RU2483109C1 - WHEAT GENES Triticum kiharae, CODING ANTIMICROBIAL PEPTIDES - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: wheat gene Triticum kiharae is presented, having nucleotide sequence SEQ ID No.1, which codes antimicrobial peptides of Kiharae wheat Triticum kiharae Tk-AMP.

EFFECT: invention makes it possible to create plants resistant to pathogenes using methods of genetic engineering.

1 cl, 3 tbl, 2 ex

Description

The invention relates to the field of plant biotechnology, specifically to plant protective genes encoding antimicrobial peptides (AMP), and can be used to create pathogen resistant crops.

Crop losses caused by plant pathogens (fungi, bacteria, viruses, viroids) and pests can reach 45% [Oerke EC, Dehne HW, Schönbeck F., Weber A. Crop Production and Crop Protection: Estimated Losses in Major Food and Cash Crops. - 1994.- Elsevier, Amsterdam]. Another negative aspect of the impact of pathogens is the decline in product quality. For example, the presence of mycotoxins Fusarium sp. in grain makes it toxic to humans and animals. In connection with the rapid growth of the Earth's population, the problem of increasing the yield of agricultural crops is becoming especially relevant. Currently, to solve this problem, they use: rotational farming, traditional breeding aimed at obtaining sustainable forms, and chemical plant protection products. Traditional breeding methods are not always effective because of their duration and the absence in some cases of sources of stability. The use of chemical plant protection products is expensive and poses a threat to environmental safety.

An alternative to increasing the resistance of crops to pathogens and insect pests, as well as to other stressful environmental factors of an abiotic nature (drought, soil salinity, etc.) is genetic engineering, which allows the integration of foreign genes that determine resistance into the genomes of cultivated plants.

The use of genetic engineering methods for plant transformation opens up new possibilities for creating crops that are widely resistant to pathogens, pests, and abiotic stress (drought, soil salinity, etc.). Work on producing resistant cultivars using recombinant DNA technologies was carried out about 20 years ago [Andrews R.E., Faust R.M., Wabiko H., Raymond K.C., Bulla L.A. The biotechnology of Bacillus thuringiensis. // Crit. Rev. Biotech - 1987. - Vol.6. - P.163-232. Vaeck M., Reynaerts A., Höfne H., Jansens S., De Beuckeleer M., Dean C., Zabeau M., Van Montagu M., Leemans J. Transgenic plants protected from insect attack. // Nature - 1987. - Vol. 328. - P.33-37]. Using the gene for the insecticidal protein of the bacterium Bacillus thuringiensis, transgenic tobacco plants resistant to the parasitic larvae of the Manducta sexta butterfly were obtained. Since then, at least 10 entomotoxin genes from Bacillus thuringiensis have been transferred and expressed in at least 26 plant species that have become resistant to certain pests [Schuler T.N., Poppy GM, Kerry BR, Denholm I. Insect- resistant transgenic plants. // Trends Biotech. - 1998 .-- Vol.16. - P.168-175]. A significant portion of the global crop today falls on Bt-transformed products (US Department of Agriculture website: www.aphis.usda.gov). However, the issue of the safety of Bt-containing products for humans, as well as their impact on the environment [Vazquez-Padron RI, Moreno-Fierros L., Neri-Bazan L., Martinez-Gil AF, de-la-Riva GA, Lopez-Revilla R. Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice. // Braz J Med Biol Res. - 2000. - Vol. 33. - P.147-155. Yu H.L., Li Y.H., Wu K.M. Risk assessment and environmental effects of transgenic Bacillus thuringiensis crops on non-target organisms. // J Integr Plant Biol. - 2011 .-- Vol. 53. - P.520-538]. While using genetic engineering methods, significant success has been achieved in creating varieties resistant to insect pests, crops resistant to fungal and bacterial diseases are still not available on the market.

