RU2631929C2 - Method for obtaining of non-marked transgenic pinnated kalanchoe plants expressing cecropin p1 gene - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a method for obtaining of non-marked transgenic pinnated kalanchoe plants expressing the cecropin P1 gene.

EFFECT: obtaining and testing of non-marked Kalanchoe plants with increased antibiotic properties for a short period of time for use in pharmacology and medicine.

9 cl, 4 dwg, 1 tbl, 12 ex

Description

The invention relates to the field of biotechnology and genetic engineering of plants, in particular to a method for producing marker-free plants of Kalanchoe pinnate, expressing the gene of the antimicrobial peptide cecropin P1.

Currently, genetic engineering methods open up the possibility of obtaining various recombinant proteins. For this, expression systems of bacteria, yeast, mammals and insects are used. However, these systems have disadvantages. In prokaryotic cells, the correct folding of polypeptide chains does not occur and their post-translational modification does not occur. In yeast, insect and mammalian cells, these drawbacks are not present, but their use is very expensive [Russel C, Clarke l. Recombinant proteins for genetic disease // Clinical Genet. 1999. V. 55. P. 389-394].

Compared with these systems, plants have a number of advantages: in higher plant cells, glycosylation occurs and the target proteins are correctly folded and their growth does not require expensive equipment. In addition, plant cells, unlike animals, do not contain viruses and prions pathogenic for humans. Transformation and regeneration of plants is much simpler than in animal cells [Daniell N., Streatfield S., Wycoff K. Medical molecular farming: productionof antibodies, biopharmaceuticals and edible vaccines in plants // Trends in Plant Sci. 2001. V. 6. P. 219-226]. In recent years, many valuable proteins have been efficiently expressed in plants. These are growth regulators, antibodies, vaccines, biopolymers, industrial enzymes, human serum proteins and reagents for molecular biology [Cabanes-Macheteau M., Fttchette-Laine A.S., Loutelier-Bourhis C. et al., N-Glycosylation of a mouse IgG expressed in transgenic tobacco plants // Glycobiology. 1999. V. 9. P. 365-372]. Some proteins synthesized by transgenic plants are already produced by foreign companies [Rukavtsova EB, Buryanov Y.I., Shulga N.Ya., Bykov V.A. // Questions of biological, medical and pharmaceutical chemistry. 2006. No2. S. 3-12].

It should be noted the promise of obtaining transgenic plants expressing antimicrobial peptides (AMP) [Zakharchenko NS, Rukavtsova EB, Gudkov AT, Yukhmanova AA, Shkolnaya LA, Kado KI, Buryanov ME AND. The expression of the artificial gene of the antimicrobial peptide cecropin P1 increases the resistance of potato plants to late blight and white rot // Doklady Akademii Nauk. 2007. T. 415, No. 1, S. 129-131]. This is due to the fact that the widespread use of antibiotics as drugs has led to the accumulation of resistant forms of microorganisms.

Cases of resistance of a number of human pathogens (Mycobacterium tuberculosis, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi, Streptococcus pneumoniae, Vibrio cholerae, etc.) to almost any of the drugs used [Walsh C. Molecular methods / Nature. 2000. V. 406. P. 775-781].

Therefore, AMP is currently being considered as an alternative to classic antibiotics. They have certain advantages: a wide spectrum of action, the ability to quickly kill target cells, activity against strains resistant to other antibiotics, as well as the relative difficulty in developing resistance. Since some antimicrobial peptides have a cytotoxic effect (act on eukaryotic cells), they can be most effectively used in the treatment of diseases of the mucous membranes and external integuments - without introducing the patient into the blood. To date, such peptides are actively used to create new drugs, for example, the peptide antibiotic Ramoplanin (Ramoplanin) - a product of non-ribosomal synthesis in fungi of the genus Actinomycetes spp. Currently, ramoplanin is in clinical trials of phase III as an antibiotic against respiratory tract infections, especially staphylococci [Breukink E., de Kruijff B., Lipid II as a target for antibiotics // Nat. Rev. 2006. Drug Discov. V. 5. P. 321-332].

All this allows us to consider AMP as the basis for creating effective drugs, especially against the background of a decrease in the potential of conventional antibiotics.

