RU2237717C2 - Recombinant plasmid pgbp450f for preparing transgenic plants and method for preparing transgenic tobacco plants with enhanced productivity and resistance to fungal phytopathogens - Google Patents

Abstract

FIELD: genetic engineering, molecular biology, agriculture.

SUBSTANCE: invention relates, in particular, to preparing tobacco transgenic plants. Method involves construction of plasmid pGBP450f comprising complementary DNA of gene CYP 11A1 of size 1754 nucleotide pairs encoding protein-precursor of cytochrome P450_scc being this DNA is a component of Eco RI-fragment of size 2535 nucleotide pairs flanked with sequences of promoter 35S and terminator of cauliflower mosaic virus, and also gene for phosphinothricin acetyltransferase (gene bar) determining the resistance to phosphinothricin that is under control of nos-promoter, gene determining the resistance to kanamycin, site for onset of replication (ori) from plasmid of broad range of hosts of pSa. Tobacco transgenic plants are obtained by co-culturing protoplasts with plasmid pGBP450f linearized by hydrolysis with site-specific endonuclease restriction Bsp 120I by unique site 5'-GGGCCC. Invention provides enhancing the yield and resistance of plants to phytopathogens and to enhance the yield of agricultural production.

EFFECT: improved preparing method, valuable properties of plasmid.

2 cl, 4 dwg, 2 tbl, 6 ex

Description

The invention relates to the field of genetic engineering, specifically to the creation of plasmid pGBP450f, as well as to a method for producing transgenic plants bearing cytochrome P450 animals.

About 150 cytochromes belonging to the class of cytochromes P450 were discovered and studied in plants. These proteins are involved in many processes of primary and secondary metabolism, including the biosynthesis of lignins, terpenoids, sterols, fatty acids, hormones, pigments, phytoalexins, etc. Plant cytochromes P450 are involved in both the regulation of growth and development, as well as in the induction of a plant defense response [CRC Crit. Rev. Biochem., 1986, Vol. 19, C.247-305]. For example, cytochrome CYP90 is a key enzyme in the biosynthesis of brassinosteroids (biologically active plant steroids) in Arabidopsis [Cell, 1996, V.65, P.171-182]. The intron-exon structure and sequence of conserved and functionally important domains of CYP90 has a high degree of homology with those of mammalian P450 monooxygenases, including steroid hydroxylases, one of which is cytochrome P450 _scc (P450XIA1) [DNA, 1993, V.12, P.1 -51]. A defect (mutation) in the cpd gene encoding CYP90 leads to a disruption in the growth of hypocotyl in Arabidopsis plants. This may be due to the lack of C23-hydroxylated derivatives of brassinolide catasterone, an ecdysone-like plant steroid hormone [Annu. Rev. Plant Physiol. & Plant. Mol. Biol., 1988, V.39, P.2352-2371].

Recently, natural plant genes that control the level of such growth hormones as, for example, indolyl-3-acetic acid have been used to create transgenic plants characterized by intensive growth, development, and increased resistance to phytopathogens [Science, 1994, V.265, P. 1699-1701], genes encoding plant lipoxygenases [The Plant Cell, 2001, V.13, N.3], as well as genes of other enzymes that catalyze the synthesis of important regulatory molecules.

Known closest to the claimed recombinant plasmid pK22rs containing the peptide antibiotic gene from Raphanus sativus, and a method for producing transgenic plants, comprising transforming the plant genome with the indicated plasmid (Pat. RF No. 2176669, MKI C 12 N 15/29, publ. 10.12.2001) . However, the use of transgenic plants containing the antibiotic gene cannot be considered completely harmless to humans.

The invention solves the problem of creating recombinant plasmid DNA pGBP450f containing cDNA of the CYP11A1 gene from bovine adrenal cortex, as well as the development of a method for producing transgenic plants expressing cDNA of the animal CYP11A1 gene, with increased productivity and resistance to fungal plant pathogens that are harmless to humans.

