Related protein for inhibiting plant leaf necrosis caused by Bax and application thereof
阅读说明：本技术 一种抑制Bax引起的植物叶片坏死的相关蛋白及其应用 (Related protein for inhibiting plant leaf necrosis caused by Bax and application thereof ) 是由 张修国 李京 朱春原 艾聪聪 于 2019-09-24 设计创作，主要内容包括：本发明涉及生物技术领域,具体的说涉及一种辣椒疫霉侵染植物相关蛋白及其应用。所述蛋白质,是如下A1)、A2)或A3)的蛋白质：A1)氨基酸序列是序列表中序列1的蛋白质或氨基酸序列是序列表1的蛋白质；A2)将序列表中序列1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与A1)所示的蛋白质具有同一性且与叶片细胞坏死和/或辣椒疫霉侵染相关的蛋白质；A3)在A1)或A2)的N末端或/和C末端连接蛋白标签得到的融合蛋白质。所述蛋白质及其应用能够为有效利用植物的抗病机制设计防治辣椒疫病的新策略提供了理论依据。(The invention relates to the technical field of biology, in particular to phytophthora capsici infected plant related protein and application thereof. The protein is the protein of A1), A2) or A3) as follows: A1) the protein with the amino acid sequence of the sequence 1 in the sequence table or the protein with the amino acid sequence of the sequence 1 in the sequence table; A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 1 in the sequence table, has identity with the protein shown in A1), and is related to leaf cell necrosis and/or phytophthora capsici infection; A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2). The protein and the application thereof can provide theoretical basis for designing a new strategy for preventing and treating the pepper phytophthora blight by effectively utilizing the disease resistance mechanism of plants.)
1. The protein is the protein of A1), A2), A3) or A4) as follows:
A1) the protein with the amino acid sequence of the sequence 1 in the sequence table or the protein with the amino acid sequence of the sequence 1 in the sequence table;
A2) protein which comprises an amino acid sequence shown in a sequence 1 in a sequence table and is related to leaf cell necrosis and/or phytophthora capsici infection;
A3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of A2), has the same property with the protein shown in A1), and is related to leaf cell necrosis and/or phytophthora capsici infection;
A4) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).
2. The protein of claim 1, wherein a2) is a21 or a22 wherein:
a21, protein with amino acid sequence shown as sequence 3;
a22, the amino acid sequence of which is shown as 16 th to 329 th positions in the sequence 3.
3. The protein of claim 1, wherein the fusion protein in A4) is A41, A42, A43 or A44, wherein:
a41, protein with amino acid sequence shown as sequence 5;
a42, protein with amino acid sequence shown as sequence 9;
a43, protein with amino acid sequence shown as sequence 7;
a44, protein with amino acid sequence shown as 16 th-340 th position in sequence 7;
a45, protein with amino acid sequence shown as sequence 11;
a46, the amino acid sequence of which is shown as 16 th-343 th position in the sequence 11; .
4. The biomaterial related to the protein of any one of claims 1 to 3, which is any one of the following B1) to B7):
B1) a nucleic acid molecule encoding the protein of any one of claims 1-3;
B2) an expression cassette comprising the nucleic acid molecule of B1);
B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);
B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;
B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;
B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);
B7) a transgenic plant organ containing B1) the nucleic acid molecule or a transgenic plant organ containing B2) the expression cassette.
5. The related biological material according to claim 4, wherein: B1) the nucleic acid molecule has the following nucleotides of B101, B102, B103, B104, B105, B106, B107, B108, B109 and B109:
b101, cDNA molecules or DNA molecules of a sequence 2 in a sequence table;
b102, cDNA molecules or DNA molecules of a sequence 10 in a sequence table;
b103, cDNA molecules or DNA molecules of a sequence 4 in a sequence table;
b104, is a cDNA molecule or a DNA molecule at the 46 th to the 987 th site in the sequence 4 in the sequence table;
b105, cDNA molecules or DNA molecules of a sequence 6 in a sequence table;
b106, cDNA molecules or DNA molecules of a sequence 8 in the sequence table;
b107, cDNA molecules or DNA molecules of 46 th to 1020 th sites in a sequence 8 in a sequence table;
b108, cDNA molecules or DNA molecules of a sequence 12 in a sequence table;
b109, and a cDNA molecule or a DNA molecule at the 46 th to the 1032 th position in the sequence 12 in the sequence table.
