39.3 / AHAS-SuR plasmid is digested and ligated to the digested pCIB200 plasmid to form the pCIB200 / Gluc39.1-AGAS-SuR plasmid.

B. Generation of plasmids pBSGluc39.1 / AHAS-SuR and pBSGluc39.3 / AHAS-SuR.

PBSG1uc39 plasmids. 1 / GUS (Example XXVI) and pBSG1uc39.3 / GUS (Example XXVII) are digested with NcoI and XbaI restriction fragments, and then the restriction fragments are separated on 0.8% gels and low gelling agarose in TAE. Plasmid pCIB1208 (Example XXIIIC) was digested with XbaI and NcoI restrictionases and the 3.3 kb fragment containing the mutant AHAS gene (sulfonylurea herbicide resistant, SuR) was isolated by agarose gel electrophoresis. The fragments containing the glucuronidase gene promoter and the pBS vector are excised, agarose melted, and the DNA ligated with the AHAS fragment to construct clones designated pBSGluc39.1 / AHAS-SuR and pBSGluc39.3 / AHAS-SuR, respectively.

4. Identification of new genes.

The following examples describe the isolation and characterization of the remaining novel PR protein genes.

Example XXXII. Isolation of other genomic and cDNA clones.

A. Isolation of a genomic clone encoding PR-1 '.

Bankgenomic is constructed and tested with the PR-1 cDNA probe as described in Example XI. After testing, the appropriate clone is isolated by labeling it lambdatobchrPR 1019. Then, the restriction map is initially determined. The restriction fragment of the Bodies is subcloned into the bluescript vector and the restriction map of the pBS-PR1019Cla plasmid is then mapped. The large restriction fragment XhoI is deleted from this plasmid to give the plasmid pBS-PR1019Cla Xho. This plasmid contains a 2.7 kb long tobacco DNA fragment containing the PR1019 gene. This plasmid is partially deleted and a series of deletions are used for DNA sequencing.

Figure 40 shows approximately 21 (X) bp of this gene. The protein encoded by PR1019 was found to be approximately 65% homologous with PR-1α, PR-1b or PR-1c, therefore this new PR-1 'gene was named.

B. Isolation of a cDNA clone encoding cucumber chitinase.

Pathogen-induced chitanases are isolated from infected cucumber leaves as described [J. P. Metraux et al., Physiological and Molecular Plant Pathology, 33, 1-9 (1988)]. From this homogeneous protein preparation, peptides are prepared essentially by methods commonly known in the art and as described in Examples 9 and 10. Two protein regions are selected and oligonucleotide probes are synthesized complementary to all possible template RNA variants capable of encoding these proteins. These sequences are:

AT A

Probe 1: 5'-CCATTTCTGNCCCCAGTA-3 '

G G G G C

Probe 2: 5'-GGATTATTATAAAATTGNACCCA-3 '

Cucumber leaves are infected with Tabacco Necrosis Virus (TNV) and 5 days post infection RNA is isolated as described in Example V. Polyadenylated RNAs are isolated by standard techniques and a cDNA bank is constructed in a lambda Zap vector (Stratagene) essentially as described by U. Gubleri BJ Hoffman , Gene 25, 263 (1983). 300,000 plaques from the plated plates are transferred to membranes and tested using either a phosphor P-labeled oligonucleotide mixture 1 (probe 1) or a mixture 2 (probe 2) as the probe. Plaques showing positive results when tested with either probe are isolated. Phage isolation and automatic excision are performed as described in the laboratory description for Stratagene Lambda Zap.

At the same time, chitazine cDNA clones are isolated in the bluescript vector and sequenced by the didesoxynucleotide method. The sequence of the chitazine gene is shown in Figure 41.

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C. isolating a cDNA clone encoding the major form of PR-R.

Nicotiana tabaccum cv Xanthi-nc leaves are infected with tobacco mosaic virus and harvested 5 days post infection. Total RNA is prepared as described in Example 5 and polyadenylated RNA is isolated using standard techniques. A cDNA bank is constructed using this polyadenylated RNA in the Lambda ongC vector (Stratagene). Approximately 300,000 plaques are tested using the sequence oligonucleotide probe

5'-GAACTTCCTAAAAGCTTCCCCTTTTATGCC-3 '

Phage from positive plaques are purified by standard techniques and phage DNA is prepared. The recombinant phage fragment containing the cDNA is subcloned into the bluescnpt plasmid. The sequence of this cDNA is determined. The sequence of one clone encoding the major form of PR-R (W.

S. Pierpoint et al., Physiol. Plant Pathol. 31, 291 (1987)] is shown in Fig. 42. The protein encoded by it has a very strong homology to the known trypsin / σ-amylase inhibitor isolated from maize [M. Trichardson et al., Nature 327,432 (1987)]. The cloned PR-R protein may be an inhibitor of either proteases or σ-amylases and thus confer upon plants expressing transgenic insecticidal, virucidal or fungicidal activity.

D. Isolation of the cDNA encoding PR-Q.

Nicotiana tabaccum cv Xanthi-nc leaves are infected with tobacco mosaic virus and harvested 5 days post infection. Total RNA is prepared as described in Example 5 and isolates polyadenylated RNA using standard techniques. A cDNA bank is constructed using this polyadenylated RNA in the lambda ongC vector (Stratagene). Approximately 300,000 plaques are tested using a labeled cDNA probe encoding the tobacco basic chitazine gene and washing the filters in a solution composed of: 0.125 mM NaCl, 1% SDS, 40 mM sodium phosphate (pH 7.2), 1 mM EDTA at 50 ° C . 24 positive plaques are purified from phage and the DNA sequence of a clone named pBSchtl5 is determined. This sequence is shown in Fig. 45.

The PR-Q protein is partially purified from the intracellular fluid of TMV infected tobacco leaves as described in Example VIII. The fractions after chromatography on Ultragel ACA54 containing the PR-O, PR-R, PR-Q and PR-R proteins are collected and concentrated by lyophilization. The proteins are further purified by polyacrylamide gel electrophoresis. The protein band containing PR-Q is excised and the protein is eluted according to Applied Biosystems User Biulletin No. 36, March 21, 1988. The protein is digested with pyroglutamate aminopeptidase and sequenced by automatic Edman degradation as described in Example IX. The N-terminal sequence of PR-Q (Xxx amino acid uncertain) was determined:

GlnGlyIleGlySerIleValThrXxxAspLeuPheAsnGluMetLeu

The protein encoded by the clone sequence shown in Figure 45 was determined to be related to the pathogenesis of protein Q on the basis of: 1 / limited structural homology to tobacco basic chitazine 2 / identity with the N-terminal sequence of the PR-Q protein as determined by its sequencing.

E. Isolation of cDNA encoding the acid form of J-1,3-glucanase.

Approximately 300,000 plaques from the cDNA bank described in Example XXXIID (above) are tested using a labeled cDNA probe encoding the basic form of β1,3-glucanase (H. Shinshi et al., As above) and washing the filters with a solution composed of: 0.125 M NaCl , 1% SDS, 40 mM sodium phosphate (pH 7.2), 1 mM EDTA at 50 ° C. 20 positive plaques are isolated and the insert is subcloned into the bluescript plasmid. Partial DNA sequencing revealed that clones 5 and 6 encode identical cDNAs that shared 55% homology with the known basic form / 1-1.3-glucanase DNA sequence. The cDNA sequence of clones 5 and 6 is determined; this sequence is shown in Figure 46. The conclusion that this cDNA encodes the acidic form of β-1,3-glucanase is drawn based on the limited amino acid sequence homology and the more acidic isoelectric point of the encoded protein.

F. Isolation of a genomic clone encoding the cucumber chitinase gene.

The cell nuclei are isolated from Cucumis sativus cv. Long Marketer and isolates DNA as described in Example 11. 3 pg of this DNA was digested with either EcoRV, EcoRI, BamHI or H ind 111 restrictionases and separated the restriction fragments on a 0.5% agarose gel. Southern analysis using cucumber chitazine cDNA (Figure 41) showed the presence of a single band in the EcoRI digested DNA corresponding to a 12 kb fragment. That's why

162 317 43 it was decided to isolate the cucumber chitinase gene using all the DNA fragments obtained after digestion with EcoRI and IIgating with the Lambda vector "replacement".

10pg of DNA isolated as described above is digested completely using EcoRI restrictase. This DNA is then extracted with phenol, ethanol precipitated and ligated at the EcoRI EMBL 4 restriction site. Approximately 100,000 plaques are tested with a labeled cucumber chitinase cDNA probe (Figure 41). 10 positive plaques are selected, phage derived from them are purified and the insert DNA is analyzed by pre-mapping by restriction digest. The same results were obtained for all clones and therefore one was selected for further analysis. The 12 kb insert of this clone is then subcloned into a bluescript plasmid to give the pBScucchi / chitinase plasmid. A restriction map for this cloned DNA is established, the fragments are subcloned, and the DNA sequence determined.

G. Isolation of a genomic clone encoding acid / 3-1,3-glucanase.

A genomic bank is constructed as described in Example 11 and tested with the acid / 51.3-glucanase cDNA clone, Example XXXII E. The lambda clone is isolated and its 1 kb EcoRI restriction fragment is subcloned into Bluescript as described above in Example XXXII A. The bluescript clone was named pBSGLóe.

5. Stable transformation and regeneration of plants.

Plant tissue is transformed with the vectors described above by each of the commonly known methods. Such methods used to transfer DNA into plant cells include, for example, directly infecting plants, plant tissue or cells, or growing them together with A. tumefaciens [R. B. Horsch, et al., Science 225, 1229 (1985). L. Marton, Cell Cultureand Somatic Cell Genetics of Plants, Vol. 1, 514-521, 1984], treatment of protoplasts with exogenous DNA by methods such as those described in the following publications: J. Paszkowski et al., EMBO J. 3.2717 (1984), European Patent Application No. O 164 575, RD Shillito et al., Bio / Technology 3.1099 (1985), I. Potrykus et al., j. w., H. Loerz, et al., Mol. Gen. Genet. 199, 178 (1985), M. Fromm et al., Ibid. British Patent No. 2,140,822 and I. Negrutiu et al., Plant Mol. Biol. 8, 363 (1987), incubation with polyethylene glycol (PEG) (I. Negrutiu et al., I. V.), Microinjection [T. J. Reich et al., Bio / technology 4, 1001-1004 (1986), T. J. Reich et al., Can J. Bot, 64, 1259-1267 (1986)], by the technique of "microprojectile bombardment [T. M. Klein et al., Nature 327, 70, (1987)].

Example XXXIII. Agrobacterium transformation.

The DNA of the plasmids pCIB270 (Example XXVI), pCIB271, pCIB272 and pCIB273 (Examples XIX D, E and F) was graded with cesium chloride / 2,7-diamino-9phenyl bromide ^ -K ^ -e ^ mp ^ 1t ^ ft ^ r ^^ and ^^^ yidinium and is used to transform A. tumefaciens A136 strain containing the helper plasmid pCIB542 according to the procedure of M. Holsters et al., Mol. Gen. Genet. 163, 181-187 (1978), to obtain strains CIB270, CIB271, CIB272, CIB273.

Using the same procedure, plasmids pCIB271, pCIB272, and pCIB273 were transformed into lrulertre strains of P. tumefaciens - 151955, A208, A281 and strain A-4 of A. rhizogenes, generating strains designated as pCIB271 X 15955, pCIB271 X A208, pCIB271, pCIB271 X A208, pCIB271 X A4, pCIB272 X 15955, pCIB272 X A208, pCIB272 X A281, pCIB272 X A4, pCIB273 X 15955, pCIB273 χ A208, pCIB273 X A281 and pCIB273 X A4.

The pCIB542 strain Agrobacterium is strain EHA101 [Hood et al., J. Bacteriol. 168, 1291-1301 (1986)] in which the selectable marker - the kanamycin resistance gene of the plasmid has been replaced with partial Tn7 - with the spectinomycin / streptomycin resistance gene.

Example XXXIV. Transformation of tobacco leaf discs.

Agrobacterium strains CIB270, CIB271, CIB272 and CIB273 are cultivated for 18-24 hours on a broth containing glutamine salts, pH adjusted to 5.6, supplemented with 0.15% manmtol, 50pg / ml kanamycin, 50 µg / ml spectinomycin and 1 mg / ml streptinomycin, prior to dilution, with the same antibiotic free medium to a Οϋβοο value of 0.2. The bacteria were then grown for 3-5 hours before dilution to an ODeoo value of 0.2-0.4 to inoculate 5-7 mm diameter discs cut from Nicotiana tabacum cv leaves. Xanthi that were grown sterile in GA7 containers according to the modified method of R. Hosch et al., Science 227, 1229-1232 (1985).

Leaf discs are maintained on 0.7% agar containing Murashi and Skoog (MS) macro- and micronutrient salts (MS), 1 mg / ml benzyladenine and 1 mg / ml ff-naphthalene acetic acid,

162,317 for two days prior to transfer to the same medium containing 50pg / ml kanamycin, 100pg / ml carbemcyline and 100pg / ml mefoxime. The shoots forming on the discs are cut and propagated to six cuttings by growing the ends of the shoots on MS medium containing 50 pg / ml kanamycin in GA7 containers.

The cuttings are rooted in a medium that does not contain any hormones and contains 50 pg / ml kanamycin, is transferred to the soil and hardened in a phytotron before being transferred to the greenhouse for induction with chemical regulators. Flower self-pollination is induced during flowering. After ripening, the seeds are harvested.

The induction experiments are described in Examples LXIII and LXVI and the results are given in Table 2.

Example XXXV. Generation of transgenic callus tissue and tobacco plants.

Agrobactenum strain pCIB270 or pCIB271 is used to transform the callus formed from leaf discs (Example XXXIV). The callus formed on the MSBN selection medium containing kanamycin is maintained on the callus growth medium composed of MS macro- and microelement salts, Fe-EDTA (Gibco No. 500-1117, 4.3 g / 1), MS vitamins, 100 mg / l myoinositol , 20 g / l of sucrose, 2 mg / l of naphthalene acetic acid and 0.3 mg / l of kinetin.

Callus can be used to regenerate transgenic plants by transferring callus pieces to MSBN media using the methods described in Example XXXIV. Kallus is also used to measure gene induction following chemical application and to test gene inducing chemicals.

Example XXXVI. Carrot transformation.

Strains CIB270, CIB271, CIB272, CIB273, pCIB271 X 15955, pCIB271 X A208, pCIB271 X A281, pCIB271 X A4, pCIB272 X 15955, pCIB272 X A208, pCIB272 X A281, pCIB272 X273 A4, pCIB272 X273 X3, pCIB272 X27 X A281 and pCIB273 X A4 Agrobacterium are grown as described in Example XXXIV. Bacteria diluted to an ODeoo of 0.2-0.4 are then used to inoculate discs cut from surface sterilized carrots.

To sterilize the surface, the carrots are peeled and then soaked for 20 minutes in a 10% chlorox solution. Carrots are washed with sterile water, cut into 5 mm pieces and placed with the base side up on a water agar medium. Then 20-50 µΙ bacteria are applied to the top surface of the discs. After 7 days, the discs are transferred to 0.7% agar medium containing MS salts, 3% sucrose, 0.1 mg / 1 2,4-D, 50 µl / ml kanamycin, 1 (X) µg / ml carbenicillin and 100 µg / ml mefoxm. The cambium callus that forms around the ring is cut and placed on 0.7% MS agar supplemented with 3% sucrose, 0.1 mg / ml 2,4-D, 50pg / ml kanamycin, 100pg / ml carbenicillin and 100pg / ml mefoxin. After the callus has grown, it is cut into small pieces and randomly spread over four plates with the same substrate. When this callus fills the plates, 3 of them are sprayed with water, 50 mM sodium salicylate, pH 5.6, or 250 pg / l benzo-1,2,3-iadiazocarboxylic acid methyl ester to induce expression of the PR chimeric gene -la / GUS.

Induction experiments and their results are described in Examples LXVIII and LXVI.

Example XXXVII. Sunflower transformation.

Strains CIB270, CIB271, CIB272, CIB273, pCIB271 X 15955, pCIB271 X A208, pCIB271 X A281, pCIB271 X A4, pCIB272 X 15955, pCIB272 X A208, pCIB272 X A281, pCIB272 X273 A4, pCIB272 X273 X3, pCIB272 X27 X A281, and pCIB273 X A4 Agrobacterium are grown as described in Example XXXIV. The bacteria diluted to an ODeoo value of 0.2-0.4 are then used to inoculate the stems of sunflower seedlings prepared as follows:

The sunflower seeds are soaked for 10 minutes in 10% captan solution, then for 10 minutes in 10% chlorox solution and washed with sterile water. The seeds are peeled and the seeds are kept on a 0.7% water agar medium for 3 days in the presence of sprouting and then placed in an incubator at 23 ° C using a 12-hour photoperiod (12 hours day and night) . The seedlings are grown for one week before decapitation and inoculation of bacteria on the surface of the cut stem.