The problem is solved due to the fact that the recombinant plasmid DNA pGBP450f contains 2535 bp of EcoRI fragment. framed by the sequences of the 35S promoter and terminator of the cauliflower mosaic virus, the complementary DNA of the 1754 bp CYP11A1 gene encoding the cytochrome P450 _scc precursor protein (P450XIA1) from bovine adrenal cortex with a leader N-terminal extrapeptide of 39 a.a. and a total molecular weight of 60.3 kDa, and consists of the phosphinotricin acetyltransferase gene (the bar gene) controlled by the nos promoter, which causes resistance to phosphinotricin; polylinker; kanamycin resistance gene; the site of the beginning of replication (ori) from the plasmid of a wide range of pSa hosts; the RB and LB sequences, as well as due to the fact that in the method for producing transgenic plants by cocultivation of their protoplasts with recombinant plasmid DNA, cocultivation is carried out using recombinant plasmid DNA pGBP450f linearized by hydrolysis of the Bsp120I restriction endonuclease at the unique 5CC-GG site.

It is known that the protein P450XIA1 is expressed in the cortex of the bull’s adrenal cortex, which catalyzes the initial limiting stage of steroidogenesis — cleavage of the side chain of cholesterol and its transformation into pregnenolone [Ann. Rev. Biochem., 1987, V.56, P.945-993].

To implement the claimed invention, total RNA is first isolated from the bark of the adrenal cortex, and then the mRNA fraction is isolated from it. Using the obtained mRNA preparation as a template, double-stranded cDNAs are synthesized using reverse transcription on it, which are then used to create clonotheque cDNAs. By hybridization with a radiolabeled probe constructed on the basis of the known amino acid sequence of cytochrome P450XIA1, clones carrying the CYP11A1 gene sequence from bovine adrenal cortex are selected. The fragment, which is a full-sized cDNA of immature P450XIA1 and containing its own initiation codon, is cloned in the pDH51 vector under the control of the 35S promoter. Using the restriction endonucleases, a cassette containing the 35S promoter, cDNA of the cytochrome P450XIA1 gene (P450scc) and terminator is cut from the obtained intermediate vector. This cassette is embedded in the vector pGreen0229. Vectors containing a head-to-tail cassette (pGBP450f) and tail-to-tail (pGBP450b) with respect to the bar gene under the control of the nos promoter, which was present in the pGreen0229 vector initially, were selected. For the transformation of tobacco plants by the method of protoplast cocultivation, DNA vectors linearized by restriction endonucleases are used. The selection of transformants is carried out on selective media. The presence of embedded genes is determined by polymerase chain reaction (PCR) and Southern hybridization. Plant resistance to phytopathogens is determined by standard methods.

The sequence of the CYP11A1 gene cDNA isolated from bovine adrenal cortex (1754 bp), which was subsequently used to create a recombinant plasmid for its expression in plants, is shown in FIG. 1.

The use of the plasmid pGBP450f described in this invention allows a fairly simple way to introduce CYP11A1 gene cDNA from bovine adrenal cortex into the plant genome to create plant lines and varieties with more intensive growth and increased resistance to phytopathogens than the original non-transformed form.

The invention is illustrated by the following examples.

Example 1. Cloning of a full-sized cDNA of the CYP11A1 gene from bovine adrenal cortex.

Total cellular RNA is isolated from bovine adrenal cortex by the guanidine thiocyanate method [Biochemistry. 1979.V.18. P.5294-5299]. 20 g of tissue is frozen in liquid nitrogen and triturated to a powder state, 40 ml of lysis buffer is extracted with the following composition: 50 mM Tris-hydrochloride, pH 7.6, 25 mM EDTA (ethylenediaminetetraacetic acid), 0.07 mM 2-mercaptoethanol, 2 % N-laurylsarcosyl. The resulting lysate was extracted three times with phenol / chloroform / isoamyl alcohol (200: 200: 4) until the interphase disappeared. The phases are separated by centrifugation for 5 minutes 5000 rpm. From the aqueous phase, RNA was precipitated with 3 M sodium acetate (pH 6.05) overnight in an ice bath. The resulting precipitate was collected by centrifugation, dissolved in TE buffer (0.05 mm Tris hydrochloride, pH 7.5, 0.01 mm EDTA) and precipitated with ethanol in the presence of 0.3 M sodium chloride.

The quality of the obtained RNA preparation is investigated by agarose gel electrophoresis in the presence of formaldehyde. Samples are dissolved in 20 μl of denaturing buffer of the following composition: 3 μl of 36% formaldehyde, 7 μl of concentrated formamide and 8 μl of electrophoresis buffer (phosphoacetate buffer). RNA is heated for 5 min at 65 ° C and introduced into the wells of 0.8% agarose gel prepared on an electrode buffer containing 1.1 M formaldehyde. The gel is stained with ethidium bromide (0.5 μg / ml) and photographed in ultraviolet light.