At present, to increase the stability of crops, an approach is considered more promising, aimed at enhancing the protective potential of the plants themselves, both by activating the expression of their own protective genes and by embedding the genes of protective compounds from other plant species, in particular wild plants, which, in Unlike cultivated plants, they are more resistant to pathogenic microorganisms. The genes of antimicrobial peptides are of the greatest interest in this direction. Antimicrobial peptides have a wide spectrum of antimicrobial activity, do not cause the appearance of resistant forms, and their genes can be directly integrated into the genome of plants sensitive to pathogens.

Known to the claimed nucleotide sequence AK333848.1 from common wheat Triticum aestivum [Kawaura K., Mochida K., Enju A., Totoki Y., Toyoda A., Sakaki Y., Kai C., Kawai J., Hayashizaki Y ., Seki M., Shinozaki K., Ogihara Y. Assessment of adaptive evolution between wheat and rice as deduced from full-length common wheat cDNA sequence data and expression patterns. // BMC Genomics. - 2009 .-- Vol.10. - P.271]. The product encoded by this sequence is unknown, and its biological activity has not been established, which excludes the possibility of using this sequence to transform plants in order to increase their resistance to pathogens.

The invention solves the problem of expanding the assortment of plant protective genes for use in biotechnology when creating new plant forms that are resistant to environmental factors using genetic engineering methods.

The problem is solved using the wheat gene Triticum kiharae with the nucleotide sequence of SEQ ID No. 1 encoding antimicrobial peptides called Tk-AMP, including antimicrobial peptides TK-AMP-L-2-2 and TK-AMP-S-2- 5, exhibiting antifungal activity in in vitro tests against Bipolaris sorokiniana, Fusarium graminearum and Fusarium oxysporum in micromolar concentrations (Table 1). This means that their genes can be used to increase the resistance of plants to pathogens using genetic engineering methods.

Table 1 Antifungal properties of the peptides TK-AMP-L-2-2 and TK-AMP-S-2-5, encoded by the nucleotide sequence of Tk-AMP and isolated from Triticum kiharae Dorof. et migusch Mushroom IC ₅₀ , μM * TK-AMP-L-2-2 TK-AMP-S-2-5 Bipolaris sorokiniana 15,5 14.5 Fusarium graminearum 6.5 7.0 Fusarium oxysporum 8.5 8.0 * IC ₅₀ corresponds to the concentration of the peptide at which 50% inhibition of fungal spores is achieved.

The nucleotide sequence of Tk-AMP (SEQ ID No. 1) corresponds to the coding portion of cDNA (mRNA) obtained from immature Kihara wheat seeds Triticum kiharae Dorof. et Migusch. Kihara wheat belongs to the family of cereals (bluegrass) Gramineae (Poaceae), the class of monocotyledonous (lilyopsids) Monocotyledones (Liliopsida), the department of angiosperms (flowering) Magnoliophyta (Angiospermae).

The nucleotide sequence of Tk-AMP encodes the precursor proteins of the protective peptides TK-AMP-L-2-2 and TK-AMP-S-2-5 with a length of 247-362 amino acid residues, consisting of an N-terminal signal peptide with a length of 25 amino acid residues; five, six or seven peptide domains 27-44 residues long, separated by sites of specific proteolysis; C-terminal prodomain 15-25 residues long.

The technical result of the invention is the encoding of several (from five to seven) protective peptides in one gene.

The invention is illustrated by the following examples.

Example 1

Establishment of the Kihara wheat cDNA structure of Triticum kiharae encoding the new antimicrobial peptides TK-AMP-L-2-2 and TK-AMP-S-2-5. Amino acid sequence of peptides TK-AMP-L-2-2 and TK-AMP-S-2-5:

TK-AMP-L-2-2:

GlyAspSerPheAspSerCysValSerGlnCysArgGlyHisGlyGlyTrpTrpGlyLysGluArgTrpAspArgCysArgArgIleCysArgGlnSerGlnGlu;

TK-AMP-S-2-5:

HisHisGlyGlySerSerCysGluGlnLysCysGlnGlnArgTyrArgHisGluTyrGluLysGluGlnCysValArgAspCysLysSerGlyGlyHisGlyGlyAlaGlyGlyArgGlyArgGlu.

