RU2457251C1 - Stellaria media starwort genes coding protective peptides - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention solves the problem of widening a range of protective plant genes applicable in agricultural biotechnology for the purpose of providing resistance of cultivated plants to environmental factors ensured by structure of a new nucleotide sequence sm-amp coding antifungal peptides of Stellaria media starwort.

EFFECT: widening the range.

Description

FIELD OF THE INVENTION

The invention relates to the field of biotechnology, specifically to plant protective genes encoding biologically active peptides with antifungal activity.

BACKGROUND

The damage caused to world agriculture by pathogens of cultivated plants (fungi, bacteria, viruses and viroids) and insect pests (two main causes of damage) is estimated at trillion. rubles annually. Losses reach half the crop. The methods used to reduce losses mainly come down to the use of synthetic pesticides, many of which are environmentally aggressive substances. The use of chemical plant protection products poses a significant environmental hazard; biological products are safer.

An alternative to long-term selection of useful traits, for example, resistance to various environmental factors, is genetic engineering. Its use for plant transformation opens up new possibilities for creating crops that are widely resistant to pathogens, pests, and abiotic stress (drought, salinity of the soil, etc.). Pioneering work on producing resistant cultivars using recombinant DNA technologies was carried out about 25 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., Höfte 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, the genes of entomotoxins from B. thuringiensis have been transferred and expressed in dozens of plant species that have acquired resistance to some pests [Schuler T.N., Poppy G.M., Kerry B.R., Denholm I. Insect-resistant transgenic plants. // Trends Biotech. - 1998 .-- Vol.16. - P. 168-175. Romeis J., Meissle M., Bigler F. Transgenic crops expressing Bacillus thuringiensis toxins and biological control. // Nat. Biotechnol. - 2006. - Vol.24 - P.63-71]. A significant part of the world crop today falls on the share of Bt-transformed products [James C. Global Status of Commercialized Biotech / GM Crops: 2010. // ISAAA Brief - 2010. - No.42]. Nevertheless, the issue of the safety of Bt-containing products for humans is still being discussed, the ecological consequences of the global spread of transgenic plants containing the genes of B. thuringiensis entomotoxins are not entirely clear, and lately the problem of developing insect resistance to toxins has been acute [Sanahuja G. , Banakar R., Twyman RM, Capell T., Christou P. Bacillus thuringiensis: a century of research, development and commercial applications. // Plant Biotechnol. J. - 2011 .-- Vol. 9. - P.283-300]. In addition, a visible violation of the interspecific barrier receives a negative response in modern society. It is worth noting that the engineering of disease resistance has proven to be a more difficult task than increasing insect resistance.

Apparently, the most promising approaches to obtaining resistant varieties are those that are aimed at enhancing the plant's own protective properties. Compounds produced by plants to protect against pathogens include the so-called phytoalexins and phytoantisipins, substances of various chemical nature, as well as some proteins and peptides [Sels J., Mathys J., De Coninck B.M., Cammue B.P., De Bolle M.F. Plant pathogenesis-related (PR) proteins: a focus on PR peptides. // Plant Physiol. Biochem. - 2008 .-- Vol. 46. - P.941-950]. It is promising to obtain resistant varieties with altered expression of their own protective genes or transfer genes from one plant to another, for example, from wild to cultivated, because it is known that during selection and selection, agricultural plants, acquiring some useful traits, lost others. Of greatest interest are polypeptide compounds with protective properties, due to the possibility of their direct use to obtain transgenic plants [Carlini C.R., Grossi-de-Sá M.F. Plant toxic proteins with insecticidal properties. A review on their potentialities as bioinsecticides. // Toxicon - 2002 .-- Vol. 40. - P.1515-1539].