The preparation of poly (A) ⁺ -PHK is obtained by chromatography on oligo (dT) -cellulose [PNAS. 1972. V.69. P.1408-1412]. The RNA alcohol precipitate is dissolved in 10 ml of 0.01 M tris hydrochloride (pH 7.5) containing 0.5 M potassium chloride and applied to a column of oligo ((dT) cellulose. The column is washed with 10 volumes of a 0.5 M solution potassium chloride RNA is eluted with a solution of 0.01 M Tris hydrochloride, pH 7.5.

Double-stranded cDNA is synthesized on the isolated poly (A) ⁺ -PHK; it is then inserted using the appropriate polylinker into the DNA of plasmid vector pUC 13 at the EcoRI site. Selected on a selective medium white colonies (recombinant clones) are analyzed by hybridization on filters with a ³² P-labeled oligonucleotide probe, complementary to both the extrapeptide and the start of the mature P450XIA1 enzyme. The radioactive label is introduced into the probe by nick translation, which includes two enzymatic reactions: introduction of randomly distributed single-strand breaks (nicks) into DNA using DNAse I, with their further expansion and simultaneous completion with E. coli DNA polymerase I. A complementary oligonucleotide is incubated in a mixture of the following composition: 0.05 M Tris hydrochloride (pH 7.5), 5 mM magnesium chloride, 10 mM 2-mercaptoethanol, 50 μg / ml bovine serum albumin, DNA (approximately 0.1 μg), all deoxynucleoside triphosphates (dATP, dGTP, dTTP, dCTP), in the presence of DNase I and E. coli DNA polymerase I (+ 15 ° C, 30 min). A labeled probe was isolated from the reaction mixture on a Sephadex G-50 column.

Correspondence of the cloned cDNA sequence of the CYP11A1 gene is confirmed by partial sequencing and restriction analysis, as well as hybridization with the bovine genomic DNA according to Southern (J. Mol. Biol. 1975. V.98. P.503-517). The resulting cDNA encodes a precursor protein P450XIA1, consisting of 520 amino acid residues (a.a.), at the NH ₂ end of which contains an extrapetid (39 a.a.), which determines the ability of the protein to penetrate the mitochondrial membrane, where it is processed and acquires catalytic activity. The plasmid carrying the cDNA of the P450XIA1 protein gene is named pUC13-P450XIAl.

The PvuII-PvuII fragment of CYP11A1 cDNA from pUC13-P450XIAl is cloned at the PvuII site into the plasmid vector pDSl. Plasmids pUC13-P450XIAl and pDSl are treated with PvuII restriction endonuclease under the conditions recommended by the manufacturer (Sibenzyme, Novosibirsk) with a four-fold excess of the enzyme. The reaction is carried out in a volume of 25-50 μl for 2 hours at 37 ° C. The incubation mixture was further extracted with phenol / chloroform (1: 1) pH 8.0, the DNA was precipitated with two volumes of ethyl alcohol in the presence of 0.1 M sodium chloride in an ice bath and collected by centrifugation for 10 min at 5000 rpm. The precipitate is dissolved in a single Tris-borate buffer, restriction fragments are separated by agarose gel electrophoresis in the same buffer [Molecular Cloning. - M .: Mir, 1984. S.240-242].