AMP isolated from transgenic plants on the market is not yet available, but it may appear in the coming years, as some of them are at the last stages of clinical trials.

One of the promising antimicrobial peptides is cecropin P1, a linear α-helical peptide that does not contain cysteine [Martemyanov K.A., Shirin A.S., Gudkov A.T. Synthesis, cloning and expression of genes for antibacterial peptides: cecropin. magainin, and bombinin // Biotechnology letters. V. 18. No. 12. 1996. P. 1357-1362]. Unlike insect cecropins, it consists of one long positively charged α-helix, in the formation of which almost all amino acid residues are involved. Cecropin P1 is highly active against pathogenic gram-negative and gram-positive bacteria, fungi, and certain tumor cells [Pillai A., Ueno S., Zhang N., Lee JM, Kato Y. Cecropin P1 and novel nematode cecropins: a bacteria-inducible antimicrobial peptide family in the nematode Ascaris suum // Biochem. J. 2005. V. 390. P. 207-214.].

The transfer of the cecropin P1 gene (CP1) into the cells of the medicinal plant Kalanchoe seems to be relevant for its pharmaceutical and medical use.

To obtain and select transgenic plants, selective marker genes for resistance to antibiotics (kanamycin, hygromycin) and herbicides (phosphinotricin) are usually used [Angenon G., Dillen W., van Montagu M. Antibiotic-Resistance Markers for Plant Transformation // Plant Molecular Biology Manual // Eds. Gelvin S.B., Schilperoort R.A. Dordrecht: Kluwer Acad. Publ. 1994. C1. P. 1-13]. Transgenic plants obtained using selective markers pose a potential biological hazard associated with the presence of these genes in their genome and the possibility of their uncontrolled transfer to other plants and organisms. Therefore, at present, the urgent task is to obtain a new generation of plants that do not contain selective marker genes.

The selection of transgenic plants is carried out by various methods: selective elimination of marker genes from the chromosomal or chloroplast genome [Puchta N. Gene replacement by homologous recombination in plants // Plant Mol. Biol. 2002. V. 48. P. 173-182], using agrobacterial genes for the synthesis of phytohormones and their subsequent removal using recombinases of mobile genetic elements, for example, corn transposon Ac [Sugita K., Kasahara T., Matsunaga E., Ebinuma N.A. Transformation vector for the production of marker-free transgenic plants containing a single copy transgene at high frequency // Plant J. 2000. V. 22. P. 461-469].

Closest to the proposed method is a method for producing and selecting transgenic tomato plants of Lycopersicon esculentum, including the removal of selective markers from the genome of transgenic plants using transposons. In this method, the nptII selective neomycin phosphotransferase gene was flanked by inert repeats of the transposon of the maize Ds element. The selective gene was cleaved during the re-transformation of tomato plants by the transposase gene [Yoder J.I., Lassner m.W. Biologically safe plants transformation system using a Ds transposon // US 5225341, C12Q 1/68, 06/06/1993]. In addition, each transformant may contain several gene inserts. Therefore, for the selection of transgenic plants, it is necessary to conduct DNA hybridization analysis according to Southern.

The disadvantage of this method is the length of the procedure, since it is necessary to carry out a second transformation, and to make the selection of transformants according to the Southern method using radioactive probes.

A known method of creating markerless transgenic potato plants Solanum tuberosum L. using selective genes for positive (gene neomycin phosphotransferase nptII) and negative (cytosine deaminase gene cod A) selection on one plasmid and the target gene on another [Kondrak M., van der Meer IM, Banfalvi Z. Generation of marker- and backbone-free transgenic potatoes by site-specific recombination and a bi-functional marker gene in a non-regular one-border Agrobacterium transformation vector // Transgenic Res. 2006. V. 15. P. 729-737]. For transformation used a strain of Agrobacterium tumefaciens AGLO. Temporary positive selection of transformed plants on a medium with kanamycin was replaced by a stage of negative selection on a medium with 5-fluorocytosine, which leads to the selection of plants containing only the target gene with a frequency of 15%.

The disadvantage of this method is the duration due to the need for additional analyzes by DNA hybridization according to Southern and PCR.

