RU2324737C1 - Process of obtaining transgene tobacco plants with increaed content of prolyl - Google Patents

Abstract

FIELD: chemistry, organic, genetic engineering.

SUBSTANCE: invention relates to genetic engineering of plants. A recombinant plasmid DNA pBi-2E of the size 14099 bps will be constructed providing transfer of target DNA sequence into the plant genome and two-chain gene PDH suppressor expression consisting of the elements as follows: - DNA of the vector plasmid of the size 14758 bps; - DNA fragment containing the first exon of the PDH gene in direct orientation of the size 545 bps; - DNA fragment containing the first exon and the fragment of the first intron of the PDH gene of the size 691 bps in reverse orientation; - promoter 35S CaMV from the genome of the cauliflower mosaics virus' genome; - 3'HTP of the nopalinsynthase gene area. The plasmid pBi-2E will then be transferred to the strain Agrohacterittm tumefaciens AGL0 with subsequent direct transformation of the Nicotiana tabacum SR1 leaf discs with cultivation together with agribacteria. Selection and generation will be performed directly on the media containing NaCl. The process enables one to obtain transgenic tobacco plants using no antibiotics, possessing increased level of stress-resistance on account of their increased content of proline.

EFFECT: obtaining of transgenic tobacco plants using no antibiotics, possessing increased level of stress-resistance.

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Description

The invention relates to the field of biotechnology, in particular to the genetic engineering of higher plants, and relates to a method for producing transgenic tobacco plants with a high content of proline.

Two methods for producing transgenic plants with a modified proline metabolism are known: by increasing the expression of the Δ1-pyrrolin-5-carboxylate synthetase (P5CS) gene responsible for the synthesis of proline (Hu C-A.A., Delauney AJ, Verma DPS // Proc. Natd Acad. Sci. USA. 1992. V.89. P.9354-9358), or a decrease in the activity of the proline dehydrogenase enzyme, which determines the rate of proline degradation (Kiyosue T., Yoshiba Y., Yamagichi-Shinozaki K., Shinozaki K // Plant Cell. 1996. V.8. P.1323-1335). The main disadvantage of these methods is low environmental safety, since the selection of transformants is carried out on media containing an antibiotic (kanamycin).

The closest to the claimed method prototype is a method for producing transgenic tobacco plants expressing cDNA of the proline dehydrogenase (PDH) gene in the antisense (reverse) orientation, responsible for proline degradation (Nanjo T., Kobayashi M., Yoshiba Y. et. Al. // FEBS Lett. 1999. V.461. P.205-210). The method consists in cloning the cDNA of the PDH gene from Arabidopsis (Arabidopsis thaliana) in the antisense orientation under the control of the constitutive 35S promoter from cauliflower mosaic virus, transferring the obtained recombinant plasmid to an agrobacterium tumefaciens strain and obtaining Arabidopsis arabidopsis transgenic plants and generation on selective medium with kanamycin.

The main disadvantage of the prototype are:

1. The need to introduce into the plant genome of the kanamycin resistance gene in the composition of T-DNA recombinant plasmids, which does not play any other functional role except the possibility of selection.

2. The use of a selective medium for the selection of transgenic plants with the addition of the antibiotic kanamycin, which does not allow the plants to be classified into high proline levels associated with stress resistance.

An object of the present invention is to simplify the method by optimizing the genetic construct for plant transformation and increasing its environmental safety.

The technical task is achieved by the proposed method, which consists in the following.

Recombinant plasmid DNA was constructed by isolating the first exon of the PDH gene from the Arabidopsis genome (Arabidopsis thaliana) using PCR with specific primers, followed by linking the fragments in the forward and reverse orientations in the T-DNA polylinker region of the pBi121 binary vector plasmid. The result is a recombinant plasmid DNA pBi-2E, size 14099 bp, providing transfer of the target DNA sequence to the plant genome and expression of the double-stranded suppressor of the PDH gene.