Separated DNA fragments are isolated from the gel by electroelution. Electrophoretic zones containing the desired fragments are cut out of the gel, placed in dialysis bags and subjected to electroelution in a single Tris-borate buffer at a voltage of 80 V for 1 h. The eluate is extracted with a phenol / chloroform mixture (1: 1) and purified on a micro column with Sephadex G -50 in TE buffer, precipitated with ethyl alcohol and checked by agarose gel electrophoresis. Ligation is carried out under standard conditions: 12 hours at + 15 ° C with a five-fold excess of the insert relative to the vector fragment [Molecular cloning. - M .: Mir, 1984. S.240-242]. The obtained ligase mixture transform cells of E. coli JM109. The transformation is carried out using CaCl ₂ [Molecular cloning. - M .: Mir, 1984. S.240-242]. Plasmid DNA was isolated from ampicillin-resistant recombinant clones by the method of Birnboim and Dolly [Nucleic Acids Res. 1979. V.7, P.1513-1517]. Transformed E. coli JM109 cells were grown in YT liquid medium with ampicillin (100 μg / ml) containing 5 g of NaCl (rh), 8 g of tryptone (Difco, USA), 5 g of yeast extract (" Difco ", USA) overnight at 37 ° C. Cells (10 ml of culture) are pelleted by centrifugation at 3000 rpm for 20 minutes, resuspended in 200 μl of the following composition: 25 mM Tris hydrochloride pH 8.0, 50 mM glucose, 20 mM EDTA, 2 mg / ml lysozyme (Sigma , USA), then 200 μl of a solution of 0.2 N NaOH with 1% SDS are added, incubated until the suspension is completely clarified in an ice bath. Add 150 μl of 3 M sodium acetate pH 4.8, mix and remove the precipitate by centrifugation (20 min, 3000 rpm). DNA from the supernatant is precipitated with two volumes of ethyl alcohol, the precipitate is dissolved in 0.3 M sodium acetate (pH 6.0) and precipitated with two volumes of ethyl alcohol. The precipitate collected by centrifugation was dissolved in TE buffer. Physical mapping of the resulting recombinant DNA is carried out using restriction endonucleases PvuII, KpnI, BamHI, SphI.

Example 2. Obtaining plasmid DNA (plasmid) pGBP450f.

 A convenient vector for molecular genetic manipulation is the plasmid pDH51. It is a derivative of plasmid pUC18 and contains the promoter and terminator of the cauliflower mosaic virus [NAR. 1986. V.14. P.5857-5868]. Between the promoter and the terminator there are recognition sites for some commonly used restriction enzymes (SmaI, BamHI, XbaI, SalI, PstI, SphI). It is also convenient that the expression cassette can be cut from the vector using several restriction enzymes (EcoRI, NcoI, SstI, HindIII). The vector is multi-copy and carries the ampicillin resistance gene.

The resulting pDHP450scc construct is convenient for creating vectors designed to transfer the CYP11A1 gene to the plant genome, because in its sequence, restriction endonuclease recognition sites are conveniently located, which makes it easy to cut out the CYP11A1 gene cassette for cloning in other plasmids.

To create the pDHP450scc vector, plasmid pDH51 DNA is hydrolyzed with a SmaI restriction endonuclease at 30 ° C in a buffer containing 33 mM Tris acetate (pH 7.9 at 37 ° C), 10 mM magnesium acetate, 66 mM potassium acetate, 0.1 mg / ml of bovine serum albumin. Then, after thermal inactivation of the restriction enzyme, the DNA of the vector is dephosphorylated by incubation with 1 unit of alkaline phosphatase at 37 ° C. The reaction products are separated on a 1% agarose gel containing 0.5 μg / ml ethidium bromide. A gel fragment containing a linearized vector is excised with a scalpel, then DNA is isolated from it using the freeze-thaw procedure. After purification by extraction with a phenol-chloroform mixture, the DNA of the vector is ligated with the PvuII fragment containing the full-length cDNA of the bovine cytochrome P450scc gene. The ligation reaction is carried out overnight at 16 ° C using T4 phage ligase DNA. The ligated DNA is used to transform strain DH5α (genotype thi-1; hsdR17; gyrA96; recA1; endAl; glnV44; relAl; phi80dlacZdelM15; phoA8; lambda-) using the standard method using calcium chloride (see example 1). Transformed cells were selected on LB agar medium containing 100 μg / ml ampicillin. To check for the presence of the plasmid, individual colonies are plated in a tube with 4 ml LB with ampicillin. Plasmid DNA is isolated as described above. The resulting preparations are analyzed using restriction endonucleases. A plasmid containing an insert of a sequence that encodes bovine cytochrome P450scc is selected. Check the insert orientation using restriction.

When constructing a vector for transfer of the bovine cytochrome P450scc gene cDNA sequence into plants, plasmid pGreen0229 was used, which is a convenient vector for plant transformation [Plant Molecular Biology. 2000. V. 42. P.819-832]. It contains, under the control of the nos promoter, the phosphinotricin acetyl transferase gene (bar gene), which causes resistance to phosphinotricin. The vector also contains a polylinker convenient for cloning, a kanamycin resistance gene for selection in bacteria, a site of replication onset (ori) from the plasmid of a wide range of pSa hosts, which allows the plasmid to be maintained not only in Escherichia coli, but also in Agrobacterium tumefaciens. The bar gene and polylinker are flanked by the RB and LB sites necessary for agrobacterial transformation.