Methods previously developed by the authors of the proposed project, obtaining markerless plants using markerless plasmids rVM [Buryanov Y.I., Zakharchenko NS, Yukhmanova AA, Pigoleva SV, Rukavtsova EB, Chebotareva E. N., Gayazova A.R. Recombinant plasmid rVM and method for producing markerless transgenic plants synthesizing target products using it. Patent No. 2410433 (RF) // B.I. 2011. No3; Zakharchenko N.S., Buryanov Y.I. Obtaining biosafety markerless transgenic plants of Kalanchoe pinnate (Kalanchoe pinnata L.) with increased resistance to biotic stress factors. All-Russian Scientific Conference “Factors of Plant Resistance in Extreme Natural Conditions and Technogenic Environment”. Irkutsk SIPHIBIR. June 10-13, 2013, pp. 89-92]. These methods make it possible to obtain marker-free plants in a short period of time by detecting the expression product of the transferred target gene.

The disadvantage of these methods is the inability to conduct a simultaneous analysis of the presence and quantitative level of expression of the transferred CP1 gene. The approximate expression level of cecropin P1 determined in these methods did not exceed 0.005% of ORB.

For practical or scientific use of transgenic plants, it is necessary to clearly determine the achievement of the level of expression of the transferred gene.

The level of expression of transferred genes in transgenic plants depends on many factors: the region of nuclear chromatin where the introduced gene is integrated, the type of promoter used, the method of transformation, the number of integrated copies of the gene, the correct design of the construct - adding transcription terminators, introducing an intron into the 5'-end of the mRNA target gene and others [G. Romanov Genetic engineering of plants and ways to solve the biosafety problem // Plant Physiology. 2000.V. 47. No. 3. S. 343-353; Jahne A., Becker D., Jorz H. Genetic engineering of cereal crop plants: a review // Euphytica. 1995. V. 85. P. 35-44].

A known method for determining the number of copies of a transferred gene in transgenic plants by real-time PCR (RT-PCR) [Stenman J., Finne P., Stahls A., Grenman R., Stenman U.-H., Patotie A., Orpana A Accurate determination of relative messenger RNA levels by RT-PCR // Nature. Biotech 1999. V. 17. P. 720-723]. In this method, using the general principles of polymerase chain reaction, the amount of amplified DNA is measured in real time after each amplification cycle. Two methods are used for quantitative determination: fluorescent dyes intercalating into double-stranded DNA molecules, and modified oligonucleotides (DNA probes), which fluoresce after hybridization with complementary DNA regions.

The disadvantage of this method is the high cost of reagents and equipment.

A known method for determining gene expression is in combination with RT-PCR and RT PCR (reverse transcription) to measure small amounts of mRNA, which allows to obtain quantitative information about the content of this mRNA in the cell and to judge the level of expression of this gene in a single plant cell [Nolan T. , Hands RE, Bustin SA 2006. Quantification of mRNA using real-time RT-PCR // Nat. Protoc. 2006. V. 1. P. 1559-1582].

The disadvantage of this method is the high cost of equipment, which so far cannot be used in every scientific laboratory.

A known method for determining gene expression by testing RNA (mRNA) and their fragments in samples (Northern blot) by RNA-DNA hybridization, where complementary DNA molecules are used as probes [Alwine J.C., Kemp D.J., Stark G.R. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes // Proc. Natl. Acad. Sci. U.S.A. 1977. V. 74. No. 12. P. 5350-5354].

The disadvantage of this method is that the data of a quantitative analysis of the synthesis of the mRNA of the target gene do not always correlate with the amount of the target protein synthesized by the cell, which may be associated with the degradation of the protein or RNA in the process of isolation and hybridization.

Closest to the proposed is a method for determining the expression of the gene of cecropin B (CB) by the final protein product and the effect that the resulting protein causes. In transgenic marker tomato plants, CB gene expression was determined by the level of antibiotic activity of the extracts against phytopathogens Ralstonia solanacearum and Xanthomonas campestris pv. vesicatoria and western blot analysis [Jan P-S, Huang H-Y, Chen H-M. Expression of a synthesized gene encoding cationic peptide cecropin B in transgenic tomato plants protects against bacterial diseases // Applied and Environmental Microbiology. 2010. V. 2. P. 769-775].

The disadvantage of this method is that the resulting tomato plants contained the nptII marker gene and expressed the antimicrobial peptide CB at a low level of 0.001% of ORB.