After one week, the stems inoculated with the strains CIB270, CIB271, CIB272 are cut and placed on 0.7% agar medium containing MS salts, 3% sucrose, 2 mg / ml of σ-naphthalene162 317 acetic acid, 1 mg / ml of 6-benzylaminopurine, 100pg / ml carbenicillin, 100pg / ml mefoxime and 50pg / ml kanamycin. Stems inoculated with pCIB271 X 15955, pCIB271 X A208, pCIB271X A281, pCIB271 X A4, pCIB272 X 15955, pCIB272 X A208, pCIB272 X A281, pCIB272 X A4, pCIB273 X 15955, pCIB272 X A4, pCIB273 X 15955, pCIB273 X, pCIB273 X 15955, pCIB273 X15 strains, pCIB273 and placed on 0.7% agar containing MS salts, 3% sucrose, 100pg / ml carbenicillin and 100pg / ml medoxin. Callus is transferred to fresh media every 2 weeks until sufficient tissue is obtained for 4 plates. Half of the callus grown from tissue infected with virulent Agrobacterium strains is transferred to hormone-free media containing 50pg / ml kanamycin. Once sufficient callus is obtained in the presence or absence of kanamycin, it is treated for gene induction as described above for the transformed carrot callus.

Example XXXVIII. Tomato transformation

Strains CIB270, CIB271, CIB272, CIB273, pCIB271 X 15955, pCIB271 X A208, pCIB271 X A281, pCIB271 X A4, pCIB272 X 15955, pCIB272 X A208, pCIB272 X A281, pCIB272 X273 A4, pCIB272 X273 X3, pCIB272 X27 X A281 and pCIB273 X A4 Agrobacterium were grown as described in Example XXXIV. The bacteria diluted to an ODeoo value of 0.2-0.4 were then used to wet the stems of tomato seedlings prepared as follows:

Tomato seeds are soaked for 20 minutes in a 10% chlorox solution and rinsed with sterile water. The seeds are then prepared on a 0.7% water-in-dark agar medium for 3 days to germinate and then placed in an incubator at 23 ° C using a 12-hour photoperiod. The seedlings are grown for one week before decapitation and incubation of bacteria on the average surface of the stem. After one week, the stalks inoculated with the strains CIB270, CIB271, CIB272 or CIB273 are cut and placed on 0.7% agar medium containing MS salts, 3% sucrose, 2 mg / ml of σ-naphthalene acetic acid, 1 mg / ml of 6-benzylaminopurine, 100pg / ml of carbenicillin. 100pg / ml of mefoxin and 50pg / ml of kanamycin. Stems inoculated with pCIB271 X 15955, pCIB271 X A208, pCIB271X A281, pCIB271 X A4, pCIB272 X 15955, pCIB272 X A208, pCIB272 X A281, pCIB272 X A4, pCIB273 X 15955, pCIB273 X273 X273 X273 X271 X273 X 15955, pCIB273 X273 X273 X273 X273 X271 X273 X 159 strains, and pCIB273 X273 is placed on 0.7% agar medium containing MS salts, 3% sucrose, 100 µg / ml carbenicillin and 100 µg / ml mefoxin. Callus is transferred to fresh media every 2 weeks until sufficient tissue is obtained for 4 plates. Half of the callus grown from tissue infected with virulent Agrobacterium strains is transferred to hormone-free media containing 50pg / ml of kanamycin. After sufficient callus tissue is obtained in the presence or absence of kanamycin, it is treated for gene induction as described above for the transformed carrot callus.

Example XXXIX. Cotton transformation.

Strains pCIB271 X 15955, pCIB271 X A208, pCIB271 X A281, pCIB271 X A4, pCIB272 X 15955, pCIB272 X A208, pCIB272 X A281, pCIB X A4, pCIB273 X 15955, pCIB273 X A273B A273B, pCIB A273 X A208, pCIB A273 X as described in Example XXXIV. The bacteria diluted to an OD value of 0.2-0.4 are then used to inoculate cotton cotyledons prepared as follows:

Cotton seeds are soaked for 20 minutes in a 10% chlorox solution and rinsed with sterile water. Seeds are kept on 0.7% agar medium in the dark to germinate. The seedlings are grown for one week before the bacteria are inoculated on the cotyledon surface.

The inoculated cotyledons are left to form a callus, then cut and placed on 0.7% agar medium containing MS salts, 3% sucrose, 100 µg / ml carbenicillin and 100 µg / ml mefoxin. Callus is transferred to fresh media every 2 weeks until sufficient tissue is obtained for 4 plates. Half of the callus grown from tissue infected with virulent Agrobacterium strains is transferred to hormone-free media containing 50 µg / ml kanamycin. After sufficient callus tissue is obtained in the presence or absence of kanamycin, it is treated for gene induction as described above for the transformed carrot callus.

Example XL. Producing a special type of callus from Zea mays, a selected inbred Funk 2717.

Zea mays from the Funk 2717 inbred line are grown to flower and self-pollinating in the greenhouse. Immature flasks containing embryos 2-2.5 mm long are removed from plants and steriles46

162 317 ingestion in 10% Clorox solution for 20 minutes. The embryos are sterilized from the seeds and placed with the axis of the embryo facing down on ONS containing 0.1 mg / liter 2,4-D, 6% (w / o) sucrose and 25 mM L-proline, 0.24% solidified (in / o) Gelrite ”(pre-substrate). (The abbreviation above indicates that these are percentages by weight / volume). After 2 weeks of cultivation in the dark at 27 ° C, the scallus developing on the disc is removed from the embryo and placed in BS [O. L. Gamborg, et al. Expemmental Cell Research 50, 151-158 (1968)] containing 0.5 mg / liter 2,4-D, solidified 0.24% (w / v) Gerlite. Callus is transferred to fresh medium every 2 weeks. After a total period of 8 weeks after the embryos are placed on the pre-medium, a special type of callus is identified by its characteristic morphology. This callus is grown on the same substrate. After a further 2 month period, the callus is transferred to N6 medium containing 2 mg / L of 2,4-D solidified Gelrite and continuously grown thereon.

Example XLI. Suspension of Zea mays culture, selected inbred Funk 2717 line.

The callus described in Example XL is grown for at least 6 months. The type of callus selected for cultivation is relatively low-slime, granular and very fine such that it is separated into small discrete aggregates of cells when placed in a liquid medium. Cultures containing aggregates with large, dilated cells are not obtained. Approximately 500 mg aliquots of the special Zea mays callus of the Funk 2717 inbred selected line are placed in 30 ml of N6 medium containing 2 mg / l 2,4-D in 125 ml Delong flasks. After one week of cultivation at 26 ° C in the dark, the medium is replaced with fresh on a rotary shaker (130 rpm, 2.5 cm swing). The suspensions are grown in this way for the next week. During this time, the cultures are controlled to retain those that do not contain large amounts of expanded cells and at the same time to eliminate those that contain aggregates of such cells. A preferred tissue consists of compacted aggregates of dividing cells having a characteristically smoother surface compared to the normal type of cell aggregates. The surviving cultures contained at least 50% of the cells present in these small aggregates. This is a desirable morphological feature. These suspensions also had a rapid growth rate with a doubling time of less than one week. Weekly, the culture suspension was passaged by transferring 0.5 ml of PCV (packed cell volume = pipette cell volume) into 25 ml of fresh medium. After 4-6 weeks of such passage, the cultures were increased 2-3 times per one-week culture. For further cultivation, cultures in which more than 75% of the cells possess the desired morphological characteristics are kept. The lines are maintained by selecting the flask which has the best morphological features for passaging. In some cases, periodic (2-weekly) filtration through stainless steel sieves with 630μτη apertures is used to increase cultivation dispersion, but is not necessary.

Example XLII. Preparation of protoplasts from a suspension of Zea mays cultures.

1-1.5 ml PCV of cells of the culture suspension obtained as in Example XLI is incubated in 10-15 ml of a filter-sterilized mixture of 4% (w / o) RS cellulase with 1% (w / o) Rhozyme in KmC salt solution 8.65 g / liter KCl, 16.47 g / liter MgCb X 6 H 2 O.5 g / liter MES, pH 5.6. The etching is carried out at a temperature of 30 ° C, on a constantly swinging, idle rotation, for a period of 3-4 hours. The process is controlled by observing prostoplast release under an inversion microscope. The released protoplasts are collected as follows: The preparation is filtered through a sieve with 100μιη apertures, then through a sieve with 50μτη apertures. The protoplasts are washed on the sieves with a KCM salt solution of a volume equal to the initial volume of the enzyme solution. 10 ml of the protoplast preparation is placed in each of the several available plastic centrifuge tubes and sublayered with 1.5-2 ml of 0.6 M sucrose solution [buffered to pH 5.6 with 0.1% (w / o) morpholinoethanesulfonic acid ( MES and KOH)]. The tubes are centrifuged at 60-100Xg for 10 minutes and the protoplast layer at the interface is collected with a pipette and placed in the following tubes. The protoplast preparation is resuspended in 10 ml of fresh KMC 1 salt solution and centrifuged for 5 minutes at 60-100 X g. The supernatant is removed and the protoplasts are carefully resuspended in the remainder of the supernatant and then 10 ml of a 13/14 concentrated KMC solution are then added gradually. After re-centrifugation for 5 minutes, the supernatant is removed again and the protoplasts are resuspended in 6/7 concentrated KMC solution. A sample is taken

162 317 for inspection and re-deposited protoplasts by centrifugation. Protoplasts are resuspended at a concentration of K) 7 (ten million) per ml in KM-8β medium (Table 1) or in 0.5 M mannium containing 6mM MgCl or other medium suitable for use in transformation as described in the following examples. The thus obtained protoplast suspension is used for transformation and cultured as described in Examples XLIII and XL1V.

Example XLIII. Transformation of Zea mays protoplasts by electroporation.

A. All steps except heat shock are performed at room temperature (22-28 ° C). In the last step of Example XLII, the protoplasts were suspended in a 0.5 M manni containing 0.1% (w / v) MES and 6 mM MgCl 2. The resistance of this suspension is measured in a Dialog Electroporator chamber (DIA-LOG G. mb H., D-4000 Duesseldorf 13, Germany) and adjusted to 1-12 kQ using a 300 mM MgCl 2 solution. Protoplasts are subjected to heat shock by immersing the tube containing the sample in a 45 ° C water bath for 5 minutes and then cooled to room temperature on ice. To 0.25 ml of this suspension is added 4pg of plasmid in a linear form containing the plant hygromycin resistance gene, which may be a selectable marker, such as described by SJ Rothstein et al., As above, or chimeric gene constructs as described in Examples ΧΙΧ -ΧΧΧΙ and 20 µg of calf thymus carrier DNA. 0.125 ml of a 24% (w / w) solution of polyethylene glycol (PEG) (MW 8000) in a 0.5 M mannium containing 30 mM MgCl ₂ are added to the protoplasts. The mixture is mixed thoroughly but carefully and incubated for 10 minutes. The sample is transferred to the electroporator chamber and subjected to three electric pulses at 10 second intervals, with initial voltages of 1500, 1800, 2300 or 2800 Vcm1, and exponential decay time of 10 / rs.

Protoplasts are grown as follows. The samples are plated in 6 cm petri dishes at room temperature. After a further 5-15 minutes, 3 ml of KM-8p medium (Table 1) containing 1.2% (w / o) SeaPlaque agarose and 1 mg / ml 2,4-D are added. Agarose and protoplasts are mixed thoroughly and the medium is allowed to solidify.

B. Example XLIII A is repeated with one or more of the following modifications:

(1) the resistance of the protoplast preparation is adjusted to 0.5-0.7 kQ, (2) PEG with a molecular weight of 4000 is used, (3) no PEG is added, or half the volume of 12% (w / w) PEG is added , (4) pulses are applied at 3-second intervals, (5) after electroporation, the protoplasts are spread on plates placed on a plate cooled to 15 ° C, (6) after the electroporation stage, the protoplasts are placed in test tubes, washed with 10 ml 6 / 7 KmC concentrated solution or W5 solution (composed of 380 mg / liter KC1, 18.375 g / liter CaCl ₂ XX 2H ₂ O, 9 g / liter NaCl, 9 g / liter glucose, pH 6.0), then collected by centrifugation at 60 g for 10 minutes, resuspended in 0.3 ml KM medium and seeded as in A, (7) no calf thymus carrier DNA was applied.

Example XLIV. Transformation of Zea mays protoplasts by treatment with polyethylene glycol (PEG).

AND/. Protoplasts suspended in the last step of Example XLII in a 0.5 M mannitol solution containing 12-30 mM MgCl ₂ are heat shocked at 45 ° C for 5 minutes as described in Example XLIII. The protoplasts are distributed in the amounts necessary for transformation in centrifuge tubes - 0.3 ml of protoplast suspension per tube. During the next 10 minutes, the following are added successively: DNA (as in Example XLIII) and polyethylene glycol (PEG) solution [MW 6000.40% (w / o) containing 0.1 M Ca / N () 3) 2 and 0 , 4 M mannitol, pH 8-9 adjusted with KOH] to give a final concentration of 20% PEG. This mixture is incubated for 30 minutes with rarely gentle shaking and then the protoplasts are placed in petri dishes (0.3 ml of the initial protoplast suspension per 6 cm diameter dish) and grown as described in Example XLIII.

B. Example XLIV A is repeated and the protoplasts are washed after 30 minutes of incubation in the PEG solution of Example VLIV A by adding 0.3 ml of W5 solution five times at 2 to 3 minute intervals. The protoplast suspension is centrifuged, the supernatant removed and the protoplasts cultured as in Example XLIII A.

C. Examples XLIV A and XLIV B are repeated with the following modification: the final PEG concentration is between 13 and 25% (w / o).

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Example XLV. Callus regeneration from protoplasts.

Plates containing the agarose protoplasts are placed in the dark at 26 ° C. After 14 days, the protoplasts form colonies. The colony-containing agarose is transferred to the surface of 9 cm diameter Petrf dishes containing 30 ml of N6 medium (Table 1) containing 2 mg / liter 2,4-D and solidified with 0.24% w / w Gerlite. This substrate is designated 2N6. The callus is further grown in the dark at 26 ° C and the callus pieces are transferred every 2 weeks to fresh 2N6 solid media.

Example XLVI. Selection of transformed callus Zea mays.

Example XLV is repeated with the following modification: 100 mg / L or 200 mg / L Hygromycin B is added to the 2N6 medium to select transformed cells.

Example XLVII. Regeneration of corn plants.

A. Kallus is stored in 2N6 media and 0N6 media (N6 media lacking 2,4-D) and N61 (N6 media containing 0.25 mg / L 2,4-D and 10 mg / L kinetin) to initiate regeneration. Callus growing on substrates 0N6 and N61 are grown in the light (16 hours of light per day of 10-100 pgeinsteinów / m ² s with a white fluorescent lamp). Callus growing on N61 medium is transferred, e.g. after 2 weeks to 0N6 medium, if the prolonged time on N61 medium is harmful. Kallus is transferred every 2 weeks to a fresh medium, even if it is of the same composition. The seedlings appear after 4-8 weeks. When they are at least 2 cm high, they are transferred to the 0N6 substrate in GA7 containers. Roots form within 2-4 weeks and when they appear well developed enough to allow seedlings to grow, they are transferred to the soil in pot peat under shaded light for the first 4-7 days. The seedlings can be hardened more easily by covering them with inverted clear cups for 2-3 days. Once the seedlings are firm, they are treated as normal maize plants and grown to maturation in a greenhouse. Plants are self-pollinated or bred with the wild type to obtain crops.

B. Example XLVII A is repeated with the following modification: 100 mg / liter or 200 mg / liter of hygromycin B is added to the medium used to hold the callus.

Example LXVIII. Preparation of an ambriogenic suspension from Dactylis glomerata L. (cocksfoot) tissue.

A. Greenhouse-grown Cocksfoot (Dactylis glomerata L.) plants are incised at the base of the youngest leaves to initiate the process of embryogenic tissue formation as described by G. E. Hanning et al., Theor. Appel. Genet. 63, 155-159 (1982). The leaves are surface sterilized by immersion in 1:10 diluted Clorox (5.25% (w / o) sodium hypochlorite, The Clorox Company, Oakland, Ca.) for approximately 10 minutes and then cut into small lengths or diameters 1-5 mm. These segments are plated on sterile SH-30 medium containing 0.8% (w / o) agarose as a solidifying agent. Callus and / or embryogenic structures appear 2-6 weeks after seeding in culture grown at 25 ° C. The embryogenic callus is stored in the dark at 25 ° C.