Recombinant plasmid consists of the following elements:

- DNA of a vector plasmid with a size of 14758 bp

- a DNA fragment containing the first exon of the PDH gene in a direct orientation of 545 bp

- a DNA fragment containing the first exon and a fragment of the first intron of the PDH gene of size 691 bp in reverse orientation;

- 35S RNA promoter from the cauliflower mosaic virus genome;

- 3'NTR region of the nopaline synthase gene.

The constructed recombinant plasmid pBi-2E, carrying the PDH gene sequence, is transferred to the agrobacterium (Agrobacterium tumefaciens) AGLO strain followed by direct transformation of tobacco leaf discs (Nicotiana tabacum) SR1 by co-cultivation with agrobacterium followed by selection and generation of NaCl directly onto media.

A physical map of the plasmid pBi-2E with genetic markers is shown in figure 1, where pdh exoni is the nucleotide sequence of the first exon of the Arabidopsis PDH gene; pdh intron - fragment of the first intron of the PDH gene of Arabidopsis; CaMV 35S pro - promoter of the 35S RNA gene of the cauliflower mosaic virus; NOS 3'UTR-3'-untranslated region of the nopalin synthase gene from the Agrobacterium Ti plasmid (Agrobacterium tumefaciens); NOS pro - promoter of the nopaline synthase gene; NPTII gene of neomycin phosphotransferase II; LB, RB - the left and right borders of the T-DNA region.

The defining distinguishing features of the proposed method in comparison with the prototype are:

1. As the vector plasmid, the pBi121 vector is used, in which the β-glucuronidase gene has been replaced by PDH gene sequences connected in direct and reverse orientations, which allowed us to obtain a smaller construct that is more optimal for transformation.

2. Use a double-stranded suppressor, which is more effective than using antisense to suppress gene activity (Wang M-B. And Waterhouse, RM // Plant Molecular Biology. 2000. V.43. P.67-82). In the prototype, the vector pBE2113 was used, where the cDNA of the PDH gene in the antisense orientation was inserted before the β-glucuronidase gene.

3. The selection of transgenic plants is carried out directly on a stressful background, namely on a medium containing 250 mM NaCl.

The invention is illustrated by the following examples of specific performance of the method.

Example 1

Construction of the recombinant plasmid pBi-2E

The construction of a double-stranded suppressor in the vector pBi121 was carried out by triple ligation. Three fragments were assembled into the construct: the first exon of the proline dehydrogenase gene, the first exon of the PDH gene with a part of the first intron, and the plasmid pBi121 fragment containing the NPTII gene in the T region, the cauliflower mosaic virus 35S RNA promoter, and the 3'-untranslated region of the nopaline synthase gene including the signal polyadenylation. The first exon of the Arabidopsis PDH gene was obtained by PCR (primers: 5'-aacaaactggatccggcgatcttac-3 '; 5'-gagatgttggtctagatttggcagc-3') and cloned in the plasmid pBlueScript KSII at the BamHI and Xba restriction sites. Then it was isolated using restriction enzymes BamHI and HindIII; The PCR fragment of the first exon of the PDH gene with a portion of the first intron was hydrolyzed with HindIII restriction enzyme, plasmid pBi121 was hydrolyzed with restriction enzymes BamHI and Ecl136II. The restriction products were separated on a 1% agarose gel, the gel was stained with a solution of ethidium bromide in water (1 μg / ml) and the necessary fragments were isolated by DNA sorption on silica gel, which were further treated with DNA ligase, and competent E. coli XL1 Blue cells were transformed with the ligase mixture . Transformants were plated on a plate with LB agar containing 50 μg / ml kanamycin. Clones carrying recombinant plasmids with an integrated gene were selected by PCR DNA analysis of individual E. coli colonies with primers for the nucleotide sequence of the first exon of the Arabidopsis PDH gene.