The objective of the present invention is to develop a method for producing marker-free plants of Kalanchoe pinnata expressing the gene of the antimicrobial peptide cecropin P1, which will be devoid of the aforementioned disadvantage and will allow to obtain a high level of expression of the peptide and at the same time determine its amount in the direct analysis of regenerants from transformed explants of feathery Kalanchoe.

This problem is solved by the proposed method, in which transformed marker-free plants are obtained and analyzed by treating the seeds of Kalanchoe pinnata (Kalanchoe pinnata) under vacuum infiltration or cocultivation of leaf explants of Kalanchoe pinnata (Kalanchoe pinnata) with a suspension of an agrobacterium strain A. gumefaciensensum tumefaciensensum tumefaciens 31acrobaceri tufaciensens (pMP90RK), A. tumafaciens LBA4404 (pAL4404), containing the markerless plasmid pBM carrying the CP1 gene encoding cecropin P1 and free of selective marker antibiotic resistance genes.

In this case, a suspension of agrobacterium Agrobacterium tumefaciens is preactivated with acetosyringone, incubated seeds or chopped leaf explants of feathery Kalanchoe on solid nutrient medium MS for 36-60 days, the seeds are treated under vacuum in the presence of a suspension of a strain of agrobacteria, and leaf explants under ordinary room conditions followed by seed incubation on solid nutrient medium without antibiotic, and after 36-60 days, explants are incubated on solid nutrient medium with antibiotic to eliminate agrobacteria. The obtained seedlings or regenerants of 4-5 weeks of age are used for Western blot analysis, plants with positive enzyme immunoassay are selected and examined for the presence of the cecropin P1 gene and its expression.

The bacterial strains Agrobacterium tumefaciens CBE21 (pTiBo542), A. tumafaciens GV3101 (pMP90RK), A. tumafaciens LBA4404 (pAL4404) contain the plasmid pBM-CP1. Moreover, the expression cassette of the plasmid pBM-CP1 includes genetic constructs selected from the following: CaMV 35S promoter - CP1 gene - pACaMV terminator.

As the target gene for transformation, the synthetic gene of the antimicrobial peptide Cecropin P1 (CP1) encoding the 31-membered amino acid sequence: SWLSKTAKKLENSAKKRISEGIAIAIQGGPR [Martemyanov K.A., Shirin A.S., Gudkov A.T. Synthesis, cloning and expression of genes for antibacterialpeptides: cecropin, magainin, and bombinin // Biotechnology letters. V. 18. No. 12. 1996. P. 1357-1362].

The co-cultivation of leaf explants of Kalanchoe with agrobacteria (transformation process) is preferably carried out for 48 ± 5 hours at 18 - 24 ± 2 ° C. At the same time, MS nutrient medium containing growth regulators 1 mg / l 6-benzylaminopurin (BAP), 0.1 mg / l naphthyloacetic acid (NAA) and 30 g / l banana powder (Banana powder, "Sigma") is used for regeneration ( BP).

Infiltration of Kalanchoe seeds with agrobacteria (transformation process) is preferably carried out under vacuum conditions (-0.1) - (-1.0) atm, for 5 ± 1 min at 24 ± 2 ° C. For seed germination, MS medium containing ticarcillin (500 or 250 mg / ml) (Duchefa Biochemien, Germany) (Tr) and banana powder (30 mg / l) (Banana powder, "Sigma", USA) (BP) is used .

A direct search for transgenic Kalanchoe plants synthesizing the antimicrobial peptide Cecropin P1 is carried out by Western blot analysis.

For example, 3 groups (30 plants) are selected that show the highest intensity of the positive signal.

In FIG. 1 shows the scheme of plasmid pBM-CP1. CaMV 35S - 35S RNA promoter of cauliflower mosaic virus; pACaMV - signal polyadenylation of cauliflower mosaic virus; CP1 - gene of cecropin P1; Km ^R - the antibiotic resistance gene kanamycin to maintain the construct in bacteria (not part of the T-element and not transferred to plants); oriV - start of replication; LB, RB — left and right borders of T-DNA; ColE1 - the beginning of replication of the plasmid ColE1; R, K, Sm, B, SI are restriction sites EcoRI, KpnI, SmaI, BamHI, SalI.