B. Cultures of the embryogenic suspension are initiated by placing about 0.5 g of wet callus embryogenic mass in 50 ml of the liquid medium described by DJ Gray et al, Plant Cell Tissue Organ. Cult. 4, 123-133 (1985), containing 45 µM dicamba and 4 g / liter casein hydrolyzate. These cultures were conducted in flasks Delong 125 ml pozatykanych metal caps and parafilm on a rotary shaker at about 130 shocks per minute, were grown at 27 ° C using fosfoperiodu: 16 hours of light (40pE / m ² s) and 8 hours dark . After about 4 weeks, the large clumps are allowed to settle on the bottom of the flask for about 30 seconds, then 10 ml of the culture supernatant containing the small cell clumps are removed and transferred to 50 ml of fresh medium. This process is repeated every 3-4 weeks, selecting for further cultivation cultures characterized by the smallest size of clumps and the presence of small, young cells in them. After 5-8 such passages, the suspensions are essentially free of non-embryogenic cells and most of these cell clumps are fine (150 to 2000 µm).

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Example XLIX.Isolation and purification of Dactylis glomerata L. protoplasts.

Protoplasts are obtained from culturing the embryogenic suspension of Example XLVIII by sterile filtering the cells on 0.2μιτι Nalgene filters and then adding 0.5 g of wet cell weight to each 12.5 ml of protoplast enzyme mixture in a Petrfe dish. The enzyme blend includes: 2% (w / o) RS ironase, 7 mM CaCl2XHzO, 0.7 mM NaHzPO4XH2O, 3mM MES (pH 5.6) and glucose (55OmOs / kg H2O, pH 5.6); this mixture is sterilized by filtration. The blend was agitated on a shaker circular at about 50 rpm and the dim light (<5 μΕ / m ² s) for about 4-5 hours. The digest mixture is sieved through a stainless steel sieve (ΙΟΟμηυ) and distributed in 12 ml centrifuge tubes which are then centrifuged at about 60-100 g for about 5 minutes. The pellet containing the protoplasts is then washed three times with the protoplast culture medium KM-8p adjusted with glucose to 55OmOs / kg H2O. The preparation can be further purified using the following procedure: The washed protoplasts are applied to 10 ml of KM-8p culture medium adjusted with sucrose to 7OOmOs / kg H2O. After centrifugation at 60-100 Xg for about 10 minutes, the protoplast layer at the interface is collected with a thin pipette. Finally, the protoplasts are resuspended in 1-2 ml of KM-8p 1 culture medium and sieved through a stainless steel sieve (20 µπι). Protoplasts passing through the sieve are harvested and resuspended in KM-8p medium or in osmotically equilibrated medium suitable for transformation according to Examples L1-III.

Example L. Cultivation of Dactylia glomerata L. and callus protoplasts.

A purified protoplasts, with a density of 5 x 10 ⁵ protoplasts per ml and seeded to a culture medium KM-8p containing 1.3% (w / v) SeaPlaque agarose ^R (FMC Corp., Marine Colloids Division. Rockland, Maine. USA) enriched with 30-40% (w / o) of the medium obtained from 3-4 week-old cultures of the Dactylis glomerata L. embryonic suspension by filtration through sterile Nalgene filters with a pore diameter of 0.2 μπι, addition of glucose to 550mOsm / kg H2O 1 re-sterilization by filtration . The plates are then placed in the dark at a constant temperature of 28 ° C. After 10-14 days, the agarose is wedged and a "bead culture" as described by RD Shillito et al., Plant Cell Reports 2, 244-247 (1983), using 20 ml of SH-45 suspension medium with 3% (w / o) sucrose on 3 ml of agar seeded stock. The plates are placed on a shaker and shaken at about 50 strokes per minute under the light (8 µΕΕ 2). As colonies grow on the agarose and release the cells into a liquid medium, a new cell suspension will be formed. This new suspension is plated on agar solidified SH-30 and placed in the dark at 25 ° C until callus formation.

B. Protoplasts are grown as described above in Example LA except that the culture medium is not enriched.

Example LI. Transformation of Dactylis glomerata L. protoplasts by electroporation.

A. Immediately after purification of the protoplasts, electroporation is performed according to the method of RD Shillit et al. Bio / Technology 3, 1099-1103 (1985), using a plasmid in linear form as described in Example XX. Protoplasts, after the last washing, suspended in electroporation buffer (0.4 M mannitol, 6 mM MgCl) of equal to 7 x 10 ⁶ protoplasts per ml of buffer. 0.7 ml of protoplasts are placed in 10 ml plastic centrifuge tubes. The plasmid DNA from Example 20 and sonicated calf thymus DNA (Sigma) are added to the tubes to give a final concentration of K ^ ing ^ mL and 50 Pg / mL, respectively. Then 0.38 ml of polyethylene glycol (PEG) solution [24% w / o) PEG 6000 in 0.4 M mannium, 30 mM MgCl _2> 0.1% (w / o) MES, pH 5.6] is added] 1 solution is carefully mixed. The protoplast suspension is transferred to the Dialog Electroporator chamber and subjected to 10 electric pulses at 30-second intervals, with an initial voltage of 3250 V / cm and an exponential decay time of 10 µΞ. The sample is removed from chamber 1 and placed in a Petri dish 10cm in diameter. 10 ml KM-8p medium containing 12% (w / v) SeaPlaque agarose is added, the protoplasts are spread in the medium and allowed to solidify.

B. Example L1 A is repeated except that a starting voltage of 3500 V / cm, 4000 V / cm, 5000 V / cm, 3000 V / cm or 2500 V / cm is used.

C. Examples L1 A and B are repeated except that PEGs with a molecular weight of 4000 or 8000 are used.

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D. Examples L1 A - C are repeated except that the final PEG concentration is 10-30% (w / o).

Example LII. Transformation of Dactylis glomerata L. protoplasts by treatment with polyethylene glycol.

A. PEG-mediated gene transfers are performed according to I. Negrutiu et al., I. V. The DNA used is the plasmid described in example XX in linear form.

The protoplasts from the last wash are resuspended in a 0.5 M mannium containing 15 mM MgCl2 at a ratio of approximately 2 × 10 ^e per ml. 1 ml of the protoplast suspension is placed in 10 ml plastic centrifuge tubes. DNA is added as described above in Example L1, and then 0.5 ml of a solution of PEG [40% (w / o) PEG in 0.4 M manni, 0.1 M CaNCO 3) 2, pH 7.0] is added. . The solutions are mixed carefully and incubated for 30 minutes at room temperature (about 24 ° C) with rarely gentle shaking. Then 1.4 ml of washing solution is added and the contents are carefully mixed. The washing solution consists of 89mM mannitol, II5mM CaClb, 27mM MgCl2, 3.9mM KCl, 1.7mM Tris-HCl, and 1.7g / liter myoinositol, pH 9.0. Four more

1.4 ml aliquots of the washing solution are added at 4 minute intervals, with mixing after each addition. Tubes are then centrifuged at approximately 60 Xg for approximately 10 minutes and the supernatant removed. The embedded protoplasts are spread in 1 ml of KM-8p culture medium and placed in petri dishes with a diameter of 10 cm. 10 ml of KM-8p medium containing 1.2% (w / v) SeaPlaque ^R agarose is added. The protoplasts are spread over the medium and the agarose is allowed to solidify.

B. Example LII A is repeated with one or more of the following modifications:

(1) the pH of the washing solution is adjusted to 5.6 or 7.0, (2) the PeG used is PEG with a molecular weight of 6000, 2000 or 8000, (3) the washing solution consists of 154mM NaCl, 125mM CaCb 5mM KCl, 5rM glucose adjusted to pH 6.0 KOH, 0.2 M CaCb, 0.1% (w / o) MES, pH adjusted to 6.0 KOH, or 0.2 M CaCb, 7 mM Tris / Hcl, adjusted pH to 9.0 with KOH.

Example LIII. Transformation of Dactylis glomerata L. protoplasts by electroporation or PEG treatment.

Transformation is performed as described in Example LI or LII except that the protoplasts are kept for 5 minutes at 45 ° C before dispensing them into transformation tubes or after separating and before adding PEG.

Example LIV. Selection of transformed colonies.

A. Culture plates (Petri dishes) containing the protoplasts obtained as in Examples L1-LIII are incubated for 10 days in the dark at about 25 ° C, and then cut into 5 equal sections for "bead culture [R . D. Shillito et al., Plant Cell Reporst 2,244-247 (1983)]. Each of the four sections is placed in 20 ml of SH-45 culture medium with 4 g / L of casein hydrolyzate and 20 g / ml of Hygromycin B. The fifth section is placed in 20 ml of the same medium but without hygromycin B for unselected control. After 4-5 weeks, putative transformed colonies of protoplast-derived cells grown in the presence of hygromycin B are excised from agarose and placed in a 19 mm diameter petri dish along with 2 ml of SH-45 liquid medium containing 20 pg / ml of hygromycin B and shakes on a shaker at 50 shakes per minute. After a further 4-5 weeks, all colonies that grow into new suspensions are transferred to 125 ml Erlenmeyer flasks and grown similar to the original suspension culture except that 20 µg / ml of hygromycin B is added to the medium.

The new suspensions are transferred to fresh SH-45 medium containing 4 g / L casein hygrolysate and 20 pg / ml Hygromycin B every 1-3 weeks. Cells from these suspensions are plated on SH-30 solid medium containing 20pg / ml of Hygromycin B and incubated at approximately 25 ° C in the dark. Callus tissue grown from these cells is transferred to fresh medium every 2 weeks. Presumably, cells grown in the presence of hygromycin B are transformants.

B. Selection is performed as described in Example LIV A except that colonies of protoplast-derived cells grown on hygromycin B-containing medium are plated on solid SH-30 medium containing 20pg / ml Hygromycin B and incubated at approximately 25 ° C in the dark.

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Example LV. Regeneration of transformed Dactylis glomerata L. plants

Protoplast-derived A. Kallus Dactylis glomerata L. (prepared as described in Example LIV) is grown on solid SH-30 medium and passaged every 2 weeks. All embryos formed are removed and plated on germination medium (SH-O) and placed in the light (45-55μΕ / m ² s). The development of these embryos lasts 1-4 weeks and the resulting seedlings are placed on the SH-O medium in the light to form a root system. At the 6-12 leaf stage, they are transferred to a greenhouse and gradually toughened.

B. Callus (obtained as described in Example LIV) derived from protoplasts is grown in SH-O medium solidified with 0.24% (w / o) Gelnte, in the light (45-55 μΕ ^ 2 s) and passaged every 2 weeks . The resulting seedlings are placed in a mixture of SH-O and OMS (1: 1) solidified by the addition of 0.12% (w / w) Gerlite and 0.4% (w / w) agar, in the light to form a root system. At the 6-12 leaf stage, they are transferred to a greenhouse and gradually toughened.

C. Obtain small seedlings as described above in Examples XLIVA and XLIV B, and place them on OMS medium solidified by the addition of 0.8% (w / o) agar in the light to form a root system. At the 6-12 leaf stage, they are transferred to a greenhouse and gradually toughened.

D. Small seedlings are obtained as described above in Example XLIV A and placed on a mixture of SH-O and OMS solidified by the addition of 0.12% (w / o) Gerlite and 0.4% (w / o) agar on light to form the root system. At the 6-12 leaf stage, they are transferred to a greenhouse and gradually toughened.

6. Short-term gene expression.

The following examples describe how chimeric genes can be introduced and used without having to be constantly incorporated into the plant genome.

Example LVI. Introduction of DNA into N. tabacum protoplasts by PEG treatment.

A. The production of N. tabacum protoplasts can be carried out according to the methods described in the following publications: J. Paszkowski et al., EMBO J. 3, 2717 (1984), British Patent Application No. 2,159,173, European Patent Application No. 0 129 668, RD Shillitu , RD and I. Potrykus, Methods in Enzymology, R. Wu and L. Grossman, Academic Press, Orlando, Florida, Vol. 153, 313-306 (1987), or by other commonly known methods.

B. Protoplasts obtained as described in Example LVI A are resuspended, after another final wash, in a solution consisting of 0.4 M mannitol, 15-30 mM CaCl 2, 0.1% (w / o) MES at a ratio of 1.6 -2X10 ⁶ protoplasts per ml. The protoplast suspension is distributed by 0.5 ml into 10 ml plastic centrifuge tubes. The DNA of examples ΧΙΧ-is added in 10 µl sterile, sterilized distilled water as described by J. Paszkowski et al., EMBO J. 3, 2717 (1984). Then 0.5 ml of a solution of PEG [40% (w / w) PEG MW 8000 in 0.4 M mannium, 0.1 M Ca (NOa) 2, pH 7.0] is added. The solutions are mixed carefully and incubated for 30 minutes at room temperature (about 24 ° C) with rare shaking. Then 1 ml of washing solution is added and the contents of the tube are carefully mixed. The wash solution consists of 154 mM NaCl, 125 mM CaCl 2, 5 mM KCl, and 5 mM glucose, adjusted to pH 6.0 with KOH. Further amounts of washing solution: 2 ml, 3 ml and 4 ml are added sequentially with 5 min intervals, with mixing after each addition. The tube is then centrifuged at about 10-100 Xg for about 10 minutes and the supernatant removed. The deposited protoplasts are spread in a K3 culture medium with 0.3 M glucose as component to provide the appropriate osmotic pressure but no sucrose in an amount sufficient to achieve a final protoplast density of 100,000 per ml and grown in a petri dish 10 cm in diameter.

C. Example LVI B is prepared with one or more of the following modifications:

(1) the pH of the washing solution is adjusted to 5.6 or 7.0, (2) the PEG used is PEG with a molecular weight of 4000, (3) the washing solution consists of 0.2 M CaCl 2, 0.1% in / o MES, pH adjusted to 6.0 KOH, or with 0.2 M CaClh, 7 mM Tris / HCl, pH adjusted to 9.0 KOH, (4) 50 μg of calf thymus DNA is added along with plasmid DNA in 25 µΐ sterile water, (5) plasmid DNA is linearized using an appropriate restriction enzyme (eg, BamHI).

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Example LVII. Introduction of DNA into N. tabacum protoplasts by electroporation.

A. Introduction of DNA into N. tabacum protoplasts is induced by acting on the protoplasts with electrical impulses in the presence of appropriate DNA, modifying the methods Μ. E. Fromma, Methods in Enzymology, R. Wu and L. Grossman, Academic Press, Orlando, Florida, Vol. 153, 307 (1987) and R. D. Shillito and L. Potrykus j., Pp. 283-306.

Protoplasts are isolated as described in Example LVI A. Protoplasts are re-suspended, after another final wash, in the following solution: 0.2 M mannitol, 0.1% (w / o MES) 72 mM NaCl, 70 mM CaCl 2, 5 mM KCl, 2.5 mM glucose, adjusted to pH 5.8 with KOH, according to the ratio 16-2 X ^{10 8} per ml. The protoplast suspension is distributed 1 ml into plastic cuvettes and 10 µg of DNA is added as described in Examples LVI B and C. The solution resistance measured between the electrodes of electrode set 471 of the electroporation apparatus described below remains in the range of 6 ohms.

The DNA of examples ΧΙΧ-ΧΧΧΙ is added to 10 µl / 1 sterile sterilized water as described by J. Paszkowski et al., EMBO J. 3. 2717 (1984). The solution is carefully mixed and then subjected to electrical pulses of 400 V / cm at room temperature (24-28 ° C) with a decay constant of 10ms in a BTX-Transfector electroporation apparatus using the 471 electrodes. The protoplasts are left undisturbed for 5 minutes, then transferred to a Petrfe dish and sufficient K3 medium as described in Example LVI to be added to obtain a final protoplast density of 100,000 per ml.

B. Example LVII A is repeated with one or more of the following modifications:

(1) a voltage of 200 V / cm or a voltage of 100-800 V / cm is used, (2) the exponential decay constant is 5 ms, 15 ms or 20 ms, (3) along with the plasmid DNA, 50 µg of calf thymus DNA is added in 25 µl of sterile water, (4) plasmid DNA is linearized using an appropriate restriction enzyme (e.g. BamHI).

Example LVIII. Introduction of DNA to the protoplasts of line 2717 Zea mays.

The protoplasts of the maize Funk 2717 inbred line are prepared as described above in Examples XL-XLVII and suspended in any of the solutions to suspend the N. tabacum protoplasts described above in Examples LVI and LVII at a density of 10 ⁷ per ml. Transformation is performed essentially as described in examples LVI and LVII. After transformation, protoplasts are grown at a density of 2 X 10 ⁶ per ml in KM-9β medium containing 1 mg / ml2,4-D, without solidifying agents.

Example LIX. Introducing DNA into Sorghum bicolor protoplasts.