The claimed nucleotide sequence of sm-amp is closest to the gene of the Ar-AMP peptide from amaranth Amaranthus retroflexus [Lipkin A., Anisimova V., Nikonorova A., Babakov A., Krause E., Bienert M., Grishin E., Egorov T. An antimicrobial peptide Ar-AMP from amaranth (Amaranthus retroflexus L.) seeds. // Phytochemistry. - 2005 .-- Vol.66. - P. 2426-2431]. Ar-AMP belongs to the same extensive family of cysteine-rich chitin-binding peptides. In the structure of the coding part of the gene (mRNA), regions corresponding to the signal peptide, mature Ar-AMP, and C-terminal pro-sequence are distinguished. The most significant difference of the claimed sm-amp sequence is the simultaneous coding of two mature peptides.

SUMMARY OF THE INVENTION

The invention solves the problem of expanding the range of plant protective genes available for use in agricultural biotechnology in order to engineer the resistance of cultivated plants to environmental factors.

The problem is solved due to the structure of the new sm-amp nucleotide sequence encoding the antifungal peptides of the stellaria media Stellaria media:

The nucleotide sequence of sm-amp corresponds to cDNA (mRNA) obtained from a natural source - the weed plant of the middle stellate or woodlice Stellaria media (L.) Vill., Which belongs to the clove family Caryophyllaceae, the class of dicotyledons Dicotyledones (Magnoliopsida), the department of angiosperms or flowering plants Magnoliophyta (Angiospermae).

The nucleotide sequence of sm-amp encodes the precursor proteins of the antifungal peptides pSm-AMP-1 and pSm-AMP-2 with a length of 163-167 amino acid residues having the following constituent elements: N-terminal signal peptide (22 residues), the first mature peptide (Sm- AMP-1.1a or Sm-AMP-2.1a, 35 residues), short spacer sequence (10 residues), second mature peptide (Sm-AMP-1.2a or Sm-AMP-2.2a, 32-33 residues) and extended C -terminal sequence (64-67 residues). The inventive nucleotide sequence sm-amp encodes the peptides Sm-AMP-1.1a, Sm-AMP-1.2a, Sm-AMP-2.1a, Sm-AMP-2.2a and their derivatives with a length of 30-35 amino acid residues exhibiting pronounced antifungal activity in against many plant pathogens, including Alternaria consortiale, Fusarium culmorum, Helminthosporium sativum (syn. Bipolaris sorokiniana, Drechslera sorokiniana), Thielatiopsis basicola, in micromolar concentrations.

The invention is illustrated by the following examples.

Example 1

Preparation of Stellaria media star cDNA encoding antifungal peptides

Total RNA is isolated from plant material using Trizol (Trizol; Life Technologies, USA) in accordance with the standard protocol proposed by the manufacturer. 1 μg of total RNA is used to synthesize the first cDNA strand according to the Smart protocol (BD Biosciences Clontech, USA).

cDNA is amplified by PCR with Smart PCR primer (BD Biosciences Clontech). Based on the amino acid sequence of the Sm-Amp-1.1a peptides (H ₂ N-SGPNGQCGPGWGGCRGGLCCSQYGYCGSGPKYCAH-COOH) and Sm-Amp-2.2c (H ₂ N-AAGQCYRGRCSGGLCCSKYG synthesis, we synthesize a 3-way synthesis and COOC-synthesis parts of cDNA.

For sm-amp-1: 5'-CAGTGTGG (T / G) CC (T / C / G) GG (T / C / A / G) TGGGG-3 '.

For sm-amp-2: 5'-GCTGCTGG (T / C / G) CA (A / G) TG (T / C) TA (T / C) CG-3 '.

Next, using primers from BD Biosciences Clontech for 3 ′ RACE-PCR and the above specific primers, 3 ′ fragments of sm-amp-1 and sm-amp-2 cDNA are amplified. Amplified fragments are cloned into a vector and sequenced on an automatic sequencer.

Based on the determined nucleotide sequences of cDNA fragments, primers are designed and synthesized for amplification of 5'-fragments of cDNA sm-amp-1 and sm-amp-2:

sm-amp-1-R1: 5'-AATCTCAGAAAGAGGAGTGTTG-3 ';

sm-amp-1-R2: 5'-CACAATATCCATATTGGGAGC-3 ';

sm-amp-2-R1: 5'-CCTGGCACATGCCTAGTCC-3 ';

sm-amp-2-R2: 5'-CGCTCCCACAATATCCATATTT-3 '.