In FIG. 2 - the results of Western blot analysis of the synthetic preparation cecropin P1. Lanes: 1 - 100 ng; 2 - 50 ng; 3 to 30 ng; 4 - 15 ng; 5 to 10 ng; 6 - 2 ng.

In FIG. 3 - results of Western blot analysis of Kalanchoe plants: 1 - synthetic cecropin P1 mol. weight 3.4 kDa, (10 ng); 2 - extract of a non-transformed plant; 3-7 - extracts of transformed plants (lines 1-5).

In FIG. 4 - results of PCR DNA transgenic plants by electrophoresis. 1 - DNA molecular weight marker (GeneRuler 100 bp, Fermentas); 2 - DNA of the markerless vector pBM :: CP1 (control +); 3, 7 - DNA of non-transformed plants (control -); 4-6, 8, 9 - DNA of transformed plants (lines 1-5).

Table 1 shows the dependence of transformation efficiency on the transformation method of K. pinnata.

The possibility of implementing the proposed method is confirmed by the presented examples, but is not limited to them.

Example 1. Sterilization of plant material

Leaf sterilization. Young, fully unfolded leaves of Kalanchoe grown in a greenhouse are cut, disinfected by treating them with a 5% sodium hypochlorite solution containing 0.5% Tween 20 for 10 minutes, then rinsing three times in sterile distilled water. Cut the leaves into pieces with a length of 5-10 mm and place them on a nutrient medium MS [Murashige T., Skoog F. A revised medium for rapid growth and bioassays with tobacco cultures // Physiol. Plant. 1962. V. 15. P. 473-497], containing a standard set of salts, 7 g / l agar, 30 g / l sucrose (pH 5.8) and including 6-benzylaminopurin 1 mg / l as growth regulators (6 -BAP) and naphthylacetic acid 0.1 mg / L (NAA), as well as an additive - banana powder in an amount of 30 g / L. Explants are incubated at 22-24 ° C, a 16-hour daylight and an exposure of 2 cells. Subsidiary shoots or regenerants form from the explant after 4 weeks. The shoots are cut off and transferred to glass tubes with fresh MS medium. Cuttings of plants are carried out 1 time per month. For this, a Kalanchoe plant 15-20 cm high is divided into 4 parts, leaving 1-2 leaves with internodes on the stem and planted in test tubes.

Seed sterilization. Before transformation, the seeds are sterilized for 1.5 minutes in 70% ethanol, then 2 minutes in 1% sodium hypochlorite solution and washed 3 times for 10 minutes in sterile distilled water. Seeds are transferred to MS medium and recultivated for 48 hours at a temperature of 22-24 ° C, a 16-hour day and an illumination of 2 cells. For the experiment, 100-500 seeds are taken per Petri dish.

Example 2. Obtaining a culture of agrobacteria A. tumefaciens CBE21, bearing the markerless construction of pBM :: CP1.

For genetic transformation, A. tumefaciens CBE21 agrobacterial cells (pTiBo542) carrying the vector pBM :: CP1 with the antimicrobial peptide cecropin P1 gene are used. To induce vir function, the agrobacterial strain is pre-induced with acetosyringone. For this, a night culture of bacteria is grown on a shaker (150 rpm) at 26 ° C in YEP liquid medium (g / l): bacto-tryptone - 10, bacto-yeast extract - 10, NaCl - 0.5, pH 7, 0, with the addition of 50 μg / ml kanamycin and 15 μg / ml rifampicin. Before inoculation of the explants, the cell suspension is centrifuged at 6000 g for 5 minutes. The suspended precipitate is washed in 50 ml of a liquid MS medium containing 12.5 mm sodium phosphate, pH 5.5, to an optical density of OD ₆₀₀ = 0.05.

After 5 hours of cultivation, 100 μM acetosyringone is added to the cell suspension [Schafer W., Gorz A., Kahl G. T-DNA integration and expression in a monocot crop plant afte induction of Agrobacterium. // Nature. 1987. V. 327. No. 6122. P. 529-532] and continue to cultivate for 12-18 hours. After that, the agrobacterial culture is used for transformation.

Example 3. Obtaining a culture of agrobacteria A. tumafaciens GV3101 (pMP90RK), bearing the markerless construction of pBM :: CP1.