Sorghum FS 562 slurry protoplasts are prepared essentially as described in Example XL for Zea mays and suspended in any of the solutions for suspending the N. tabacum protoplasts described above in Examples LVI and LVII at a density of 10 ⁷ per ml. Transformation is performed essentially as described in Example LVIII. After transformation, protoplasts are grown at a density of 2 × 1) 6 per ml in KM-8β medium without the addition of solidifying agents.

Example LX. Introduction of DNA into protoplasts of Nicotiana plumbaginifolia, Petunia hybrida and Lolium multiflorum.

N. plumbaginifolia, P. hybrida or L. multiflorum protoplasts are obtained as described by Shillito and Potrykus, ibid. And treated as described in Examples LVI and LVII. They are cultivated in the medium described by Shillito and Potrykus J. V. Without adding agarose or any other gelling agents.

Example LXI. Introducing DNA into protoplasts Glycine max.

Glycine max protoplasts are obtained by the methods described by DM Tricoli et al., Plant Cell Reports, 5, 334-337 (1986) or VK Chowhury and JM Windholm, Plant Cell Reports4,289-292 (1985) or AS Klein et al., Planta 152, 105-114 (1981). DNA is introduced into these protoplasts essentially as described in Examples LVI and LVII. Protoplasts are grown as described by A. S. Klein et al., J. W. Chowhury and J. M. Widholm, J. W., or D. M. Tricoli et al., As above, without adding algiman to solidify the medium. 7. Chemical regulation of transgenic plant tissues.

The following examples describe procedures for the chemical regulation of genes so regulated in representative transgenic plant tissues. Although these examples relate to induction, similar procedures can be used for repression systems.

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A variety of techniques are available for treating transgenic cells with chemical regulators. For example, the cells can be suspended in a regulator-containing liquid medium. Callus can be grown or transferred to a medium containing a regulator. The regulator can also be used with a sterile brush or plant sprayer. The same can be done with the solutions and suspensions for plants or plant parts.

Expression of the phenotypic trait in transgenic plants containing chemically regulated chimeric genes is proportional to the amount of regulator applied to the plant or plant tissue.

Example LXII. Chemical induction in transgenic callus cultures.

A. In the callus obtained according to the previously described procedures, the chemical induction induced by the use of a filter-sterilized solution of 0.1-50 mM salicylic acid, adjusted to dopH 6.0-8.0 by adding NaOH, using a sterile brush or plant sprayer.

After induction, the callus is incubated for 2 days, harvested, frozen in liquid nitrogen and stored at -80 ° C until gene induction is determined as described in Example LXV and / or LXVI.

B. Alternatively, the following solutions are used for induction:

(2) 0.1-50 mM benzoic acid, adjusted to pH 6.0-8.0 by adding NaOH.

(2) 0.1-50 mM polyacrylic acid, adjusted to pH 6.0-8.0 by adding NaOH.

(3) Benzo-1,2,3-thiadiazole-7-carboxylic acid methyl ester. The solution is prepared by suspending the wettable powder containing the reagent in sterile water for a few minutes at a concentration of 1 mg / ml. The insoluble solid residue is removed from the solution by sterilization filtration.

(4) 0.1 to 50mM benzo-1,2,3-thiadiazolecarboxylic acid-7, pH 6.0-8.0. This compound is dissolved in a minimal volume of dimethylsulfoxide (DMSO) and then diluted with sterile water to the desired concentration. The resulting suspension is used without filtering.

(5) 0.1-50 mM Benzo-1,2,3-thiadiazole-7-carboxylic acid n-propyl ester, prepared as described in (4).

(6) 0.1-50mM Benzo-1,2,3-thiadiazole-7-carboxylic acid benzyl ester, prepared as described in (4).

(7) OJ ^ 51mM N-II. noun benzo-1,2,3-thiadiazole-carboxylic acid butylhydrazide, prepared as described in (4).

(8) 0.1-50mM 2,6-dichloroisonicotinic acid, prepared as described in (4).

(9) 0.1-50mM 2,6-Dichloro-isonicotinic acid methyl ester, prepared as described in (4).

Example LXIII. Chemical induction of transgenic plants.

A. Gene expression in plants obtained according to the previously described procedures is induced by fine dust spraying, plant sprayer, on leaves 0.1-50 mM salicylic acid solution adjusted to pH 6.0-8.0 with NaOH.

After induction, plants are incubated for 2 days, harvested, frozen in liquid nitrogen and stored at -80 ° C until gene induction is determined as described in Example LXV and / or LXVI.

B. Alternatively, the following solutions are used for induction:

(1) 0.1-50 mM Benzoic acid adjusted to pH 6.0-8.0 by adding NaOH.

(2) 0.1-50 mM Polyacrylic acid adjusted to pH 6.0-8.0 by adding NaOH.

(3) Benzo-1,2,3-thiadiazole-carboxylic acid methyl ester. The solution is prepared by suspending the wettable powder containing the reagent in sterile water for a few minutes at a concentration of 1 mg / ml. The insoluble solid residue is removed from the solution by sterilization filtration.

(4) 0.1-50 mM Benzo-1,2,3-thiadiazolecarboxylic acid-7, pH 6.0-8.0. This compound is dissolved in a minimal volume of dimethylsulfoxide (DMSO) and then diluted with sterile water to the desired concentration. The resulting suspension is used without filtering.

(5) OJ 50 mM Benzo-1,2,3-thiadiazoI-carboxylic acid n-propyl ester, prepared as described in (4).

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(7) 0.1-50mM benzo-1,2,3-thiadiazole-7-carboxylic acid N-tert-butylhydrazide, prepared as described in (4).

(8) 0.1-50 mM 2,6-dichloroisomotinic acid, prepared as described in (4).

(9) 0.1-50mM 2,6-Dichloroisomotinic acid methyl ester, prepared as described in (4).

Example LXIV. Induction of the expression of the introduced DNA in the protoplasts of N. tabacum, Zea mays, N. plumbaginifolia, P. hybrida L. multiflirum and Sorghum bicolor.

A. Protoplasts treated as described above in Examples LVI-LXI are grown at 26 ° C in the dark for 2 days. After this time, salicylic acid is added to a concentration of 0.1-50 mM as a neutralized solution, pH 6.0-8.0. Protoplasts are cultivated for a further 2 days, harvested by centrifugation, snap frozen and assayed as described in the following examples.

B. Alternatively, neutralized solutions or suspensions of the following compounds are added to the protoplasts to a final concentration of 0.1-50 mM:

(1) benzoic acid, (2) polyacrylic acid, (3) benzo-1,2,3-thiadiazole-7-carboxylic acid methyl ester, (4) benzo-1,2,3-thiadiazo-7-carboxylic acid, (5) ester n-propyl benzo-1,2,3-thiadiazole-carboxylic acid-7, (6) benzo-1,2,3-thiadiazole-carboxylic acid-benzyl ester, (6) benzo-1,2 J-thiadiazoloc acid benzyl ester ^ and ^ benzo-1,2,3-thiadiazole-7-carboxylic acid N-second-butylhydrazide, (8) 2,6-dichloro-olzonicotinic acid, (9) 2,6-dichloroisonicotinic acid methyl ester,

C. Alternatively, induction is performed as described in Examples LXIV A and B except that the protoplasts are cultured for 1 day, 2 days or 3 days prior to incubation.

D. Induction is performed as described in Examples LXIX A, B or C, except that the protoplasts are grown 1 day, 3 days, or 5 days after induction and prior to the harvesting step by centrifugation.

Example LXV. Determination of chemically inducible DNA sequences: mRNA synthesis.

In induced and harvested plant material as described in Examples LXII and LXII1, the induction of mRNA species is determined by RNA isolation and primer extension assay as described in Example 5.

Callus or regenerated plant material obtained from transgenic plants containing chemically regulated chimeric genes encoding GUS, AHAS or BT and induced with salicylic acid, methyl ester of benzo-1,2,3-thiadiazole-7-carboxylic acid, benzo-1,2, 7-thiadiazole-carboxylic acid, n-propyl ester of benzo-1,2,3-thiadiazolecarboxylic acid-7, benzyl benzo-1,2,3-thiadiazolecarboxylic acid-7, N-2nd order. Benzo-1,2,3-thiadiazole-7-carboxylic acid butylhydrazide, 2,6-dichloroisonicotinic acid, 2,6-dichloro-isonicotinic acid methyl ester or TMV show elevated mRNA levels in the GUS, AHAS or BT primer extension assay, respectively. The level of mRNA is proportional to the amount of regulator used.

Example LXVI. Determination of inducible DNA sequences: enzymatic activity of β3-1, 3-glucuronidase.

A. Labeling on ELISA plates.

The frozen leaf tissue is ground in a mortar in the presence of liquid nitrogen to form a fine-grained powder. The leaf extract is prepared by mixing a given weight (in g) with an equal volume (in ml) of GUS extraction buffer (50 mM phosphate buffer, pH 7.0, 0.1% Triton Χ-100, 0.1% sarcosyl, 10 mM / 5-mercaptoethanol) as described by RA Jefferson et al., Proc. Natl. Acad. Sci. USA 83, 8447-8451 (1986).

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Reactions are performed in an ELISA well plate by mixing 5-25 µl of extract with 120-100 µl of G US assay buffer (50 mM phosphate buffer, pH 7.0, 0.1% Triton®-100, 10 mM / 3-mercaptoethanol) containing 4-methylumbeliderole glucuronide (MU) at a final concentration of 2 mM in a total volume of 125/1. The plate is induced at 37 ° C for 1-5 hours and the reaction is stopped by the addition of 150 µl of 3M Na2CO3. The concentration of the released fluorescent tracer is determined by reading the plate in a Flow Labs Fluoroskan II ELISA plate reader.

B. Results.

Table 2 shows the specific activity in Kat MU / mg protein

Table 2

Plant' AND B C. D E ° 1 2 3 4 5 6 Η 1 5.39 + 1.9 13.9 ± 1.2 13.5 = 0.8 20.1 5J 33.6 = 2.6 H 3 0.21 ± 0.02 6.92 ± 0.88 11.95 = 0.85 1.66x0.02 6.54 = 1.43 H 4 8.39 + 1.31 27.5 = 0.7 18.52 ± 1.07 2.28 ± 0.78 25.1 x0.9 H6 0.10 = 0.04 4.08x0.58 2.58 ± 0.0l 0.09 ± 0.06 2.8 ± 1.2 H 9 0.23 10.8 9.1 0.5 7.5 Alu 1 5 6 20.0 206.9 20.5 70.5 Alu 2 2.0 6.6 83.2 8.9 155.3 Alu 3 0.1x0 07 2.8zx 0.9 2.0 = 0.9 2, O ± O, 5 5 8x3. 8 Alu 4 2.1x1 43.0 ± 19 7.1x2.6 6.1x3.2 7.5 = 3.5 Alu 5 0.6 = 0.2 4.4x 2.3 1.2 ± 0.6 0.5x0.3 8 3x3.7 Alu 6 0.8 28.8 67.0 3.1 36.4 Alu 7 1.7 38.7 29.1 21.7 63.0 Alu 8 0.6 0.4 0.4 0.6 0.2 Alu 9 0.9 0.6 0.4 0.3 n / a Alu 10 4.0 39.2 52.0 8.1 37.9 Alu II 1.6 ± 0.8 1.9 ± 0.9 2.8 ± 1.3 2.2 ± 2.6 2.8 ± 2.9 Alu 12 0.7 ± 0.4 57.1 ± 22 73.4 2.3 ± 1.2 78.0 ± 41.4 Alu 13 0.1 19.1 13.2 1.9 10.6 Hae 1 0.6 1.6 2.6 1.7 3.9 Hae 2 2.3 8.8 11.8 0.4 0.8 Hae 3 1.7 11.1 11.2 3.0 1.6 Hae 4 2.0 8.1 10.8 1.7 0.82 Hae 5 18.1 59.4 132.5 36.6 14.0 Hae 8 0.4 30.6 28.9 0.6 29.0 Hae 9 2.6 63.9 129.7 12.5 55.2 Hae 10 1.6 70.5 98.2 13.5 94.5 Hae II 2.6 81.8 58.5 3.9 46.0 Hae 12 0.2 2.5 5.8 0.6 14.5 Hae 13 0.01 0.7 0.1 - - Hae 14 0.1 1.8 8.6 0.1 3.3 Hae 15 0.1 6.4 7.2 3.4 7.4 Hae 16 0.1 2.3 4.3 0.8 2.7

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Reference explanations a / H 1-9 are tobacco plants obtained from leaf discs transformed with strain CIB27I -Mu 1-13 are tobacco plants obtained from leaf discs transformed with strain CIB272 Hae 1-16 are tobacco plants obtained from leaf discs transformed with strain CIB273 b / A Water

B salicylic acid

C benzo-1,2,3-nadiazole-carboxylic acid methyl ester-7 D buffer E TMV

Transformed Tobacco Callus (Example XXIII): The following GUS activities are obtained for the transformed callus samples collected 21 days after treatment. CIB271-transformed H 2 O spraying: 1.8 nKat MU / mg protein: 50 mM sodium salicylate spraying: 3.8 nKat MU / mg protein. CIB271 transformed (other experiment): H2O spraying: 2.8 nKat MU / mg protein; spraying with 1,2,3-thiadiazole-7-carboxylic acid methyl ester (250pg / liter): 13.7 nKat MU / mg protein. CIB273 transformed: H 2 O spraying: 0.46 nKat MU / mg protein; spraying with methyl ester of benzo-1,2,3-thiadiazolecarboxylic acid-7: 31.2 nKat MU / mg protein.

Transformed Carrot (Example XXXVI); for samples of transformed pCIB27I callus collected 21 days after treatment, the following GUS activities are obtained. H2O spraying: 5.2 pKat MU / mg protein. Spraying with 50 mM sodium salicylate: 1 L, 6 pKat MZ / mg protein. Spraying with 1,2,3-thiadiazole-7-carboxylic acid methyl ester (250 µg / liter): 22.1 pKat MU / mg protein.

Transformed Tomato (Example XXXVIII): The following GUS activities are obtained for the transformed callus samples harvested 21 days after treatment. Transformed CIB271. spraying with H2O I7.2pKat MU / mg protein; spraying with 1,2,3-thiadiazole-7-carboxylic acid methyl ester (250pg / liter): 56.1 pKat MZ / mg protein. CIB273 X A4 transformed: H 2 O spray: 4.6 pKat MU / mg protein; spraying with methyl ester of benzo-1,2,3-thladiazole-carboxylic acid-7 (250pg / liter): 22.1 pKat MU / mg protein.

Short-term expression in tobacco protoplasts (example LVI) treated with pCIB269. H2O · 36.6 ± 13pKat MU / mg of protein; Benzo-1,2,3-thiadiazole-carboxylic acid methyl ester: 66.6 ± 20 pKat MU / mg protein.

Example LXVII. Determination of chemical induction of an endogenous gene.

The mRNA of endogenous PR-1? Gene is transcribed in tobacco-derived plant material induced as described in Examples 62 to 64 with salicylic acid, benzo-1,2,3-thiadiazolecarboxylic acid-7 and TMV methyl ester by the methods described in Example 5. Elevated mRNA levels are detected after induction with all three inducers.

Example LXVIII. Determination of inducible chemical DNA sequence: enzymatic acetohydroxy acid synthase (AHAS) activity.

A. Sample preparation for AHAS activity assay.

The weight of the tissue material of the leaf and / or root to be determined is determined. The tissue is placed in liquid nitrogen and ground to a fine powder. A volume (in ml) equal to twice the weight (g) of tissue of cold homogenization buffer (50 mM NaHaPOa buffer, pH 7.0; 0.5 mM EDTA pH 7.0; 0.5 mM MgCl2; 1 mM sodium pyruvate) is added , pH 7.0; 10μΜ flavin-adenine dinucleotide; 1 mM phenylmethylsulfonyl fluoride; 10% glycerin) and the mixture is transferred to a cell press (French pressure celi). All subsequent steps are performed keeping the material cool (about 4 ° C). The cells are broken under the pressure of the IIOMP and then collected in an ice-cooled container. Alternatively, tissue is ground with homogenization buffer in a mortar instead of treatment in a cell press. The disrupted cells are centrifuged for 5 minutes at 10,000 rpm to clarify the homogenate. The volume of the supernatant is measured and the ammonium sulphate is added of enzyme purity up to 25% saturation (144 mg / NIWSO4 / ml of supernatant). The mixture is stirred on ice for 15 minutes then centrifuged for 5 minutes at 10,000 rpm. The precipitate is removed and the supernatant is brought to 50% saturation with ammonium sulfate (158 mg / NH4 / 2SO4 / ml of supernatant). After stirring on ice for 15 minutes, the precipitate is collected by centrifugation at 10,000 rpm for 5 minutes. The resulting pellet is dissolved in 1 ml of column buffer for every 2 g of leaf material or 5 g of roots. The extract is desalted on a Sephadex G50 column equilibrated in column buffer (50 mM NaH2PO4 buffer, pH 7.0: 0.1 mM

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EDTA; 0.5 mM MgCb, ΙΟμηι flavin ademnic dinucleotide; 1 mM phenylmethyl sulfonic fluoride, 10% glycerin). The sample is eluted with buffer onto the column, collecting 2 ml of sample for each ml of sample applied to the column.