Plasmids pGreen0229 and pDHP450scc are hydrolyzed with an EcoRI restriction endonuclease at 37 ° C in a buffer containing 10 mM Tris-Hcl (pH 8.5), 10 mM magnesium chloride, 100 mM potassium chloride, 0.1 mg / ml bovine serum albumin. After thermal inactivation of the restriction enzyme, the DNA of the vector pGreen0229 is dephosphorylated with 1 unit of alkaline phosphatase in the same reaction mixture. Purification of fragments on an agarose gel, ligation and transformation are carried out in the same way as described above. Transformants were selected on LB agar medium containing 50 μg / ml kanamycin. After isolation of plasmid DNA by alkaline lysis, it is analyzed using restriction endonucleases. Clones containing plasmids with inserts in the “head to tail” (pGBP450f) and “tail to tail” (pGBP450b) orientations with respect to the bar gene under the control of the nos promoter are selected [Molecular Cloning. M .: Mir, 1984]. A diagram of the resulting structure is shown in FIG. 2.

Example 3. Transformation of plants using plasmid DNA pGBP450f and analysis of the inclusion of cDNA of the CYP11A1 gene in the genome of transformed plants and its expression in them.

DNA preparation of plasmids pGreen0229 (empty vector) and pGBP450f (contains the desired gene) for transformation is carried out as follows. DNA is isolated from E. coli strain DH5α [pGBP450f] according to the procedure described in example 1, in terms of a larger volume. For transformation, the isolated and purified plasmid is first linearized using the appropriate restriction endonuclease. The pGBP450f vector, which contains the CYP11A1 gene sequence under the control of the 35S promoter of the cauliflower mosaic virus, is linearized by hydrolysis with restriction endonuclease Bsp 120I at 37 ° C in a buffer containing 10 mM Tris-Hcl (pH 7.5 at 37 ° C), 10 mM chloride magnesium, 0.1 mg / ml bovine serum albumin. For plasmid pGreen0229, HindIII restriction enzyme hydrolysis was used at 37 ° C in a buffer containing 10 mM Tris-Hcl (pH 8.5 at 37 ° C), 10 mM magnesium chloride, 100 mM potassium chloride, 0.1 mg / ml bovine serum albumin . Treated plasmids (2-3 μl) were separated on a 0.8% agarose gel to confirm complete restriction. Plasmid DNA was precipitated with ethanol, washed with 75% ethanol and dried in a sterile air stream. The plasmid is dissolved in sterile distilled water, the concentration is adjusted to 1 μg / μl.

Obtaining transgenic plants is carried out on tobacco plants. Other dicotyledonous and monocotyledonous plants can be used.

For transformation, an aseptic culture of the Nicotiana tabacum cv tobacco line is used. petit Havana SRI. Tobacco is cultivated at 24/18 ° C, a 16/8-hour photoperiod, illumination of the order of 2000-3000 lux.

For transformation use the leaves of 6-8-week-old tobacco plants grown under sterile conditions. The leaves are cut in an enzyme solution and left to isolate protoplasts for 16-18 hours at a temperature of 24 ° C in the dark. Isolation of protoplasts is carried out in an enzymatic solution of the following composition: 0.4 M K3 medium (K3 macro salts, B5 micro salts, chelating agent, vitamins K3, xylose, sucrose, 2.4 D, NAA, BAP (pH 5.8), Sea agarose plag ”), cellulase R-10, macerozyme R-10, pH 5.6.

The next day, the mixture of the enzyme with protoplasts is filtered through a metal mesh with a diameter of 100 μm. The remaining tissue is additionally washed with K4 medium (K3 macro salts, B5 micro salts, chelating agent, vitamins K3, xylose, sucrose, 2.4 D, NAA, BAP, pH 5.8) to completely isolate the remaining protoplasts. W5 medium (NaCl, CaCl ₂ , KCl, glucose, pH 5.8) is layered on protoplasts and centrifuged for 10 min at 80g. Good protoplasts are collected from interphase and washed twice from the enzyme in W5 medium, centrifuged for 5 min at 70g. The total number of protoplasts in the W5 medium is counted. To do this, 100 μl of protoplasts are dissolved in 900 μl of W5 medium, 10 μl of a suspension are taken and viable protoplasts are counted in a hematocytometer.