Obtaining a culture of agrobacteria A. tumafaciens GV3101 (pMP90RK), bearing the markerless construction of pBM :: CP1 is carried out according to the method described in example 2.

Example 4. Obtaining a culture of agrobacteria A. tumafaciens LBA4404 (pAL4404), bearing the markerless construction of pBM :: CP1.

Obtaining a culture of agrobacteria A. tumafaciens LBA4404 (pAL4404), bearing the markerless construction of pBM :: CP1 is carried out according to the method described in example 2.

Example 5. Transformation of leaf explants using agrobacteria Agrobacterium tumefaciens A. tumefaciens CBE21 (pTiBo542, pBM-CP1).

Co sterile 3-4-week-old plants, two days before the transformation, young leaves are cut, their edge and central vein are cut off. Then the leaves are cut into small pieces 0.5 × 0.5 cm ^{2 in} size, which are laid out on the surface of a solid MS medium containing 1 mg / l BAP, 0.1 mg / l NAA and 30 g / l BP. Incubation is carried out at a temperature of 22-24 ° C and a 16-hour daylight for 48 hours. Then the explants are transferred into a suspension of agrobacteria Agrobacterium tumefaciens A. tumefaciens CBE21 (pTiBo542, pBM-CP1) for 20 minutes. After this, the explants are laid out on plates with MS medium with 1 mg / L BAP, 0.1 mg / L NAA and 30 g / L BP for 48 hours. The resulting explants are transferred to Petri dishes with solid MS medium containing 1 mg / L BAP, 0.1 mg / L NAA, 30 g / L BP, 500 mg / L thiracillin to eradicate agrobacteria. The regeneration of shoots begins after 3 weeks of cultivation of transformed explants.

солей МС и с уменьшенной концентрацией тиракциллина (250 мг/л). Укоренение протекает в течение 2-3 недель. Полученные укорененные растения используют для дальнейшего молекулярно-генетического анализа.The resulting shoots are separated from the original tissue of the explants and transferred for further growth on Wednesday with

salts of MS and with a reduced concentration of thiracillin (250 mg / l). Rooting takes place within 2-3 weeks. The resulting rooted plants are used for further molecular genetic analysis.

Example 6. Transformation of leaf explants using agrobacteria Agrobacterium tumefaciens A. tumefaciens GV3101 (pMP90RK, pBM-CP1).

Transformation of leaf explants using agrobacteria Agrobacterium tumefaciens A. tumefaciens GV3101 (pMP90RK, pBM-CP1) is carried out according to the method described in example 5.

Example 7. Transformation of leaf explants using agrobacteria Agrobacterium tumefaciens A. tumefaciens LBA4404 (pAL4404, pBM-CP1).

Transformation of leaf explants using agrobacteria Agrobacterium tumefaciens A. tumefaciens LBA4404 (pAL4404, pBM-CP1) is carried out according to the method described in example 5.

Example 8. Transformation of seeds using agrobacteria A. tumefaciens CBE21 (pTiBo542, pBM :: CP1).

Sterile seeds pre-recultured for 48 hours on MS medium are immersed in a flask with a suspension of A. tumefaciens CBE21 agrobacteria (pTiBo542, pBM :: CP1) (10 ⁹ cells / ml) (100 ml). The flask with seeds is placed in a vacuum desiccator and incubated for 5 min at a negative pressure of 0.8 atm. After the vacuum is interrupted, the seeds are transferred onto sterile filter paper to remove excess moisture and transferred to an MS agar medium. After two days, the seeds are transferred to fresh MS medium containing 500 mg / l of the antibiotic ticarcillin (Tr) and 30 g / l of banana powder (BP). Seeds germinate 4 weeks to the length of seedlings 3-5 cm and used for analysis. Further in vitro cultivation of plants is carried out on MS medium with the addition of Tr 250 mg / L. To obtain seeds, plants are transplanted into the greenhouse.

Example 9. Transformation of seeds using agrobacteria Agrobacterium tumefaciens A. tumefaciens GV3101 (pMP90RK, pBM-CP1).

Seed transformation using agrobacteria Agrobacterium tumefaciens A. tumefaciens GV3101 (pMP90RK, pBM-CP1) is carried out according to the method described in example 8.