B. Determination in the tube

5-50μ1 of the extract obtained according to example LXVIII A is dissolved in a total volume of 0.5 ml of the reaction mixture (20 mM NaH2PO4 buffer, pH 7.0; 20 mM sodium pyruvate, pH 7.0, 0.5 mM thiamine pyrophosphate; 5 mM MgCL, ΙΟμιη flavin-adenine dinucleotide). The mixture is incubated for 30 minutes at 37 ° C. The reaction is stopped by adding 6N H2SO4. The mixture is incubated for 15 minutes at 60 ° C and then treated with 625μιη of naphthol reagent (500μL 5% σ-naphthol in 2.5M NaOH 1 125μΐ creatinine at a concentration of 25 mg / ml 1 incubated for another 15 minutes at 60 ° C C. If a precipitate is formed, it is removed and the absorption is measured at 520 nm The concentration (pmole) of acetoin forming is calculated from a standard curve in the range 0.4 µg - 10 µg acetoin The specific activity is expressed as pKat / mg protein.

C. Labeling on ELISA plates.

1-20 µl of sample is placed in the wells of an ELISA plate in a total volume of 0.15 ml of the reaction mixture. The reaction is run for 30 minutes at 37 ° C. The reaction is stopped by adding 50/1 of 2.4 M H2SO4 containing 1% creatinine. The mixture is incubated for 30 minutes at 60 ° C. Any precipitate present is separated by centrifugation. 150/11 of the assay mixture is transferred to a new ELISA plate, treated with 100 µl of 10% σ-naphthol in 5 M NaOH and incubated for a further 20 minutes at 60 ° C. The absorbance is measured at 520 nm and the specific activity is expressed as in Example LVIII B.

LVIX Example. Determination of chemically inducible DNA sequences: Bacillus thuringiensis endotoxin.

A. Plant extraction procedure.

About 100 mg of plant tissue is homogenized in 0.1 ml of extraction buffer (50 mM Na2CO3, pH 9.5; 10 mM EDTA; 0.05% Triton Χ-100; 0.05% Tween; 100 mM NaCl; 1 mM phenylmethylsulfonic fluoride (added just before use) and 1 mM leupeptin (added just before use) After extraction, 2M Tris, pH 7.0 is added to adjust the pH to 8.0-8.5. The extract is then centrifuged through 10 minutes in a Backman microcentrifuge 1 the supernatant is used for ELISA analysis.

B. ELISA.

The ELISA plate is pretreated with ethanol. The affinity purified rabbit antiserum against Bt (50μ1) at a concentration of 3μg / ml in borate buffer salt (100mM boric acid, 25mM sodium borate, 75mM NaCl, pH 8.4-8.5) is added to the wells of the plate and left to stand. for incubation overnight at 4 ° C. Antisera is prepared by immunizing rabbits with gradient purified Bt (Bacillus thuringiensis) endotoxin crystals [B. J. Ang and K. W. Nickerson, Appl. Environ. Microbiol. 36, 625-626 (1978)] dissolved in sodium dodecyl sulfate. The plates are washed with a wash buffer (10 mM Tris-HCl, pH 8.0; 0.05% Tween 20, 0.02% sodium azide) then treated at room temperature for 1 hour with blocking buffer (10 mM phosphate buffer , pH 7.4; 140 mM NaCl; 1% bovine serum albumin, 0.02% sodium azide). The plant extract is added in an amount to give 50 µg protein (typically 5 µg extract; protein is determined by the method of M. Bradford, Anal. Biochem. 72, 248 (1976) using commercial Bio-Rad kit) and incubated overnight at 4 ° C. After washing, 50 μl of affinity purified goat anti-Bt serum is added to a concentration of 3 μg / ml in ELISA diluent (10 mM phosphate buffer, pH 7.4, 140 mM NaCl, 0.05% Tween 20, 1% serum albumin bovine, 0.02% sodium azide) and incubated for 1 hour at 37 ° C. After washing, 50 μl of rabbit anti-goat alkaline phosphatase-bound antibodies (Sigma Chemicals, St. Louis, Mo., USA) diluted 1: 500 in ELISA diluent are added and incubated for 1 hour at 37 ° C . After washing, 50 µl of substrate (0.6 mg / ml p-nitrophenol phosphate, 0.05 mg / ml MgCl 2 = 10% diethanolamine, pH 9.8 adjusted with HCl) is added and incubated for 30 minutes at room temperature. The reaction is terminated by adding 50 µl of 3 M NaOH. Absorbance is read at 405 nm in a modified ELISA reader (Hewlett Packard, Stanford, Ca., USA).

162 317

Atobchr PR1013 kb arm Λ

-19kb onm .- <9.2kb

PR- 1A τ-Τ7ι and mr

R PHR R R RPC

Fig 1

Atobchr PR1013

C C | xCla I | x LGT agarase l_

Jx Cla I jx LGT agarose j

jmame J ligation Jtransformation

Fig 2

| x EcoRI jx EcoRI jx a ^g arose U3T jx Agarose LGT \ ___ /

J mixing 1 ligation j transformation

Fig 3

^j xagarose LGT J and mixing J ligation

J transformation

j transformation

Fig. 5

Fig ί

| mixing | ligac ^j a { ^tr ans ^f ormac ^j a

PBS-PR1013 EcoAPst AXho

Fig 6

Fig 7 pohlinker

JxAsp 718 jxPst I {x LGT agarose

P | xAsp 718 Jx Pst I jx agarose

LGT {mixing and ligation łrans ^{j f j} and ORMAC

Insert in both orientations

Fig 8 u ΚΧ B P single strand ------ lj-1_i _____

M13mp18-PR1013 EcoAPst AXho {primer heating | Klenow fragment elongation I ligotion

5 'Β P 3 ’

- ⁵ - CTCCTATAGTCATGGGATTT- · - ¹

-y ---- G ^ GAAAATC ^ C ^ TACCCT ^^ --.---- and transformation and isolation of the c * B P v 'mutant

- i-CCATGG-1 - ³ - M13m ^p 18-PR 1013

-j --- GGTACC -> - EcoAPst A XhoNco

Ncol

Fig. 9

RFM13, (mp18- PR1013I EcoPst Xhca. .. * p ~ N | xBst Eli JxPst I k_ ^p BS-PR1013 \ EcoAPst

AXho lxBstEII JxPst I 'mixing and ligation | transformation of X

Fig.10

X

jx Nco I

Jxagarose LGT k_.

| LGT xagarose

J mixing | ligation and transformation

Fig.11

jxSal I jxSnaBI JxSall | xSnaBI jxEcoRI jx EcoRI | xagarose LGT ixcg) arozaLGT jxagaro> for LGT \ / mixing ligation transformation

Fig 12

Fig. 13 Fig U

5 'deletion of the PRla promoter sequence (Alul-Ncol) at the beginning of the GUS gene in pCIB 200

Fig 15

5 'deletion of the PR la promoter sequence (Haelll-Nco I) at the beginning of the GUS gene in pCIB 200

Fig.16

PR1a promoter at the beginning of the spliced Bacillus thuringensis endotoxin gene cloned into pCIB7lD digested with Sal I / BamHI

Fig.17

6759

2000

EcoRl <12320 / Sst I

Pstl

Xhol

AND

Promoter sequence from the PR1a gene at the beginning of the truncated Badllus thuringensis endotoxin gene

800!

θςΙΙ

Hmd ni vul vu II St I Sstll Xba I • 000

3000 Ncol Sst II Hindlll

Hindlll '4000 coRI BamHI

Hindlll

Fig 18

Fig 19

The PR1a promoter at the beginning of the Arabidops wild-type AHAS gene cloned into a plasmid

Bluescript

Fig 20

Promoter sequence from the PR 1a gene at the beginning of the Arabidopsis wild-type AHAS gene cloned into pCIB 200

Fig 21 Fig 22

Promoter sequence from the -1,3-glucoromdase gene clone in pBSGIuc 391 at the beginning of the GUS gene from pBSUS! 2

Fig 23

EcoRI <12884 / Sst I PvuL

12,000 EcoRI, EcoRI Nco I.

Promoter sequence from the β-1,3-glucuronidase gene clone in pBSGluc 39 1 at the beginning of the truncated Bacillus thuringensis endotoxin gene

Fig 24

Promoter ^g lu ^{y k} Anaz 1 to ³⁹ g poczq ^{early ene} AHA ^S, wild-type Arabidopsis cloned into the plasmid Bluescript

Fig. 25

162 317

KpnK Ssti / 1i.0U <EcoRl 1ŁO1

EcoR EcoRI

Sst I ¹ Hind 12000 Hind Ul BamH Hmdll.

H ind Ul HindUSj Sst / i · BomHr 10000

Xbal <BamH Pstl <Sol HindllKSfWI

Ρνυ I

mdlii <Ρνυ l

Pstl

Xhol Cla I Sma I Hmdlll

Promoter sequence from clone ii-1,3-glucuronidase in pBSGIuc39 1 at the beginning of the wild-type AHAS gene in Arabidopsis

Fig. 26

Fig. 27

Promoter sequence from the ft-1,3-glucurontdase clone in pBSGIuc 39 3 at the beginning of the GUS gene from pBSGUS12

Fig 28

EcoRl <130 36 / Sstl

6000

Promoter sequence from the β-1,3-glucuromdase clone in pBSGIuc 39 3 at the beginning of the truncated Bacillus thuringensis endotoxin gene

Fig 29

Glucanase 393 promoter at the beginning of the gene

Wild-type AHAS, cloned into a plasmid

Bluescript

Fig. 30

Pvul

Kpnl <Sstl / 14196 <EcoRI 14000

Zn

EcoRI.

Ncol Sstl.

Hind III1200Γ 'Pvu I

ΧοαΚθα / ηΗ

PstKSal

Hind III <Sphl

Ńdei

6000

Sequence of the promoter from the clone / L -1.3 -glukuoonjdase in pBSGluc 39 3 at the beginning of the wild-type AHAS gene - from Arabidopsis

Fig 31

8759

Hmd II Hmdlll

2000

Hindll ul νυ II ^S tVt.l Xbal 1000

3000 Ncol SstH Hind III

Hind III 4000 EcoRI! On HI

Herbicide resistant Arabidopsis AHAS gene in a Bluescript plasmid

PR1a promoter at the beginning of the herbicide resistant Arabidopsis AHAS gene, cloned into the Bluescript plasmid

Fig 32

Fig 33

Kpnl <Sst I / 13482 * EcoRI

Ρνυ I

Ncol

4000

Pstl Ndel «OO vul Sst ll Sphl

Bal l <2000 Sst ll indlll'Pvui

10,000

X ba KBamH PsIKSall

HindllKSphi Ncol

Promoter sequence from the PR1a gene at the beginning of the herbicide resistant Arabidopsis AHAS gene, cloned into pCIB 200

Fig. 34

Glucanase 391 promoter at the beginning of the herbicide resistant Arabidopsis AHAS gene, cloned into the Bluescript plasmid

Kpnl <Sst l / 14044 <EcoRI

Ecq ^{r | U001} '

EcoR

Nco Sst I

indlll <Pvu l

Xbal <BamH

PstKSol Hind II! <Sph

Pstl Xhol Clal Sma I

Hmdlll

Fig 35

Sequence of the promoter from the? -13 -glucuronidase clone in pBSGIus 39 1 at the beginning of the herbicide-resistant Arabidopsis AHAS gene

Fig. 36

162 317

8772

8000. ΡνυΙ

Kpnl <Sst l / 14196 <EcoRl 14000 Sphl

EcoRI.

Ncol,

Sstll

Xbal <BamHI

PstKSoll HmdllK Sphl

vu I Pstl 000

Pstl Xhol Jol Sma and Hindlll

Glucanase 39 3 promoter at the beginning of the herbicide resistant Arabidopsis AHAS gene, cloned into the Bluescript plasmid

Sequence of the promoter from the? -1.3-glucuronidase clone in pBSGIuc39 3 at the beginning of the herbicide-resistant Arabidopsis AHAS gene

Fig. 37

Fig. 38

30 50

CTTGAGGATTTCGAGTTTTGGCTTTTTCAAAACTTGAGCTTTCTTTTTCATTAATTTCGG

00 110

AGAGCAATGTAAACGTAATCTATTTGTAAAGGATCTAAAAAAACTCCATTTGAATCTAAA

130 1500 170

TTCAGATCGTGATTGGGATAGGGAGACGTAAATTTTTTTATATTTTGAGTCAAGTTGTGA

2W 200 ttggtacgagataatataaaattactgtatcgcgtttgtagcattagatcggaaattagg

250 700 200

GTAAATTTATTTAGAGTTGAGAACTCATAAATTTTTTGATTTTCTTTACGTCITTTATCTGAT

310 300 500 agttatctaacgtcatcggggtgacggccatgttcaagttttccgcgagtattgaggaag

370 300 110 ggggaagaggatttaaactgtgtttggtattattatagtttttttttttagtaagttga

430 400 470

TTGTACATATTGAGGATGTGGTGTGTTATTCTATAGTGTGATTATTAGTGGGGCTGGACG

430 H 0 503 ttaaggtgtttattttaaattcttcctttttttgacgttcccattctgagagggggaaag

Fig 39 (I)

550 570 590

TATAAATATGGAAGTA / AAATTAATCACG \ TCGTTAAATGTA (G \ AAATTAATTAACACA 610 630 650

TTAACnATAAnCAGTCTACTTTATTTTTCTTTmnTCATCTGATAGTTCTATAAAGTGT 650 600 710

TTATCTTCTTGCATTGACcr (TTT / ATrTTTATTTTGCAACATCGGGTTATTAT; TATTTTTCA 530 700 500

AnAATTGGTAACTGCATATATAAGTTTTATTTGGTTTnnTTGAAAATAGGATTTTATTATn 590 100 300

TATATnAGGATATATTACATTGATATTTnCTT’GTnAAAAAATTTTGTATGTACATGTATA 850 870 890

ATCACCGTGA, TTrCTTTAAGATTTCTCCTATAAATAra: TTGGTAGTAAATCTAGTπTT f_ "910 930 950 n (CATTnATGATACAA (nTΠ'TCTCCTATAGT (nATGGGATTTGTTCTCTΓTTCACTATTGnC

MetGlyPheValLeuPheSerGl nLeuPr 950 990 1010

TTCATTTCTTCTTGTCTCTACACTTCTCTTATTCCTAGTAATATCCCACTCTTGCCGTGC oSerPheLeuLeuVal SerThrLeuLeuLeuPheLeuVal 11 eSerHi sSerCysArgAl

Fig. 39 (Π)

1030 1050 1070

CCAAATTTCTCAACAAGACTATTTGGATGCCCATAACACAGCTCGTGCAGATGTAGGTGT aGt nAsnSerGI nGl nAspTyrLeuAspAlaHisAsnThr AlaArgAlaAspVal GlyVa

1090 1110 1130

AGAACCTTTGTCCTGGGACGACCA (GGTAGCAGCCTATGCGCATTTrΓTATGCTTCCCAATT I GI uProLeuThrTrpAspAspGl nVal Al aAlaTyrAI aG I nAsnTy r Al aSerGI nLe

1150 1170 1190

GGCTGnAGTTTGTATnCTnGTACTTTCTCATGGT (A (ATACGGCGAAAACCTAGCTGAGGG uAI aAlaAspCysAsnlLe ^ al HisSerHi sGlyGI nTyrGl yGl uAsnLeuAlaGl uGl

1210 1230 1250

TTGTGGCGTTTTCATGACGGCTGCTTAGGCTGTTGAGATGTGGGTCGTTGAGATACAGTT y SerGl yAspPheMet ThrAl a Ala Lys Al aVa IGl uMetTrpValAspGI uLysGl nTy

1270 1290 1310

T TATGTCCATGACTCAAATTCTTGTGCA <AAAG (: T ^ CTG & TGTGTGGACTC TATACTCAGGT r Tyr AspHisAspSerAsnThrCysAla Gl nGl yGl nValCysGlyHi sTy rThrGlnVa

1330 1350 1350

GGTTTGGCGTAACTCGGTTCGTGTTGGATGTGCTAGGGTTTAGTGTAACAAT (GTAC & ATT l Val TrpArgAsnSerVal ArgValGl yCysAlaArgVal Gl nCssAsnAsnGlyGlyTy

Fig 39 (ΙΠ)