Total RNA is isolated from seeds in the stage of milk ripeness using the Trizol RNA Prep 100 reagent kit (Isogen, Russia) in accordance with the protocol of the manufacturer. 2 μg of total RNA is used as a template for cDNA synthesis using the Mint kit (Eurogen, Russia).

The determination of the cDNA structure is carried out in several stages: determination of the 3'-terminal part of cDNA, determination of the 5'-terminal part of cDNA, obtaining the coding part of the gene. For this purpose, primers were designed and synthesized based on the known amino acid sequences of the peptides TK-AMP-L-2-2 and TK-AMP-S-2-5. For TK-AMP-L-2-2, these are primers Dir1-1 and Dir1-2; for TK-AMP-S-2-5, primers Dir2-1 and Dir2-2 (Table 2).

The 3'-terminal region of the cDNA is amplified using "nested" PCR, for the first round, use the universal T7Car primer and specific primers Dir1-1 and Dir2-1, for the second round, use the universal T7Car primer and specific primers Dir1-2 and Dir2-2. Amplified fragments are cloned and sequenced on an automatic sequencer.

Based on the established nucleotide sequences of cDNA fragments, primers were designed and synthesized for amplification of the 5'-regions of Tk-AMP cDNA: Rev1 and Rev2 (Table 2). 5'fragments of cDNA are amplified using a universal T7Car primer and specific primers Rev1 and Rev2.

To obtain the cDNA coding region, two primers were designed and synthesized: 5e3 and 5r (Table 2), the use of which for PCR on the cDNA matrix allows the amplification of the coding part of the genes to be fully amplified. The resulting fragments are cloned and sequenced, resulting in the nucleotide sequence of SEQ ID No. 2 (Tk-AMP-L1), SEQ ID No. 3 (Tk-AMP-L2), SEQ ID No. 4 (Tk-AMP-M1), SEQ ID No. 5 (Tk-AMP-S1), SEQ ID No. 6 (Tk-AMP-S2), SEQ ID No. 7 (Tk-AMP-S3), SEQ ID No. 8 (Tk-AMP-S4).

The sequences Tk-AMP-L1, Tk-AMP-L2, Tk-AMP-M1, Tk-AMP-S1, Tk-AMP-S2, Tk-AMP-S3 and Tk-AMP-S4 are homologous (66% homology) and can be combined by the General formula SEQ ID No. 1.

Example 2

Analysis of the products of the protective genes of Kihara wheat Triticum kiharae The identified nucleotide sequences (SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8) encode homologous ( 68% homology) precursor proteins (preproteins) Tk-AMP-L1 (SEQ ID No. 9), Tk-AMP-L2 (SEQ ID No. 10), Tk-AMP-M1 (SEQ ID No. 11), Tk-AMP- S1 (SEQ ID No. 12), Tk-AMP-S2 (SEQ ID No. 13), Tk-AMP-S3 (SEQ ID No. 14), and Tk-AMP-S4 (SEQ ID No. 15), respectively.

The nucleotide sequence of SEQ ID No. 2 encodes a Tk-AMP-L1 precursor protein (SEQ ID No. 9), which contains the following components: N-terminal signal peptide (25 residues), spacer sequence (8 residues), mature TK-AMP peptides -L1-1, TK-AMP-L1-2, TK-AMP-L1-3, TK-AMP-L1-4, TK-AMP-L1-5, TK-AMP-L1-6, TK-AMP-L1 -7 (27-44 residues), separated by short (0-11 residues) spacer sequences, C-terminal prosequence (25 residues). The sequences of mature peptides are presented in Table 3.