Using primers from BD Biosciences Clontech for 5 'RACE-PCR and the above specific primers, 5'cDNA fragments are amplified by sequential two-step PCR. Amplified fragments are cloned into a vector and sequenced.

The resulting nucleotide sequences sm-amp1 and sm-amp-2 encoding pSm-AMP-1 and pSm-AMP-2 are obtained by combining overlapping sequences of 5'- and 3'-fragments of cDNA:

sm-amp-1 (826 nucleotides):

sm-amp-1 and sm-amp-2 are closely related sequences (~ 92% of identical nucleotides) and can be combined by a general formula.

Example 2

The structure of the coding part of the genes of antifungal peptides of the stellaria stellaria media

sm-amp-1 and sm-amp-2 encode the closely related (~ 86% identical amino acid residues) precursor proteins (preproteins) pSm-AMP-1 and pSm-AMP-2, respectively. Their amino acid sequences are obtained using in silico translation of the sm-amp nucleotide sequences:

pSm-AMP-1 (167 residues):

pSm-AMP-1 contains the following components: N-terminal signal peptide (22 residues), mature Sm-AMP-1.1a peptide (H ₂ N-SGPNGQCGPGWGGCRGGLCCSQYGYCGSGPKYCAH-COOH; 35 residues), short spacer sequence, mature (10) Sm-AMP-1.2a peptide (H ₂ N-DAGRCSGRGTCSGGRCCSKYGYCGTGPAYCGLG-COOH; 33 residues) and an extended C-terminal prosequence (67 residues).

Similarly, pSm-AMP-2 contains the following components: N-terminal signal peptide (22 residues), mature Sm-AMP-2.1a peptide (H ₂ N-YDPNGKCGRQYGKCRAGQCCSQYGYCGSGSKYCAH-COOH; 35 residues), short spacer sequence, 10 residues) (10 mature Sm-AMP-2.2a peptide (H ₂ N-AAGQCYRGRCSGGLCCSKYGYCGSGPAYCGLG-COOH; 32 residues) and an extended C-terminal sequence (64 residues).

Thus, sm-amp-1 simultaneously encodes the mature peptides Sm-AMP-1.1a and Sm-AMP-1.2a (and their derivatives), and sm-amp-2 encodes the Sm-AMP-2.1a and Sm-AMP-2.2 a (and their derivatives).

Claims (1)