Protoplasts are incubated in an incubator for 30 min at 24 ° C, centrifuged for 5 min at 70 g, completely removing W5 medium, and resuspended in MMM medium (mannitol, MgCl ₂ , MES, pH 5.6.) To obtain 1,000,000 protoplasts in 0.7 ml of solution. A linearized plasmid (10 μg), a thymic DNA carrier (40 μg) and polyethylene glycol (0.7 ml) are added to protoplasts (~ 1 million per variant). After 15-20 minutes, washing is carried out in W5 medium. After centrifugation at 70g for 5 min, the supernatant was removed and the protoplasts resuspended in 0.5 ml of K3 medium. Protoplasts are sealed in K3 medium containing 0.6% agarose (Sea Plaque). Cultivation of sealed protoplasts is carried out at 24 ° C for 1 day in the dark, then at 24/18 ° C, a 16/8-hour photoperiod, illumination of about 500 lux (scattered light) for 5 days. Colonies from transformed protoplasts germinate after 5-6 weeks at 24/18 ° C, a 16/8 hour photoperiod, illumination of about 500 lux in A-med medium (A-med macrosalts, B5 microsalt, KZ vitamins, NAA, BAP, sucrose , mannitol, pH 5.6) with a selective agent phosphinotricin (PPT) of 20 μg / ml. Cultivation of colonies for callus formation and regeneration is carried out at 24/18 ° C, a 16/8 hour photoperiod and illumination of the order of 2000-3000 lux.

After 5-6 weeks, when the colonies reach a size of 2-3 mm in diameter, they are transferred to MS-morpho medium (MS macro salts, B5 micro salts, chelating agents, CaCl ₂ , BAP, NAA, pH 5.6) with 0.6 % agar for callus formation and plant regeneration. The transformation efficiency of the pGBP450f and pGreen0229 constructs is 32 independent plants per 1,000,000 protoplasts. Seedlings 3-4 cm in size are transplanted onto a T-med medium for rooting. Rooted plants are planted in the soil for further molecular genetic, morphological and pathophysiological analyzes.

The primary selection of plants is carried out according to the results of a test for resistance to phosphinotricin. Plants rooted in the soil are treated with the phosphinotricin 40 and 80 mg / L herbicide to check for bar gene expression.

Isolation of DNA from selected plants is carried out as follows. Plant tissue, frozen or fresh, is ground in a mortar with liquid nitrogen. The homogenate is poured with S buffer (100 mM tris HCl pH 8.5, 100 mM NaCl, 50 mM EDTA pH 8.0, 2% SDS) in a 1: 2 ratio. After thawing, proteinase K (10 mg / ml) is added directly to the mortar - 50-100 μg per 1 ml of the mixture, incubated at 65 ° C for 1.5-2 hours. After that, an equal volume of phenol-chloroform mixture (1: 1) is added. , mix and centrifuged at 4.5 thousand revolutions for 20-30 minutes Remove the upper phase and transfer to a new tube, add an equal volume of chloroform, shake, centrifuged for 20-30 minutes at 4.5 thousand revolutions. The upper layer is transferred into small 50 ml glasses, isopropanol is added in a ratio of 0.6-0.9 V from the sample. Using a stick, they wind the DNA until it is completely extracted. The sticks with the DNA wound on them are washed with 75% alcohol and dried for about 40 minutes. The dried DNA was dissolved in 300 μl of sterile TE buffer (10 mM Tris HC1, 1 mM EDTA, pH 8.0).

After dissolution, the DNA is treated with RNase for 45-60 minutes at 37 ° C. Then carry out a standard purification with phenol-chloroform. DNA precipitated with ethanol, washed with 80% ethanol. After the DNA is dried, it is dissolved in TE buffer. DNA concentration is determined using agarose mini-gel electrophoresis and spectrometry.

Example 4. Phenotypic analysis of transgenic plants.