Example 2

Obtaining transgenic plants bearing the construction of pBi-2E

The pBi-2E construct carrying the PDH gene sequence obtained in Example 1 was used for direct transformation of Agrobacterium tumefaciens cells of the AGLO strain by the known method described in (Horsch R., Fry J., Hoffman N. et al. // Plant Molecular Biology Manual Dordrecht. Kluwer Academic Publishers. 1988, P. A5 / 1-A5 / 9). For agrobacterial transformation, leaves of three-week tobacco plants (Nicotiana tabacum) SR1 were used as initial explants. Transformants were selected on culture medium supplemented with 250 mM NaCl. Figure 2 presents the results of the selection of leaf tobacco discs transformed with the construct pBi-2E and the control pBi101, which represents the base vector, without the target gene, where

A - pBi101 construction transformation, selection on kanamycin;

B — transformation with the construction of pBi101, selection on a medium containing 250 mM NaCl;

B - pBi-2E transformation, selection on a medium containing 250 mM NaCl.

After agrobacterial transformation, leaf disks were transferred to Murashige and Skoog (MS) media (Murasige T., Skoog F. // Physiol. Plant. 1962, V.15, P.473-497) containing 250 mM NaCl. Figure 2B shows that callus formation on leaf disks with pBi-2E construction on a medium with 250 mM NaCl was somewhat inhibited. Leaf discs with the control design pBi101 on a medium with 250 mM NaCl (Fig. 2B) were completely necrotic, and on a medium with kanamycin they developed normally (Fig. 2A).

Figure 3 presents the electrophoregram of PCR analysis of the genomic DNA of transgenic plant seedlings for the presence of the 35S insert of the cauliflower mosaic virus promoter with a PDH gene fragment in the antisense orientation. Trace application: 1 - marker DNA; 2 - vector pBi-2E; 3 - non-transgenic tobacco SR1; 4-12 - transgenic tobaccos SR1. Figure 3 shows that PCR analysis with primers for the nucleotide sequence of the first exon of PDH confirms the presence of the PDH nucleotide sequence of Arabidopsis in the genomes of selected plants.

Transformants carrying the recombinant plasmid pBi-2E were analyzed for the content of free proline in leaf tissue. For measurement, plants of the same age (2-3 weeks) were used, grown sterile under the conditions of a climatic chamber, leaves were selected from approximately the same size from one plant level in each experiment. The proline content in leaf extracts was estimated by the Bates method (Bates LS, Waldren RP, Teare ID // Plants and Soil. 1973. V.39. P.205-207) in micrograms (μg) of free proline per gram of wet weight (gsm) sample. The measurement results are shown in the table.

Table №№ Transgenic plant mcg / gsm Experience The control one 2E No. 10 433 2 2E No. 11 715 3 2E No. 13 818 four 2E No. 14 1128 5 2E No. 16 958 6 2E No. 17 511 7 pBi No. 1 98 8 pBi No. 2 240

From the data presented in the table, it is seen that the obtained transgenic tobacco plants carrying the recombinant plasmid pBi-2E have a several times higher level of proline content compared to control plants.

Thus, the method allows to obtain transgenic tobacco plants bearing the sequence of the first exon of the PDH gene in direct and reverse orientations, with a high content of proline.

Claims (1)

A method for producing transgenic tobacco plants with increased proline content, comprising constructing a recombinant plasmid DNA containing the proline dehydrogenase (PDH) gene sequence from Arabidopsis thaliana, transferring the obtained plasmid DNA to an Agrobacterium tumefaciens strain, infection of the plant material with the latter, selection of transgenic plants on selective nutrient medium with subsequent testing of the selected transformants for a high content of proline, characterized in that they are constructed and transferred to the strain of the indicated ba terii recombinant plasmid DNA pBi-2E, consisting of the following elements: DNA of the vector plasmid pBI121 not containing the beta-glucuronidase gene; a DNA fragment containing the first exon of the PDH gene in a direct orientation of 545 bp; a DNA fragment containing the first exon and a fragment of the first intron of the PDH gene of 691 bp in reverse orientation; 35S CaMV promoter from the cauliflower mosaic virus genome; 3'NTR region of the nopalin synthase gene, while the selection of transgenic plants is carried out on a nutrient medium containing 250 mM NaCl and not containing antibiotics.

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