1390 1410 14 30

TGTTGTCTCTTGCAACTATGATCCTCCAGGTAATTATAGAGGCGAAAGTCCATACTAATT rVal Val SerCysAsnTyr AspProProGI yAsnTy r ArgGl yGl u Ser Pro Ty rEnd

1450 1470 1490

GAAACGACCCACCTCCATTTCACGGTAATATGTATGGATTGTTCTGCTTGATATCAAGAA

1510 1133 1550

ACCCCCAACCTGCTCTAAACCGAAACTTAAAGTCAAGTATATAGTAATCTTAATACACCT

1500 1150 1610

GCCCTCACCTTAAGTGGATACCCCAAAAGAAACAGTGGTCATAACC AAGTGGAACACCAC

1630 1150 1600

TATTTTATGTCCACTAACTCGTATGATCTGATACTATTATGAACACTCTTTCAATCACCC

1600 UTOI 1730

TCATACTTTGTTGATGGTCTAGCCACCTTATATATTAAGCGAAACATTACTCACCAAATT

1750 1770 1790

CACCC (TTACCAAGTCCACACAGTTACCCCCACCCAATAGAGTTAAAAAAAAATGACAACC

1810 1830 1850

CACAACTCATATTGTAGGAGGATATCGACCTTTTAGCAGCTAGTTTTCCTAAIcΓGCACTC

Fig 39 (IY)

1870 1800 1010

TCTTCCTCAACπTAAGGTCTAAAAATAπTACATCATCATTCAACCCACAAGTTAACCA 1930 1900 1970

TCTTTAAATATCTATGCATAATTCTAAATAGTAATCTGCCCACCACCCrGCCAACCAAACC 1900 0010 2030

TCCCAAACCAACTAATATACTCTGTCCTACCCCATTCCTAATGTCA'ΠΆGCAGACCCC

Fig. 39 (V), 0 30 50 πACCTCCCAAGTATCATTAACATCCTATCCTCAAACAATCATATAGAGATTCTACACCA 70 00 110

TACTCAACTATATATCΆCATTCGTCCCCTATACCTCGTATATAGATTTTTAACTAAAAGAA 130 150 170

TTTAACA T TACCAAATAAATCATCGTTTAAATCAAATTCCGACTATACATCCGATACACT 100 HO 200

ATCπTTCGTACTCGACCACCAAAT <TTCCTGCπCCATTΆCCCCTATCGCAGATAACCCA 250 700 290

CTTTATAAGGATGTATTCACTTTCTACCCCCATCATACTCTGTπCATTTTTCCCGAACC 310 300 500

TTCTATACGTTCGACAG (AcTCCCTCCAATCGAACAGGTCCATCTCCACAACTTCCCGCAC 370,000 HO

AATCACCCCCATCACACTCTTTCCTCGCAACACACCCATGCTCAAATACACTGGTAACCA 430 450 4TO

CTTATCACCACTACCTTCCTGTATATACTACAAATCACCGATCTCTAAACGTCGTTCAAAC 400 510 300

CATGATCATGTCCCCCCCCGTTGCTCCTCAATATCTCTACTCCTCCACTGTAGGCTTTCT

Fig. 40 (1)

550 570 590

TTTCTTTAACGAATATGATCTAACGTTCTTTGMTTAACGGAGCTATAATTTTATACTAT

610 630 650

TCTTTTCAAATATGGCTAAA (AGTTTGCTAACAAGTTGC.AAACTTTGTAACTCAGCTATA

670 900 700

TTCTTCCCTATAAAAATCATCCTTATTTTGCTTTGTTTCTCACCAAAAGCIAAAAAA-GGG

MetGI

730 750 770 τπATTTTCTTCAATATTTGCTTTTTTCATTTACTTTGATATATATTAATTCACTCATCAAA yPheLeuThr Thr II eVal AlaCysPhell eThrPheAla I leLeu I le His Ser SerLy

790 8Ό 830

TTCTCAATTCTCACAAAAATATTATCTTAAACCTCACTVTTGAATATATTAGACAAGTTTT sAlaGt nAsnSerProGl nAspTyrLeu AsnProHis AsnAla AlaArg ArgGl n ValGI

850 870 890

TTTTGTCCCAATTAAATGGGACTATTA (G3ATAGCATAATATGAAAAAAAπATGAAAATAT yVa I Gl y Pr oMetThr T rpAspAsnArgLeuAlaAl aTyrAI aGl nAsnTyrAI aAsNGI

Fig 40 (H)

9I0 930 950

TTGATTTTGTGAGTGAGGGATGATAGACTATATTGTAAGTTAATGATAAAAAAATTGCATC nArglleGlyAspCysGlyMet 11 eH i sSerHi sGI yProTyrGl yGI u AsnLeuAl aAI

970 990 I0I0

ATGCTTCAATAAAATTAAAGATGATTGTTATTTTAAAATTTGGGTAGATGAGAATCTTTT aAloPheProGI n LeuAsnAlaAlaGI yAlaVal LysMetTrpValAspG I uLysArgPh

I030 7050 I070

CTTTGATTTAAATTAAAATTCTTGTGTAGGAGGATTATGTGGAAAAATATACT (AGGGTGTT eTyrAspTyrAsnSerAsnSerCysVal Gly Gl y Val CysGI yHi sTy rTh r GI nVal Va

I090 III0 II30

TTGGAGTTTTGTGAGTAAGTGTATGTTTTGCTTGTTTTA (GTTGGATT (ATATITTTTTGTTTTT lTrpArgAsn SerVa lAgLeuGlyCysAla Arg ^ / aA ArgSerAsnAsnGlyTrpPhe

II50 II70 I190

ATTAAATTGAAATTATGiATAAAAAATGTAATTTTTTAGGAAAAAGTAAATTTGGATATAT eIleThrCysAsnTyrAspProProGlyAsnPheI I eGI yGI n ArgProPheGI yAspLe

I2I0 7230 I250

TTACTGTTAATCACTTGATTCCATTATTGGMCTTCCMCTGATGTATAATGAGTGCAATT uGI uGI uGI nProPheAspSerLysLeuGI at LeuProThrAspVal End

Fig. 40 (IH)

1270 1290 1310

ACATGMCAAATTATCATGAATAAAGGAAAATAAAATGCAGTGCTATGCTATGTCGATTTT 1330 1350 1370

AGCTTCCCAGTTGGATAATAATCTGATGGTGTAGTAACTGGCCGAGTGTTTCGACCCTAC 1390 1410 1430

CπTCATGTTGGTAGATGCCATCATTTTATATATGAGTTTTCTGTTGGCTTATTTTTTTT 1450 1470 1490

TTTTTTTAATCTTATTCTAGTTTTTCAGTTTTTTATTTGTACTATATGTGTAT (GG3TCCA 1510 1500 1S50

TTTTTTTTCTTCCACGACCTTTTTCGTTTTCTTCATTCGACCGGGTTCCTTCGAATCTGC 1570 1500 16W

TTCCCAATGGCATAC (TT: CCTAGGGCCATTGAATGTGTCGTATGACTCTTGTATTTTTTTT 1630 1650 1670

TTCCCCTTTAGGGAATATT / TACTTATTCCCTCGGATATCAATTGTATTTTTTTTCTTTCA 1650 1700 1700

TTTCTTAATGGTTTCTCTCTAGTATATMAGGTTTCTTTAAA (XTTGTAAGT (T3C «; TTT 1750 1770 1500

TTTCTCTTTTAAGCTTACTAATCTTG (τ ^ T (GTAJATGAAATATCTCTCTTCTCTTTTTCTTTT

Fig 40 (IY)

1810 1830 1850

TT7TTTCTTTTTTTTATTATCTTCAGCTACGATTTACCCCTTCTTTTTTGATTTTTTTTT 1370 1500 19Ί0

TCTAAAATGTTTTAACATCTTTTGAGT (TTAAAT ^ TTTGGCNCCGTCTTTGGGGTTTTCTT 1930 1500 1570

TATCTTAATCATAGTTCTCATCTTGTTTTCTGAATTTATTTTTTCTTTTCTTCTATCCT 1950 0000 0000

CTTAATTATCATCAATGTCSCCATTGGTTTTGTAGCTATτ ^ TGCTTTASACTTCTTCTCTA 2050 2070 0590

TTAACTCCTTAACTCCCTCATACCAτACTCCGGCAGTGTACTCAATTTGAATT

Fig. 40 (Y)

30 50

TAAGTMGCTCπTAAGCMTGGCTGCCXA (CTAATLTTCTACAAQKTTTCCATCπCTT

MAAHK ITTTLSIFF 70 50 110

CCTCCTTTCCTCTTTTT1 ^^ C ^ C ^^ (^ TC ^ (T ^ (^ GC ^ CTGTATU ^^ TC ^ TCTATTGGTGTCA LLS SIFR SSDAAG IAIYWGO

130 150 170

ATACTTCAACGACT3GCTCTCTTGCATCCTCCTTCTCAACTGGTAACTACGAGTTCGTCAA

NGNEGSLASTCATGNYEFVN

150 210 230

CTTTTCATTTCTCTCTTCCTTTGGCACCCGTCTTTCTCCATTTCTCTACCTTGCTCGTCA

AND AFLSSFGSGO APYLNLAGH

250 270 250

CTGCATCCCTTACAACTACTGTTGCGCTπTTTTTTCTACGTTATAATCTCTTGCTAAAG CNPDNNGCAFLSDEINSCK S

Fig. 41 (I)

310 330 350

TCAAAATGTCAA ^ GGTCCTCCTCTCTATCGGTGGTG ^^ G ^^ (GG ^^ C ^^ TkTT ^. ^ (CTlCT ^^ TC

QNVKVLLSIGGGAGSYSLSS 370 390 410

CGCCGACGATGCGAAACAAGTCGCAAACTTGATTTGGAACAGCTACCTTGGCGGGCAGTC

ADOAKQVANFIWNSYLGGQS

430 450 470

GGATTCCAGGCCACTTGGCGCTGCGGGTTTOGATGGCGTTGATITCGATATCGAGTCTGG

DSRPLGAAVLDGVDFDIESG 490 510 530

CTCGG ^ (C ^> ^ (3TTiCTG ^ (^ G ^ (^^ Tr ^ (^ Tr ^ C ^ C ^ TC / ^ G ^ GAGGCTAA ^ GGAATTTTGGAC ^ GT ^ TTTT

SGQFWDVLAQELKNFGQVIL

550 570 590

ACCTGTTGTGCTGTGGCGTCCAATACCAGGTGCCTATTTAGACGCCGTGGCTAGGGTCGG

SAAPOCPIPDAHLDAAIKTG

Fig. 41 (II)

610 630 650

ACCGCCCGATCCCGCCCGGGCTTAGTCTCGCGGTGG (TCTGTTGCGTACGCCCGTAGGCGA

LFDSVWVQFYNNPPCMFADN

670 690 710

TGCG (CGTGGCTCTTCAGTC (T \ TGGCGGCAGTGGACGGCGTTTTCGATATCGGA (CTTTCA

ADNLLSSWNCIWT AFPTSK LY

730 750 770

CATGGGACCGTCGGCGGCATGGGAGGIτGGCGC £ GAGCGGGGGATTCACCCTGGTGGGCGC MGLPAAREAAPS G G F I P A D V

790 610 830

GTCCG'CπC T TGGGCTCCCTTGGTCGCCG A AGCTT T ^ TTGGTCAC ^ GGGGGGGCGGCGCC

LISQVLPTIK ASSNYGGVML 850 870 890

ATGGGGT / AGGGCGΓΓTCcACAATGGCTACAGCGATTT (TACCGGAGCCAGTGCCGGTCGGGG

WSKAFDNGYSDSIKGSIG ·

Fig. 41 (III)

910 330 900

GA AGC ^ dA AGTT T AAT T T TAGCTAGAGTTGTGAATGGGTTTTGGGGCATCGTGACGGC 970 990 1010

TAA> GGGGT (CGGAC ^ TΓCATCTCCCTCACTCAGTCCCGCATTAAATTAGTGTGACGTGGCAG 1030 1050 1070

ΓTGACATCCTCCTTTTCATTACTATACTACCGGCΓGTTTGAGACTG (GGGGCGTTGATCCTG

1090

GTGGGG ^ (^ TT ^ (^ TG ^ (^ TTT ^ TCT

Fig 41 (IY)

AAAAAGAAAA AAAAAATGAA

CCTTTATTTT GGCCAATACT ACATTGTCAA CAAATGCACC 101 151 201 GGAGGCAGGC GGCTCGACTC AGGAACAGTC CAGGCTCGCA GCAGTGGCCG AGGTAATTGT 251 301 351 TGTCAAGGCT ATGGAAAAGC TCAACCCAAT CAGGACTTTG 401 TCCCCATGGA ATTCAGCCCG ACAGCACCTA TTAACGAACA 451 501 551 ATGTAACAAC CCATGTACTG ATGGGCCTGG ATCATGTGGG 601 AGATGCCCTG ATGCTTATAG

CTTCCTCAAA AGCTTCCCCT TTTTTGCCTT TTGTAGCTGT TACTCATGCT GCCACTTTTG TACACAGTCT GGGCCGCGGC CTCTCCAGGT AGGCCAATCT TGGAGCATTA ATGTGAACCC TTTGGGGTCG AACCAATTGC AACTTCGATG GAGACTGGAG ACTGTAACGG GATGTTAGAG ACCTAACACT TTAGCTGAAT TTGCACTTAA TCGACATCTC TCTTGTTGAT GGATTTAACA ACCAATGGAG GATGTCGTAA TCTCAGATCG ATGCCCAGCA CAGTTGAAAA CACAAGGTGG TGATAAAAAC CAATGAATAT TGTTGTACAA CCTACTGATT TGTCGAGATT TTTTAAGGAA TTATCCACAG GATGATCCAA CCTGTTTGTT

Fig. 42 (I)

551 TACGTGTCCT TCTGGTACTA ATTACAGGGT TGTCTTCTGC CCTTGAAATT 701 GAAGCCTGCA AAATTATGAC TATGTAATTT GTAGTTTCAA ATATATAAGC 751 TACACAAGTA GTACTAAGCA CTATTAAATA AAAAAGAGAG TGACAAAGAG 801 GAGAGGCTGT GGGTCAGATT CTCTTGTTCG CTGTTGTCGT TGTTGTAGCA 851 TTCTGGTTTT AAGAAATAAA GAAGATATAT ATCTGCTAAA TTATTAAATG

Fig. 4 2 (II)

30 50

AAAAAGAAAAAAAAAATGAACTTCCTCAAAAGCTTCCCCTTTTTTGCCTTCCTTTATTTT

Met AsnPheLeuLy s Cheese PheProPhePheAl aPheLeuT yrPhe 70 90 110

GGCCAATACTTTGTAGCTGTTACTCATGCTGCCACTTTTGACATTGTCAACAAATGCACC Gl yGI nTyrPheVal AlaVal ThrHisAl aAI aThrPheAspIleVal AsnLysCysThr

130 150 170

TACACAGTC TGGGCCGCGGCCTCTCCAGGTGGAGGCAGGCGGCTCGAC TCAGGCCAATCT TyrThr Val T rpAl aAI aAlaSerProGI yGI yGI yArgArgLeuAspSerGI yGI n Ser

190 210 230

TGGAGCATTAATGTGAACCCAGGAACAGTCCAGGCTCGCATTTGGGGTCGAACCAATTGC TrpSer 11 eAsnVal AsnProGlyThrValGl nAlaArg I leTrpGlyArgThrAsnCys

250 270 290

AACTTCGATGGCAGTGGCCGAGGTAATTGTGAGACTGGAGACTGTAACGGGATGTTAGAG AsnPheAspGI y SerGI y ArgG ly A snCysG I uThrGlyAspCysAsnGI yMetLeuGI u

Fig. 42a (I)

310 330 350

TGT (CAAX5CtATGGAAAAGCACCTAACACTTTAGCTGAATTT (G: ACTTAATCAACCCAAT CysGI nCI yTyrCl y LysAla ProAsnThrLeuAlaCI uPheAla LeuAsnCI nPro Asn

370 390 410 tA (GArCAAAGAC (AAATATATAGGAGATCGATTAAACATtttCAAGGAAAAtAGCtCC Cl nAspPheVal AspIleSerLeuVal AspGly PheAsnlleProMetCI uPheSerPro

430 450 470

ACAAAACGAGACTGT (ASATCA'CT (A ^ GATGCΔCAG (TACCTATTAACAAACAATCttCAGAA ThrAsnClyCI y CysArgAsnLeuArgAysAhrAI a Pro I l e AsnCI uCI nCysProAl a