Analysis of changes in the phenotype of bar-transgenic tobacco clones and transgenic plants containing both the bar gene and the cDNA of the CYP11A1 gene is carried out by assessing and comparing morphological parameters such as rooting rate, plant height, flowering start time, and plant productivity with control plants. Phenotypic analysis was performed by independent observers at the beginning, middle and end of the growing season. The results are shown in table 1; in FIG. Figure 3 shows a photograph of control and transgenic tobacco plants in the field. The numbers denote: 1-control tobacco plant (height 15 cm), 2-transgenic tobacco plant containing cDNA of the CYP11A1 gene (height 72 cm).

Transgenic plants containing cDNA of the CYP11A1 gene are characterized by faster rooting processes, earlier flowering and fruit formation (these processes occur 2 weeks earlier compared to control plants), higher productivity (3 times higher than the parameters of control plants), more the area of the leaf blade (2-3 times more than the control).

Example 5. Biotests on protein extracts from transgenic plants carrying cDNA of the CYP11A1 gene from bovine adrenal cortex.

Plants are tested for resistance to non-specific nightshade pathogens - Botrytis cynerea fungus - by counting the number of spore germinated in a drop of juice from leaves. Biotests are carried out as follows. 20 μl of Botrytis cynerea spore suspension (10 ⁵ spores / ml), 20 μl of crude protein extracts from control (non-transformed and transformed by an empty vector) and transgenic plants containing the studied gene are applied to the wells of the microcuvette. Microcuvettes are incubated for 24 hours at 24 ° C, after which the number of germinated spores is counted under a microscope. The results are shown in table 2.

The arithmetic average of the number of sprouted spores for the control transgenic (with an empty vector) and non-transgenic tobacco lines vary from 80 to 96%. For transgenic tobaccos containing cDNA of the CYP11A1 gene, the number of germinated spores varies from 7 to 21%. These results indicate a suppression of the growth of pathogenic fungi with protein extracts isolated from transgenic plants containing the studied gene.

Example 6. Biotests for the resistance of transgenic plants containing cDNA of the CYP11A1 gene to fungal phytopathogens.

The experiments are carried out on isolated leaves of transgenic tobacco containing cDNA of the CYP11A1 gene. As a test culture, phytopathogenic fungi of the species Botrytis cynerea, causing spotting and early rot, are used. Under provoking conditions, leaves of middle age and one size are placed in Petri dishes on filter paper moistened with water. 10 μl of a spore suspension of the pathogen containing 2000 spores / ml is applied at 6 points per leaf, after which the cups with leaves are hermetically sealed to create high humidity and kept in a climatic chamber at a temperature of 24 ° C and 16 hours of light day. As a control, the leaves of non-transformed plants and plants containing an empty vector are used. The results of a Botrytis cynerea resistance test, evaluated by the number and size of necrotic spots 10 days after infection, are shown in FIG. 4.

The numbers denote: 1 - control non-transgenic plant (defeat 7 points); 2 - control transgenic plant with an empty vector (defeat 7 points); 3-5 - independent lines of transgenic plants containing cDNA of the CYP11A1 gene (3 - damage 1 point; 4 - damage 0 points; 5 - damage 2 points); and - damage to the leaf.

In resistant transgenic tobacco lines containing CYP11A1 gene cDNA, the average area of leaf lesions on the 10th day was 12 times smaller than in control plants, or there were no areas of necrosis on the leaves.

Claims (2)

1. Recombinant plasmid DNA pGBP450f to obtain transgenic plants with increased productivity and resistance to fungal plant pathogens, containing 2535 bp EcoRI fragment. framed by the sequences of the 35S promoter and terminator of the cauliflower mosaic virus, the complementary DNA of the 1754 bp CYP11A1 gene encoding the cytochrome P450scc precursor protein (P450XIA1) from bovine adrenal cortex with a leader N-terminal extrapeptide of 39 a.a. and a total molecular weight of 60.3 kDa, and consisting of the phosphinotricin acetyltransferase gene (the bar gene) controlled by the nos promoter, which causes resistance to phosphinotricin; polylinker; kanamycin resistance gene; the origin of replication (ori) from the plasmid of a wide range of pSa hosts. 2. A method for producing transgenic tobacco plants by cocultivation of their protoplasts with recombinant plasmid DNA pGBP450f, linearized by hydrolysis of restriction endonuclease Bsp 1201 at the unique 5'-GGGCCC site.

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