The nucleotide sequence of SEQ ID No. 3 encodes a Tk-AMP-L2 precursor protein (SEQ ID No. 10), which contains the following components: N-terminal signal peptide (25 residues), spacer sequence (12 residues), mature TK-AMP peptides -L2-1, TK-AMP-L2-2, TK-AMP-L2-3, TK-AMP-L2-4, TK-AMP-L2-5, TK-AMP-L2-6, TK-AMP-L2 -7 (28-43 residues), separated by short (6-13 residues) spacer sequences, C-terminal prosequence (25 residues). The sequences of mature peptides are presented in Table 3.

The nucleotide sequence of SEQ ID No. 4 encodes a Tk-AMP-M1 precursor protein (SEQ ID No. 11), which contains the following components: N-terminal signal peptide (25 residues), spacer sequence (12 residues), mature TK-AMP peptides -M1-1, TK-AMR-M1-2, TK-AMR-M1-3, TK-AMR-M1-4, TK-AMR-M1-5, TK-AMR-M1-6 (28-43 residues) separated by short (6-13 residues) spacer sequences, C-terminal prosequence (25 residues). The sequences of mature peptides are presented in Table 3.

The nucleotide sequence of SEQ ID No. 5 encodes a Tk-AMP-S1 precursor protein (SEQ ID No. 12), which contains the following components: N-terminal signal peptide (25 residues), spacer sequence (12 residues), mature TK-AMP peptides -S1-1, TK-AMP-S1-2, TK-AMP-S1-3, TK-AMP-S1-4, TK-AMP-S1-5 (28-43 residues), separated by short (6-11 residues ) spacer sequences, C-terminal prosequence (25 residues). The sequences of mature peptides are presented in Table 3.

The nucleotide sequence of SEQ ID No. 6 encodes a Tk-AMP-S2 precursor protein (SEQ ID No. 13), which contains the following components: N-terminal signal peptide (25 residues), spacer sequence (12 residues), mature TK-AMP peptides -S2-1, TK-AMP-S2-2, TK-AMP-S2-3, TK-AMP-S2-4, TK-AMP-S2-5 (28-42 residues), separated by short (6-9 residues ) spacer sequences, C-terminal prosequence (15 residues). The sequences of mature peptides are presented in Table 3.

The nucleotide sequence of SEQ ID No. 7 encodes a Tk-AMP-S3 precursor protein (SEQ ID No. 14), which contains the following components: N-terminal signal peptide (25 residues), spacer sequence (8 residues), mature TK-AMP peptides -S3-1, TK-AMP-S3-2, TK-AMP-S3-3, TK-AMP-S3-4, TK-AMP-S3-5 (27-44 residues), separated by short (0-11 residues ) spacer sequences, C-terminal prosequence (15 residues). The sequences of mature peptides are presented in Table 3.

The nucleotide sequence of SEQ ID No. 8 encodes a Tk-AMP-S4 precursor protein (SEQ ID No. 15), which contains the following components: N-terminal signal peptide (25 residues), spacer sequence (12 residues), mature TK-AMP peptides -S4-1, TK-AMP-S4-2, TK-AMP-S4-3, TK-AMP-S4-4, TK-AMP-S4-5 (27-43 residues), separated by short (2-10 residues ) spacer sequences, C-terminal prosequence (25 residues). The sequences of mature peptides are presented in Table 3.

Thus, the nucleotide sequences of Tk-AMP-L1, Tk-AMP-L2, Tk-AMP-M1, Tk-AMP-S1, Tk-AMP-S2, Tk-AMP-S3 and Tk-AMP-S4 (SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, respectively) encode the precursors Tk-AMP-L1, Tk-AMP-L2, Tk- AMP-M1, Tk-AMP-S1, Tk-AMP-S2, Tk-AMP-S3 and Tk-AMP-S4 (SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15) containing from five to seven mature peptides (and their derivatives).

Claims (1)

The wheat gene Triticum kiharae, having the nucleotide sequence of SEQ ID No. 1 encoding the antimicrobial peptides of Kihara wheat Triticum kiharae Tk-AMP.

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