490 510 530

TAGAAGAAAACACAAGAA (AACAAAATAACtCATAAAGTGAGAAAAAAACCAAAGAATAA Cl nLeυLysThrGtnGlyGlytysAsnAsnProCysThrVal 11 eLysThrAsnCI uTyr

550 570 590

ACAAATACAAATGGGCtTG <ATT <ArTGTGGGGttACTGATTTGACGAGATTATTTAAGATA CysCysThr AsnCI yProClySerCysCly ProThrAspLeuSer ArgPhePheLysCI u

Fig A2a (II)

610 630 650

ACAAGtATAAAAGC AAAAAGA AATCCAtACGAAGATCAAAtTACπ TCT T TATGTCTtt T ArgtysProAspAlaAyrSerTy rProd nAspAspProAhrSerLeuPheT yrCysPro

670 690 710

TtAGGAACTAAAAATAAAATΆGACAACACTtCAAGACATTCAACCTTCCAAAATTATATT SerCI yAhrAsnAyΓAΓgValValP ^ e? CysProEnd

730 750 770

AAAGAACATTCTACTTAtAAAAATATATAtTACAtAACTACTAtTAAGCAtTATAAAATC

790 810 830

AAAAAACGCGAGAAArAAT3 (AAAAACtAATAGAAACGTTATAATGAAtCTTGAACAtGA

850 870 890

ACAAGAACTAAAtACCTATArAAG> AA ^ ArAAAAAGAAAAAAATtACtAAAAAAATAAAATC

Fig. 2a (III)

101

151

201

251

301

351

401

451

501

551

601

651

701

GACtAtTAtT

AAAAGAtCAA

AAAATTAACA ttCAAttCAA

AAAAGAAAGA

TTtAAACACt

AAGAtCGAAt tAATACTtAT

CCCTCtAAGt

AAACATtTtA

AAATAtCACA

ATACACAtAG

AACAACAAAA

AATAAAAATA

AACAAtAAAt

AGCCGGCACA

CAGAAAGAAA

CAAATACACA

AAAtCAAAAA tgacaaaaca

AAtAAAAAAG

AAAAAAAAAA

CTAAGTGAAA

TTCAGAAAAT

ATAAAAAAAA

AAAGAAAAAA tGAGCCATAA

TAACGAAAAA

ATAGCTAAAA

ATAAAAAAAA

GCCAAATCAA AAACtAAATC GAAAAAAAAA AACCAACTtA AATACAAAAA ATAAACCGCA AAAAAtATAT GGACCCAAAA ^^^^ TAT GACCAAAATC AACAAGAAAA AAAtATTTAT CAAACCAGAA AACAAAAGTA AAAAAAAAAt CAGCCAttAC AAAACAAAAG CCAtTAAAAT tCCCAAAAAA AAAATGAAAA ATACAAtATT AAAtAtTAtC AATAtAATGC TGtAATAACA ttAGAACAAT ATtAAACAAT AACAtAATAA AGAAAAAAAA ATCCAGtAAA ACAAtAACAA ATCACACCCA TAACAAAAAC AATCCtAAAT ACACAAAAAG CACGAAAAGA AtAACCAAAA GAAATACGGA AATCATTAAA AAtAAAATTA ACGAAAAAAA aaaaaaaaaa AAATACAGAA AGAAGAATAG tAAAAAACAA ACttACCAtA

Fig. Λ3 (I)

751 801 851 901 951 1001 1051 1101 115Ί 1201 12 ^ 1 1301 1351 µm U 51

1501

1551

1601

1651

1701

1751

1801

1851

1901

1951

2001

2051

2101

2151

2201

GTTAGAATCC AGTTTCCTCT AATAAATGCA CATTAAATCA ATTCATATAA TTTAAAAATA CGATTCGAGA CTGTTATGGA AGGCGTTGCC ATTGCTTTCG TGCAATAGCA ATATGACATC AAATGAAAGT CATATAGAAC TTTAAAATCT TTCAAAATAC GACCAATATT TATTACTTAC TATGTATTTT ATCTTGAAAT TGAATTGAAA ATATAAACAA TAGATATCGT TAAGTATTTA GTAACGATGA TTAAGCAGGA ATTCCTCTGG TAACTAGCTA TAATTTCTTT TGTAAAGTCA GAGAAAATAA ATAACTTTAC ATCATCAAAC AATGATCAAG TTGACATGTT AATAGCCAGG CTTCATTAGC CTAC6AATAA CAAATCCAAT TATATATATC TCAATATAAA TAGCTCGTTG AAGTCTTCCC ATCATGTCTA CCTCACATAA

Fig. 43 (II)

GCGAAATATT AGAAGACTTT CTAGACATGG aaaaaaaaaa TTCGATGTAG ATTCTCATCC TTATTCTTAG AACTACTTTT CTCAACTAGT TTTAAGGGAA TTATGGATAA ATTTAAATAA ATATTAAATT ACTTGGTTTA CCACAAACAT TTTATTAGTT TTGTGCAGGA TGAAAGAAAC AGATAGTACG GCACCTTATG TCATCTTCTT TTTTCCACAA TCACCGGGGG CGGCTCTTAA ATTATATTTA CACAAGGCTA TTCATCTTAA TTCTCCCAAC ACATAATACT CCTCAAATGG

CTGCTATCAC ACTCCTAGGA TTACTACTTG TTGCCAGCAG CATTGACATA GCAGjTTTCT ggtcaaatat ttgaacttcc cagccaaaaa tattgtctta TAATTTTGTG TGCGCAAAAT TTTAATTTAG TTGATAGTTA TTTGCTTATT TTTCTTTTCA AATTGCTTGT GTTTTTTTCT CAAATTAACT TGCACCGTAT TCATTTAGCG ATAGTTATTT GCTCTATTTT TGTGTAACAC TCACTCACAA ACTTTTCAAT TTGAGGGGAG GACAGTGAAT CTAAGATTGA AATTTATGAG TTTAATTAGA CTAATTCCCA TTTGATTTAT TGGCTAGAAG TCAATTATTT GCATAGTGAG TCTTTTAACA CACAGATTTG AGTTAAAGCT ACTACGTTCG TATTAACCCA TAACATATAC ACCTTCTGTT CTAATTTCTT TGACACTTTT TGTTAGTTTG TTCCAAAAAG GACGGACATA TTTGATATTT GAGAATACTT TACCTTAACC TTAATAGAAT TTTTTATGAC ATCACATATA TTATGGAATA TATACGACCA TAATTTTCAA ATATCTTATA GTCGTACAAA TATTATAGCA TGTTTAATAC CACAACTTTC AAATTCTTCT TTTCCTTAAA AACAAAATAT GTCACATAAA TTAAAATAGA GGAAGTATAC TACATCAATC AGCCCCTAGT GGAGGGGACC CTACTGTAAG TTTT.TAAGTT TTCAAGAATT CAGTAATTGA

Fig. 43 (III)

2251 TTAGGAGCCC GTCTGGACAT AAAAAAAAAT TCCTTTTTTT CCAAAAAATG 2301 CCCACTAAAT TTCTAACACT ATTTTGTAAT TCTTATTGAG CAGGGGGCTC 2351 AATCGATAGG TGTTTGCTAT GGAATGCTAG GCAACAACTT GCCAAATCAT 2401 TGGGAAGTTA TACAGCTCTA CAAGTCAAGA AACATAGGAA GACTGAGGCT 2451 TTATGATCCA AATCATGGAG CTTTACAAGC ATTAAAAGGC TCAAATATTG 25C1 AAGTTATGTT AGGACTTCCC AATTCAGATG TGAAGCACAT TGCTTCCGGA 2551 ATGGAACATG CAAGATGGTG GGTACAGAAA AATGTTAAAG ATTTCTGGCC 2501 AGATGTTAAG ATTAAGTATA TTGCTGTTGG GAATGAAATC AGCCCTGTCA 2551 CTGGCACATC TTACCTAACC TCATTTCTTA CTCCTGCTAT GGTAAATATT 2701 TACAAAGCAA TTGGTGAAGC TGGTTTGGGA AACAACATCA AGGTCTCAAC 2751 TTCTGTAGAC ATGACCTTGA TTGGAAACTC TTATCCACCA TCACAGGGTT 2801 CGTTTAGGAA CGATGCTAGG TGGTTTGTTG ATCCCATTGT TGGCTTCTTA 2851 AGGGACACAC GTGCAOCTTT ACTCGTTAAC ATTTACCCCT ATTTCAGTTA 2901 TTCTGGTAAT CCAGGCCAGA TTTCTCTCCC CTATTCTCTT TTTACAGCAC 2951 CAAATGTGGT GGTACAAGAT Fig GGTTCCCGCC . 43 (IY) AATATAGGAA CTTATTTGAT

3001 GCAATGCTGG ATTCTGTGTA TGCTGCCCTC GAGCGATCAG GAGGGGCATC 3051 TGTAGGGATT GTTGTGTCCG AGAGTGGCTG GCCATCTGCT GGTGCATTTG 3101 GAGCCACATA tgacaatgca gcaacttact tgaggaactt aattcaacac 31 ^ 1 GCTAAAGAGG GTAGCCCAAG AAAGCCTGGA CCTATTGAGA CCTATATATT 3201 TGCCATGTTT GATGAGAACA ACAAGAACCC TGAACTGGAG AAACATTTTG 3251 GATTGTTTTC CCCCAACAAG CAGCCCAAAT ATAATATCAA CTTTGGGGTC 330Π TCTGGTGGAG TTTGGGACAG TTCAGTTGAA ACTAATGCTA CTGCTTCTCT 3351 CGTAAGTGAG ATGTGAGCTG ATGAGACACT TGAAATCTCT TTACATACGT 3401 ATTCCTTGGA TGGAAAACCT AGTAAAAACA AGAGAAATTT TTTCTTTATG 3451 CAAGATACTA AATAACATTG CATGTCTCTG TAAGTCCTCA TGGATTGGTA 3501 TCCAGTGACG ATGCAACTCT GAGTGGTTTT AAATTCCTTT TCTTTGTGAT 3551 ATTGGTAATT TGGCAAGAAA CTTTCTGTAA GTTTGTGAAT TTCATGCATC 3601 ATTAATTATA CATCAGTTCC ATGTTTGATC AGATTGGGAT TTGGTAACTT 3651 CAATGTTTAG TTATTTATTA ATTAGTGTCT TTATCATTTG ACTATCAATT 3701 AATCTTTATT TGGCAAGGCT TGATATATTT GAGTTACTCT TAGGTATTTG Fig. 43 (V)

3751 CAAGCAACTG ATCTTTCTTT TATCCCGTTT CTCCGTTAAA CCTCATTAGA 3801 AATATATTAT AATGTCACCT ACTCTGTGGT TTAAGACATT CCCTTACATT 3851 ATAAGGTATT TCACGTCGTA TCAGGTCGAA AAAAATAATG GTACGCTCTT 3901 TCTTATCACA AATTTCTCTC AACTTCTAGA CCAATTGAAT CTTGTCTCCA 3951 ATAAGCATTG CTTTTACTGT ATGTTTCTCT CTATCAATTC AAGGCTCAAG 4001 CCATCAAATC GCTTGGTATT TCTCGCTCCC ATTTAACCAA TCGAGCTGTT 4051 AGCTCTGCTA AAGTCTCATT CTTCAGCTTC TCAAACCACT CAGAGCTTCT 4101 CCAACCAAAA ATGTAGAAAA AATCTTTGAT CCACATGTAA AACCCCAGAA 4151 TTGTCATTCG AGTGAAGGAA GGTGCTGGAA ACGCGACATC AAACACGGAT 4201 TTGCCAGCTG ATTGTCATAG GAAAAAGAAA AGTTGATCAG ATAGTTCGTG 4251 CGGGGTATTT CCTACCTCGC CTTTGATCAT AGCTCTAGGC TTGCGAAACT 4301 AAGTTATGCA CGAGGCCCCC TGCGAATCCA TAAATCTAAG AAGCATGCCA 4351 CAACAAACGG GATAACTTCC AGCTAAGAGA TCTATTTTGA TCTTACCCAC 4401 AAACTAGCTC CGTGTGAATT GTCCATTTCA TCAACTCCAA AGGCAGGAAA 4451 GAAGACTCTA TTCAGAGAGC AGCAAAAGAG Fig 43 (VI) CTC

GAGCTCCCTT GGGGGGCAAG GGCAAAACTT TTTGCTAAAT GGAAAAATAT 51 TATACCAAGT GTTTGTAATA GTTACTCAAT TTGAATTAAC AAAGGGGCAA 101 ATTTGACTAT TTTGCCCTTA TATCTTTTGG TCACAAAAAC ATAAAATATC 151 CCATCCGAAA TTCCAAATGG TCCATTATCG GCAAGTAGCT TTCTTTAATT 201 ATAGTTAGTT GACAAAACAC TATCAAGATA TCATTATTAT AATAATAACT 251 TCAAAGTCCA TCATCTTAGC TGCCTCCTCA GTAGAGCCGC CAGTAAAATA 301 AGAGCGATCA AATAAAAGCC GCCATTAAAA TAATGAATTT TAGGACTCTC 351 GATTGGCACG TAAGTGCCAA AACTCTTCCA ATACTTTGCT GCAACTTGGG 401 GCTGCTAGGT TCTGAGCTTC CAGATATGGG ATATTTCTAA GTTTATCTCC 451 TAATTTACAT CTCAACTAAT ATTAAGAAAT TAAACAGGTA CAGCAAATCA 501 TAAAATTTTC CTCTAAAGAA GACAATGAAT CCGGTTACTG ATTCATTGGC 551 CTTTTCAGAG TCTGCATGCC ATATTCACTA AGGGGTCGTT TGGTACAAGA 601 AATAATAATA ATAATTTCGG GATAGAATTT GAGATTGCAT TTATCTTGTG 651 TTTAATTATA AGTATTAGCT AATTTCAGAA TAAATTTTAC ACTAAAATAG

Fig 44 (I)

TAAAATCAAC TATCACATGT AGAAGGTGGA ATGGAATAGC GCCACTCACA TAGAATATCC TTATTTATCT CACTATTTTA GGTTAGTCTT CATGAGAATC CAGTATCCTC AATAAATGCA AGAAAATTTT CATTAAATCA ATTCATATAA TTTAAAAATA AGCACTTAAG ATACAATAAA AGATGTACCG TTAATAATAA AGAGTTTTAA ATAGGAAAAA AAAAACGGTT CGAGACACTC CGTTGTCTTC AAAGTAGATT CTCATTCATT GCTCTGGTGC TGACATCTTA CTCTTAAGAT ACAGCGAGCC ACTCTACAAT ATACTCAAAT GAAAGTTTTA GAGAACTTTC AAATCTCTCA AGGGAATTCA AAATACGACC AATATTTATT ACTTACTTAC ATGATATGAA TTTTATTTTA AATTTGAATT GAAAATATTA TTTAATATAA ACAATAGATA TCGCTAAGTA TTTACCACAA ACTACAGAAG ATTTTATTAT TTGTAACGAT GATTAAGCAG GGTTGTGCAG GATGAAAGAA AGTAACTAGC TATAATTTCT

Fig A (f

TAATCCCATA

CCAAATGATC

GTAAGAAGTT

TTAGATATGG

AAGATAAGAT

TTATGGAAGG

AATAGCAAAA

CTTCTATTGT

ACTACTTTTA

TTATAGTTAA

AATTACTTGA

ACATGGAGAT

CTATTCATCT

TTTGTAAAGT

CAAGATAGTA CGGCACCTTT ACTCATTTTC TTTTTTCCAC GGTCAGCCGG GGCGSCTCTT CCAATAATAT TCCACTCGAA TAAATAGCTC ATTGTTCATC TCTACCTCAG ATAAACATAA AGGATTACTA CTTGTTGCCA ATATTTGAAC TTCCGAGCCA AAATCTTAAT TTAGTTGATA TTGTGTTTTT TTCTCAAATT ATTTGCTCTT TTTTCGGGTA GGGAGGGCGG TGAATCTAAA CATTTGATTT ATTGGCTAGA CACATACACC GACCTGAGTC

GGANNAAATA TAAAACTTGT AAAATGATCA AGTTGACATG AACTTCATTA GCCTACGAAT TATTTACATT TACACAAGGC TTAATTCTTC CAACAAGTCT TACTCCTCAA ATGGCTGCTA GCACCATTGA GATAGCAGGT AAAATATTGT CTTATAATTT GTTATTTGCT TATTTTTCTT TACTTGCACT ATATTCATTT ACACTCACTC ACAAGCTTTT ATTTGAAATT TATGAGTTTA CGTCTATTAG TTGTATAGTA AAAGCTATTA GGTTCGTATT

ACATCATCAA

TTAATAGCCA

AACAAATACT

TATCTCAATA

TCTCATCATG

TCACACTGCT

TTCTGGTCAA

TGTTGGTGCA

ATCAAATTGC

AGCGATAGTT

CAAATTTGAG

GACTAGTGTC

AATCTTTTAA

AACACATAAC

Fig LL (III)