Example 10. Transformation of seeds using agrobacteria Agrobacterium tumefaciens A. tumefaciens LBA4404 (pAL4404, pBM-CP1).

The transformation of seeds using agrobacteria Agrobacterium tumefaciens A. tumefaciens LBA4404 (pAL4404, pBM-CP1) is carried out according to the method described in example 8.

Example 11. Calibration of synthetic cecropin P1.

To quantify cecropin P1 expressed by plants, a commercial preparation of cecropin P1 is calibrated (Sigma, USA). For Western blot analysis, take a different amount of peptide diluted in water: 100 ng, 50 ng, 30 ng, 15 ng, 10 ng, 2 ng.

Example 12. Western blot analysis of plants

To search for transformed plants, one leaf explant is taken from each of the regenerated or grown from seeds after the genetic transformation of the feathers of Kalanchoe. A total of 500 plants are analyzed. Leaf explants from each individual shoot are combined into groups of 10 explants in one group and used to prepare the initial test samples of plant extract. All 10 leaf explants are placed in one tube. Only 50 groups - 50 test tubes.

Next, the protein is extracted from the plant tissue, the samples are aligned according to the total protein product content (about 100 μg of total protein in each sample), the obtained samples are used for electrophoretic separation and Western blot analysis.

The Western blot analysis was carried out according to the following scheme:

1. Protein extraction is carried out: 1 g of plant tissue is triturated in liquid nitrogen, an equal amount of water heated to 70 ° C is added and placed in a thermostat for 1 hour. Then centrifuged at 12000 rpm Trichloroacetic acid (TCA) is added to the supernatant to 5% and left for 10 min at 4 ° C. Centrifuged again at 12,000 rpm, the precipitate was washed three times with cold acetone and dissolved in 200 μl of 0.1 N. NaOH. Electrophoresis is carried out with the obtained protein solution in a 12% polyacrylamide gel (PAGE). For electrophoresis take 10 μl of the peptide solution.

2. For the transfer of proteins using a PVDF membrane ("Porablot", Macherey-Nagel, Germany).

3. Electric transport is carried out for 2 hours at 200 mA or overnight at 20 mA in buffer (25 M Tris-HCl, 250 mm glycine) with the addition of 20% methanol.

4. The membrane is then incubated in a 5% skim milk solution (1 g per 20 ml of TBS-t buffer) with moderate swaying for 1 h at room temperature and washed in TBS-t buffer.

5. After that, the membrane is incubated with antibodies to cecropin P1, then with secondary rabbit polyclonal antibodies conjugated with horseradish peroxidase.

6. Immunodetection is carried out using a set of ECL reagents ("Amersham", UK).

According to the result of the initial Western blot analysis of the 50 initial plant groups, for example, 3 groups (30 plants) are selected that show the highest intensity of the positive signal. Groups that gave a low signal intensity are not used in the work, as well as groups that are completely negative according to the results of immunoblotting.

Three groups are used to identify the most productive individual transgenic plants. For this, plants of selected groups — 30 plants in total — are transplanted into separate tubes, grown for 3 weeks, and repeated Western blot analysis of extracts from the leaves of each individual plant is carried out.

In FIG. Figure 2 shows the results of a Western Western blot analysis of the synthetic preparation cecropin P1. Lanes: 1 - 100 ng; 2 - 50 ng; 3 to 30 ng; 4 - 15 ng; 5 to 10 ng; 6 - 2 ng.

In FIG. Figure 3 shows the results of a Western blot analysis of extracts of five plants (lines 1-5) expressing a peptide with a molecular weight of 3.4 kDa corresponding to the mature form of cecropin P1. Lanes: 1 - synthetic cecropin P1, mol. weight 3.4 kDa (10 ng); 2 - extract of a non-transformed plant; 3-7 - extracts of transformed plants (lines 1-5).

Comparing with the calibration analysis of the synthetic peptide (Fig. 2), it is noted that the level of cecropin P1 synthesis in plants of lines 2 and 3 corresponds to about 15 ng, which corresponds to an expression level of 0.02% of the total soluble protein (ORB).

The presence of the CP1 gene in transgenic Kalanchoe plants is confirmed by polymerase chain reaction (PCR) with primers specific for the CP1 gene. When the PCR products of DNA of transgenic plants are separated by electrophoresis, the presence of a 102 bp band corresponding to the CP1 gene is identified (lanes 4-6, 8, 9) (Fig. 4).