The nucleotide sequence encoding antifungal peptides of the stellate Slellaria media ':
5'-X ₁ ATAAACATAAASSTTASATASAAAAGCTAGTCACAH AAAAATSAAGTGAAAATGTTGAACATGAAGAGCTTTGCACTTX ₂ CGX ₃ TTATGCTATTCGCCACCCTAGTAGGGGTGACAATAGCATX _{4 5 6} CCX AATGGCX AGTGTGGTSKH _{7 8 9} TX GGTH ATGTSGTGH _{10 11 12} SH ₁₃ SGGH GTGTTGSTSSSAATATGGATATTGTGGTTC AGGCX ₁₄ CTAAGTACTGX GCCCACAACACTCCTCTTTCTGAGATTGAGCCTACTGX _{15 16 17} CGCX GGCCX GTGTX _{18 19 20} TX CGCGGAAX ATGTAGTGGTGGGCX _{21 22 23} TTGTTGCTCCAAATATGGATATTGTGGGAX GGCCCTGCGTACTGCGGACTAGGCATGTGCCAGGGCAGCTGTGCCTGATATGCCTAATCACCCAGCTCAGATCCAAGCTCGGACTGAAGCTGCTCAGGCTGAAGCTCAGGCTGAGGCTTATAACCAX GCAAATGAAGCTGCTCAAGTTGAGGCTTATTAX _{24 25 26} CAGX GCTCAGACACAGGCTCAACCTCAGGTTGAGCCTGCTGTX ₂₇ ACCAAAGCTCCTTGAAAGCTGGCX TAX _{28 29 30} CTATGGGTTACGTGGCAAATAAACTTTGCTGGTTAATTGTAGTTAATTAGX TTCAAGGTTAAATCGATTGCATGCATGTTAGTACGX ₃₁ TAGATAGATGGTTAAATAAGTTATGC ATGC AAGTGTX CTTTGX _{32 33 34} TTATX TTTTGTTX GTCATTTTCCTTTAX _{35 36 37} TGTAATAGX TCGATCGTATTTCX ATCATAX _{38 39 40} ACAAGTCTAX GGAATGAAATAAAGAGTGCCACX TTX _{41 42 43} TX AAAAA AAAAAAAAAAAAAAAAH ₄₄ -3 ',
where X _{1 is} absent (for sm-amp-1) or = ATS (sm-amp-2), X ₂ = A (sm-amp-1) or CT (sm-amp-2), X ₃ = C (sm -amp-2) or G (sm-amp-1), X ₄ = A (sm-amp-2) or C (sm-amp-1), X ₅ = AC (sm-amp-2) or GT ( sm-amp-1), X ₆ = A (sm-amp-2) or C (sm-amp-1), X ₇ = A (sm-amp-2) or C (sm-amp-1), X ₈ = CGGGC (sm-amp-1) or GTCAG (sm-amp-2), X ₉ = AT (sm-amp-2) or GG (sm-amp-1), X ₁₀ = AA (sm-amp- 2) either GG (sm-amp-1), X ₁₁ = C (sm-amp-2) or G (sm-amp-1), X ₁₂ = A (sm-amp-2) or G (sm-amp -1), X ₁₃ = A (sm-amp-2) or T (sm-amp-1), X ₁₄ = C (sm-amp-1) or T (sm-amp-2), X ₁₅ = C (sm-amp-1) or T (sm-amp-2), X ₁₆ = A (sm-amp-1) or C (sm-amp-2), X ₁₇ = A (sm-amp-2) or T (sm-amp-1), X ₁₈ = A (sm-amp-2) or G (sm-amp-1), X ₁₉ = AG (sm-amp-1) or TA (sm-amp-2) , X _{20 is} absent (sm-amp-2) or = GGA (sm-amp-1), X ₂₁ = C (sm-amp-1) or G (sm-amp-2), X ₂₂ = G (sm- amp-1) or T (sm-amp-2), X ₂₃ = CT (sm-amp-1) or G C (sm-amp-2), X ₂₄ = A (sm-amp-2) or G (sm-amp-1), X ₂₅ = C (sm-amp-2) or T (sm-amp-1) , X _{26 is} absent (sm-amp-2) or = GCAACTCAG (sm-amp-1), X ₂₇ = C (sm-amp-2) or T (sm-amp-1), X ₂₈ = A (sm- amp-2) or G (sm-amp-1), X ₂₉ = A (sm-amp-1) or G (sm-amp-2), X ₃₀ missing (sm-amp-1) or = T (sm -amp-2), X _{31 is} missing (sm-amp-2) or = TAGATAC (sm-amp-1), X ₃₂ = A (sm-amp-2) or G (sm-amp-1), X ₃₃ = C (sm-amp-1) or T (sm-amp-2), X ₃₄ = C (sm-amp-2) or G (sm-amp-1), X ₃₅ = C (sm-amp-1 ) or T (sm-amp-2), X ₃₆ = A (sm-amp-1) or C (sm-amp-2), X ₃₇ = A (sm-amp-2) or C (sm-amp- 1), X ₃₈ = A (sm-amp-1) or G (sm-amp-2), X ₃₉ = A (sm-amp-1) or C (sm-amp-2), X ₄₀ = C ( sm-amp-2) or T (sm-amp-1), X ₄₁ = C (sm-amp-2) or G (sm-amp-1), X ₄₂ = CAT (sm-amp-1) or TCA (sm-amp-2), X ₄₃ = A (sm-amp-1) or G (sm-amp-2), X _{44 is} missing (sm-amp-2) or = AAAAA (sm-amp-1).