2101 ACATATTCCC TCTGTTCTAA TTTATGTGAC ACACTTTCTG TTAGTTTCTT 2151 CCAAAGAGAA TGACATATTT GATATTTAAA AATATTTTAA CTTTAAACTT 2201 TTTATTCTCA ACCTTTTATA ACATCACAAA TATTATGGAA CATATTAGAC 2251 TACAAGTTCC AAATATCTTA TAGTCTGTAC AAATATTATA GCATGTTTAA 2301 TAACACAATT TTCACATTCT TCTTTTTCTT AAACTTTGTG CCGAATTAAA 2351 TTATGTCACA TAAATTAAAA TGGTTACATC ATCCCCTAGT GGAGGGACCT 2401 ACCATGTCTA CTGTAAGTTT TTAACTTTTC AAGAATTACA TAATTGATTT 2451 AGTTTCTAAC ACTAATTCTA ATTCTTATTG AGCAGGGGCT CAATCAATAG 2501 GTGTTTGCTA TGGAATGCTA GGCAACAACT TGCCAAATCA TTGGGAAGTT 2551 ATACAGCTCT ACAAGTCAAG AAACATAGGA AGACTGAGGC TTTATGATCC 2601 AAATCATGGA GCTTTACAAG CATTAAAAGG CTCAAACATT GAAGTTATGT 2651 TAGGACTTCC CAATTCAGAT GTCAAGCACA TTGCTTCCGG AATGGAACAT 2701 GCAAGATGGT GGGTACAAAA AAATGTTAAA GATTTCTGGC CAGATGTTAA 2751 GATTAAGTAT ATTGCTGTTG GGAATGAAAT Fig. 44 (IV) CAGCCCTGTC ACAGGCACAT 2801 CTTACCTTAC CTCATTTCTT ACTCCTGCCA TGGTAAATAT TTACAAAGCA 2851 ATTGGTGAAG CTGGTTTAGG AAACAACATC AAGGTCTCAA CTTCTGTAGA 2901 CATGACCTTG ATTGGAAACT CTTATCCACC ATCACAGGGT TCGTTTAGGA 2951 ACGATGCTAG GTGGTTTACT GATCCAATTG TTGGGTTCTT AAGGGACACA 3001 CGTGCACCTT TACTCGTTAA CATTTACCCC TATTTCAGCT ATTCTGGTAA 3051 TCCAGGGCAG ATTTCTCTCC CCTATTCTCT TTTTACAGCA CCAAATGTGG 3101 TAGTACAAGA TGGTTCACGC CAATATAGGA ACTTATTTGA TGCAATGCTG 31 ^ 1 GATTCTGTGT ATGCTGCCCT CGAGCGATCA GGAGGGGCAT CTGTAGGGAT 3201 TGTTGTGTCC GAGAGTGGCT GGCCATCTGC TGGTGCATTT GGAGCTACAT 3251 ATGACAATGC AGCAACTTAC TTGAGGAACT TAATTCAACA CGCTAAAGAG 3301 GGTAGCCCAA GAAAGCCTGG ACCTATTGAG ACCTATATAT TTGCCATGTT 3351 TGATGAGAAC AACAAGAACC CTGAACTGGA GAAACATTTT GGATTGTTTT 3401 CCCCCAACAA GCAGCCCAAA TATAATCTCA ACTTTGGGGT CTCTGGTGGT 3451 GTTTGGGACA GTTCAGTTGA AACTAATGCT ACTGCTTCTC TCATAAGTGA

Fig 44 (V)

3501 3551 3601 3651 3701 3751 3801 3851 3901 39 51 4001 4051 Z 101 and 151

GATGTGAGAT

GAAAGCTTAG

TAACATTGCA

GCAACTCTGA

GCAAGAAACT

TGAATTTCAT

AGGATTTGGT

ATTTGACTCG

CGACTCTTAG

CGTTAACCCT

TAGAGCTTCA

GGACATTCTG

AAATAATGGT

AATTGAATCT

GAGACACTTG TAAAAACAAG CGTCTCTGTA GTGGTTTTAA TTCTGTAAGT GCACCATCAA AATTGCAAAG ATCAATTAAT GTAGTGGCAT CAT TAGAAAT AAATCGATTG TTACATTATA ACGCTCTTTC TGTCTCCAAT

Fi

AACTCTcm

AGAAAmAT

AGTCCTCATG

Arcccrrrrc

ΠΟΓΟΑΑΙΤΓ

TTATACATCT mAG ^ ATT

CmAATTGG tcaactgatc

ATATTATAAT

ACAACAGTTT

AGOTAmCA rrATCACAAA

AACTATTGCT g 44 (VI)

ACATAAGTAT

TCTTCATGCA

GATTGTTATC mG ^ A ^

CATGACTTTC mCCATGU

Μ ^ ΑΑ ^ ΑΟ

TAAGGCTTGA mCTeKAT

GTCACCTAAA

TGGAGTTACA

CGTCGTATCA mCTCTCAA mAGmAT

TGCTTAGATG

AGACACTAAA

CAGAGACGAT

TG ^ OmG tgatcacatt τGrcrττArc

TATATCGGAG

CCCATGTCTC

TCAGAGGTTT crownA

AGGTCGAATA

CTTCTAGACC

Grrrcrcrcr

1201 4251 4301 4 351 4401 4 451 4501 4 551 z 601 4651

ATCAATTCAA

TTAACCAATC

AAACCACTCA

ACATGTAAAA

ATGCAACGAA

ACTTGATTAG

AGCTTTAGGC

AAATCTGAGG

CTATTTGATC

AACTCCAAAG

GGCTCAAGCC

GAGCTGTTAA

AAGCTTCTCC

CCCCArAACC

CAAACACGGC

ATAGTTCOTO rTGCGAAACr

AGCATGCCAC rTACCCACTA

GCAGGAAAGA

ATCAAATCGC

CTCTGCTAAA

AACAAAAAAA

ATG ^ ATTCG mOCCACTC

CGGGGCAm

AAGTAGAACC

AGCAAAAAGG

ACTAGCTCTG

AGACTATTGA rτGGrArrrc

GrcτcArrcr

GTAGAAAAAA

AGTGAAGGAA

HOWED

CCTACCTCGC

TAAGGCCCCA rrAGcrτrcA

TGrGAArτGr

GAGAGCAGCA

TCGCTCTCAA reAGCneTC

ACmGATCC

GGrGcrrGAA

GTAAAAGAAA ^ ΚΟΑ ^ Η

GCGAArCCGr

GArAAGAGAr

CCATTTCATC

AAAGAGCTC

Fig 44 (ΥΠ)

1- 50 AAAAAAAAAA

-100 ATCACCAATG 101-150 GCTTGGCACA 151 -200 ATGCTGAAGA 201 -250 nAWA ^ CA 251 - 300 CTOGTOATGA 301 - 350 ACTTCTCACG 351-4OO AGGGTATTGC 401- 450 GAGGACCCAT 451 - 500 GCAATTOGAC 501-550 TACTATCATC 501-550 TACTATCATC 651 601 - 550CCG 651 601 ACAGATCA 551 - 700 CARAACOGT

AAAAACATAA GAAAGTACAG GCAnOTm orrGrorGrr AGGCArroor υπΑκο ^ Α ATAGGAACGA CGGrAGArGr KCA CTC ^ oc ^ ^ A ^ C rAcrGCCCGr aggaaagaaa AAACTACTGG TOGATCCCTO mGTTAGGC AAAATGACCA CCAATTGACA AACCGAAATA AAGAGCTAGT TAACAACCCT TTCAAAACAG CTAT ^ H TTCCCACGAC GTTATCATCG CGGCGAArCG AOTACCAGOT GGAArrGAAr GTOGCATAGO

AGGAAAATGG AGTTTTCTGG TTTCCTGTTC TTAACAGGGA CGAGTGACTT GTTCAACGAG CCTGCCAATG GCTTCTACAC CTTTCCTGGT TTTGGAACTA TTGCTGCCTT TTTCGGTCAA AGTGCAGAAC CATTTACAGG GAGTGACAGA TATTATGGTA ACTATGAGAA AGCTGGAACT GATTTAGTGG CCACAGATGC TTGGATGACA CCACAGGACA GTCGTTGGAC TCCGTCTGCC TACGGTGTAA TTACCAACAT ACGGTAATGAC GCAGTGGAAG

Fig. 45 (1)

701-750 ATCGAATTGG ATACTACAGG AGGTATTGTG GTATGTTAAA TGTTGCTCCG 751 -800 GGGGAAAACT TGGACTGTTA CAACCAAAGG AACTTCGGCC AGGGCTAGGC 801-850 TTCGTTACAT AGAATGCAGA TCATGTTATG TATACAAGTT ATATTTGTAT 851-900 TAATTAATGA ATAAGGGGAT TGTGTATCCA TTAAGAATTA GGTGAAATAT 901-950 TTCTGTTATT TGTCTTCTTG GGAAGAACCA ATAGCTCCTA TATATGAGGC 951- 0 GCTTTTAAGT GATGAGGCTA CTGCATTGAT GAAAACGAAA TTTCTATCCA

1001- 20 GAAATAAAAG TTCCTTGTCT

Fig. 45 (11)

1- 50 CAGGTGCTCA AGCAGGAGTT TGTTATGGAA GGCAAGGGAA 51-100 TCTCCAGCAG ATGTTGTGTC GCTATGCAAC CGAAACAACA 101-150 GAGAATATAT GATCCTGACC AGCCAACTCT CGAAGCGCTT 151-200 ACATTGAGCT CATGCTAGGT GTCCCGAATC CGGACCTTGA 201,250 GCTAGCCAAG CCAATNCAGA TACTTGGGTC CAAAACAATG 251- 300 TGGTAATGTC AAGTTCAGGT ATATAGCAGT TGGAAATGAA 301- 350 TAAATGAAAA CTCTAAGTAT GTACCTGTCC TTCTCAACGC 351- 400 ATTCAAACTG CCATATCTGG TGCTGGTCTT GGAAACCAGA 401 - 450 CACAGCTATT GAAACTGGAC TTACTACAGA CACTTCTCCT 451 - 500 GGAGATTCAA AGATGATGTT CGACAGTTTA TAGAGCCTAT 501- 550 CTAGTGACCA ATCGCGCCCC TTTGCTTGTC AACCTTTATC 551- 600 AATAGCAAAC AATGCAGATA TTAAGCTTGA GTATGCACTT 601 - 650 CTGAAGTTGT TGTAAATGAT AACGGAAGAG GATACCGAAA 651- 700 GCCATCTTAG ATGCCACATA CTCGGCCCTT GAAAAGGCTA

TGGATTACCA

TTCGTAGGAT

AGAGGCTCCA

GAATGTTGCT

TTAGGAACTA

GTTAGTCCCT

CATGCGAAAC

TCAAAGTCTC

CCATCAAATG

CATCAACTTC

CTTACTTTGC

TTTACATCCT

CCTTTTTGAT

GTGGCTCGTC

Fig. 46 (1)

701-750 TTTGGAGATT GTTGTATCAG 751- 800 AATTAACATC CATTGACAAT 801-850 CACGTGAAGG GAGGGAGTCC 851-900 GTTTTCGCTC TGTTTGATGA 901- 950 TTTTGGACTA TTTTCAGCAA 951- AGCA ACTAGTAGTAA

1001- 50 GTATGAAGAG AAAGTAGTCC 1051-100 TGCTCATAAA GAAAGCAGTT 11 (01- 107 CCATCAA

AGAGTGGTTG GCCTTCAGCT GGAGCAGGAC GCCAGGACTT ataacaacaa cttgattagt CAAAAGGCTT CCGGTCCAAT AGAGACCTAC AGATCAGAAA GA (XCTGAAA TTGAGAAGCA ACATGCAACC AAAGTACCAG ATCAGTTTTA GAGCATTAAT AGGAATAAGG ACTTTCCTTT ATTGGCACTA TGTACTGAAA CTATATATCA ATTACAATAA TGAAACACTT ACAAGAAAAG

Fig. 46 (11)

Publishing Department of the UP RP. Circulation of 90 copies Price PLN 10,000

Claims (10)

Patent claims A method for regulating the transcription of a chemically regulated gene in plant material, characterized in that A) isolating a substantially pure, chemically regulable DNA sequence from a chemically regulable gene present in a natural system containing the gene by activating RNA expression from a chemically regulated gene in this system, the isolation of this RNA, the differential screening of a genomic RNA bank generated from RNA isolated from this activated system, as well as an RNA bank generated from RNA isolated from a control system that was not activated, isolating a genomic clone, subcloning giving chemically regulate the gene from this genomic clone and isolate the desired chemically regulable DNA sequence, B / produce a chemically regulatable chimeric DNA sequence containing the first non-coding, chemically regulatable component DNA sequence isolated in step A / having sentence the ability to regulate the transcription of an associated DNA sequence in a plant or plant tissue, where regulation is dependent on a chemical regulator, and a second component DNA sequence capable of being transcribed as RNA having the ability to regulate expression of the phenotypic trait according to a mechanical anti-sense, or capable of being transcribed and translated in a plant or plant tissue to produce a phenotypic polypeptide by ligating a first, non-coding, chemically regulable component DNA sequence with a second component DNA sequence, C / transforming plant protoplasts, plant cells, plant tissues or whole plants with of the chimeric DNA sequence produced in step B / using transformation methods involving Agrobacterium-mediated transformation, direct gene transfer, microinjection or microprojectile bombardment, transformed cells or plant tissues are selected and E / the regulator is used to plant tissue, plant or seed containing the chimeric DNA sequence of step B /. 2. The method according to p. A method according to claim 1, characterized in that the chemically regulated DNA sequence is obtained from the natural system constituting the plant by the following steps: the plant activates the expression of polyA + RNA from the chemically regulated gene, this polyA + RNA is isolated, a cDNA bank is constructed from this poly + RNA, this cDNA bank is differentially screened for cDNA generated from RNA derived from control plants that have not been activated, cDNA clones that are chemically regulated are isolated, a genomic clone of the genomic bank of this plant is isolated, using as cDNA clone probes from the previous step, a chemically regulated gene from that genomic clone is subcloned, and the desired chemically regulable DNA sequence is isolated. 3. The method according to p. The method of claim 2, wherein the chemically regulated gene is a gene for a PR protein. 4. The method according to p. The method according to claim 3, characterized in that the gene for a PR protein is activated by a chemical inducer or pathogen in the first step as set forth in claim 3, 2. 5. The method according to p. A method according to claim 4, characterized in that benzoic acid, salicylic acid, acetylsalicylic acid, polyacrylic acid, benzo-1,2,3-thiadiazole carboxylic acid, benzo-1,2,3-thiadiazolothiocarboxylic acid, cyanobenzo-1,2,3-thiadiazole are used as the chemical inducer, benzo-1,2,3-thiadiazole carboxylic acid amide, benzo-1,2,3-thiadiazole carboxylic acid hydrazide or dichloroisonicotinic acid or derivatives thereof. 6. The method according to p. A method according to claim 1, characterized in that benzoic acid, salicylic acid, acetylsalicylic acid, polyacrylic acid, benzo-1,2,3-thiadiazole carboxylic acid, benzo-1,2,3-thiadiazolothiocarboxylic acid, cyanobenzo-1,2,3-thiadiazole are used as the chemical regulator, benzo-1,2,3-thiadiazole carboxylic acid amide, benzo-1,2,3-thiadiazole carboxylic acid hydrazide or dichloroisonicotinic acid or derivatives thereof. 162 317 7. The method according to p. The method of claim 1, wherein the regulator is applied to plant tissue, plant or seed, wherein the first DNA component sequence in the chimeric DNA sequence is a chemically inducible sequence in the PR gene or a sequence substantially homology to the sequence. 8. The method according to p. The process of claim 1, wherein the chemical regulation of the chemically regulated gene encodes a phenotypic trait such as stage of development, sterility, disease resistance, pest resistance, herbicide tolerance or resistance, secondary metabolite production, and production of an enzyme marker. to be marked. 9. The method according to p. A method according to claim 8, characterized in that the gene encoding the production of an enzyme marker is regulated, which is luciferase (LUX), chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (NPT), nopaline synthetase (NOS), octopine synthetase (OCS), β-1, 3-glucuronidase (GUS), acetohydroxy acid synthetase (AH AS) or Bacillus thuringiensis endotoxin (BT). 10. The method according to p. The method of claim 9, wherein the regulation regulates the gene encoding the production of an enzyme marker which is β-1,3-glucuronidase (GUS), acetohydroxy acid synthetase (AHAS) or Bacillus thuringiensis endotoxin (BT).