The data presented in Table 1 show that the transformation efficiency depends on the method of transformation. Transformation by the method of vacuum agroinfiltration of seeds is higher (35.1%) than by the method of cocultivation of leaf explants with agrobacteria (25.9%), while the maximum expression level is 0.03% and 0.02% of ORB, respectively.

The proposed method for producing marker-free plants Kalanchoe pinnata expressing the gene for the antimicrobial peptide cecropin P1, allows you to get transgenic plants with a high level of expression of the gene cecropin P1, reduce the time of receipt and selection of transgenic markerless plants Kalanchoe to 1-2 months, compared with other methods of producing markerless plants - 4-8 months [Truesdale MR, Toldi O., Scott P. The effect of elevated concentrations of fructose 2.6 bisphosphate on carbon metabolism during deacidification in the crassulacean acid metabolism plant Kalanchoe daigremontiana // Plant physiology. 1999. V. 121. P. 957-964], due to direct organogenesis (shoot regeneration not through the callus stage, but directly from explants and seed germination), due to the use of a shortened by 2600 bp constructs (vector pBM-CP1) that does not contain the neomycin phosphotransferase II marker gene, which involves selection on the antibiotic kanamycin when the plant is subjected to additional stress, as well as a quick Western blot analysis on the CP1 gene product, regardless of the age of the plant, the method provides environmental and biological safety transgenic plants expressing the cecropin P1 gene without additional selective genes.

Claims (9)

1. A method of obtaining markerless transgenic plants of Kalanchoe pinnate, expressing the cecropin P1 gene, comprising genetic transformation of plants with the construction of pBM-CP1 by vacuum infiltration of seeds with agrobacterium species Agrobacterium tumafaciens or cocultivation of leaf explants with agrobacteria, elimination of agrobacteria, which is differentiated by selection before transformation, agrobacteria are treated with an inducer of the vir region with acetosyringinone, explants are incubated for 48 hours on MS medium with regulators and growth and the addition of a banana "powder before transformation and after transformation, the elimination of agrobacteria is carried out by ticarcillin, transgenic plants are obtained by shoot regeneration not through the callus stage, but directly from explants and seed germination, and the presence and quantitative content of the cecropin P1 western blot peptide is determined analysis of extracts of Kalanchoe pinnate seedlings, while for the quantitative determination of the synthesized peptide cecropin P1 in plants, a comparison of calibres month old data with different amounts of synthetic cecropin P1 with the data of Western blot analysis of extracts of sprouts Kalanchoe pinnate. 2. The method according to p. 1, characterized in that as a strain of a bacterium of the species Agrobacterium tumafaciens, a strain selected from the range of A. tumefaciens CBE21, A. tumafaciens GV3101 (pMP90RK), A. tumafaciens LBA4404 (pAL4404) containing a markerless recombinant is used rVM with the gene for the antimicrobial peptide cecropin P1. 3. The method according to p. 1, characterized in that the agrobacteria activate in the nutrient medium MS acetosyringone 100 μm. 4. The method according to p. 1, characterized in that the seeds of Kalanchoe pinnata plants and leaf explants recultured for 48 hours on MS nutrient medium with banana powder (30 g / l) before and after transformation. 5. The method according to p. 1, characterized in that the seeds of Kalanchoe pinnata plants are subjected to vacuum treatment for 1-15 minutes 6. The method according to p. 1, characterized in that for the elimination of agrobacteria using the antibiotic ticarcillin in an amount of 250-500 mg / L. 7. The method according to p. 1, characterized in that for the quantitative determination of the synthesized cecropin P1 peptide in plants, calibration experiments are carried out by Western blot analysis with different amounts of the synthetic preparation cecropin P1, which allows one to determine the expression level of the CP1 gene in individual transgenic plants. 8. The method according to p. 1, characterized in that for the preparation of extracts of experimental plants of Kalanchoe use the method of sedimentation of a peptide of 5% TCA and dissolution in 0.1 N. NaOH. 9. The method according to p. 1, characterized in that the level of expression of cecropin P1 in the resulting transgenic plants of Kalanchoe was 0.02-0.03% of the total soluble protein of the cell.

Images