6. A method for inhibiting phytophthora capsici infection in a plant, comprising introducing into the plant a recombinant vector comprising B101, B105, B106 or B107 as defined in claim 5 or introducing into the plant a recombinant microorganism comprising B101, B105, B106 or B107 as defined in claim 5.
7. The method of inhibiting phytophthora capsici from infecting a plant according to claim 6, for use in regulating phytophthora capsici infection in a plant, preparing a product for the plant to resist phytophthora capsici infection, growing a plant to resist phytophthora capsici infection, or breeding a plant.
8. Use of any one of the following P1-P9 of the protein of any one of claims 1-3, or the biomaterial of claim 4 or 5:
use of P1, a protein according to any one of claims 1 to 3, or a biomaterial according to claim 4 or 5 for modulating leaf cell necrosis in a plant;
use of P2, a protein according to any one of claims 1 to 3, or a biomaterial according to claim 4 or 5 in the manufacture of a product for reducing necrosis of plant leaves;
use of P3, a protein according to any one of claims 1 to 3, or a biomaterial according to claim 4 or 5 for the cultivation of a plant for the reduction of leaf necrosis in a plant;
use of P4, a protein according to any one of claims 1 to 3, or a biomaterial according to claim 4 or 5 for the manufacture of a product for reducing leaf necrosis in a plant;
use of P5, a protein according to any one of claims 1 to 3, or a biomaterial according to claim 4 or 5 for modulating phytophthora capsici infestation in plants;
use of P6, a protein according to any one of claims 1 to 3, or a biomaterial according to claim 4 or 5 in the manufacture of a product for increasing the resistance of a plant to phytophthora capsici infestation;
use of P7, a protein according to any one of claims 1 to 3, or a biomaterial according to claim 4 or 5 for growing plants resistant to phytophthora capsici infestation;
use of P8, a protein according to any one of claims 1 to 3, or a biomaterial according to claim 4 or 5 for the preparation of a product for combating phytophthora capsici infestation in plants;
use of P9, a protein according to any one of claims 1 to 3, or a biological material according to claim 4 or 5 in plant breeding.
9. Use according to claim 6, characterized in that: the plant of claim 6, which is a monocotyledonous plant or a dicotyledonous plant.
10. Use according to claim 6, characterized in that: the plant of claim 6, which is Nicotiana benthamiana or Capsicum annuum.
Technical Field
The invention relates to the technical field of biology, in particular to phytophthora capsici infected plant related protein and application thereof.
Background
Phytophthora capsici belongs to the genus oomycete, and is a destructive filamentous plant pathogen with relatively strong pathogenicity in plants, particularly dicotyledonous plants (Margulis and Schwartz, 2000).
Phytophthora capsici belongs to the order of Oomycetes of Oomycota of the order of Peronosporales of the family Phytophthora,
phytophthora capsici usually overwinter with oospores in soil or disease residues, and diseases are transmitted by wind, water and other agricultural activities (zheng et al, 2007). After the disease occurs, new sporangia can be generated, and zoospores are formed for re-infection. The temperature range of germ growth is 10-37 deg.C, and the optimum temperature is 20-30 deg.C. Under the conditions of continuous cropping, low-lying land, poor drainage and the like, the regions with overlarge density, weak plants and the like are all beneficial to the occurrence and spread of the disease.
The survival time of pathogenic oospores of the pepper phytophthora blight can reach about 3 years at most, and pathogenic bacteria mainly live through the winter in soil and on diseased residues in the form of the oospores so as to pass seasons with low temperature and unsuitable environment. After spring comes, the oospores will begin to germinate under appropriate temperature and humidity conditions, and zoospores can be rapidly produced to invade the roots, stem bases, leaves, etc. of the peppers (Lamour and Hausbeck, 2001). During the growth of pepper plants, pathogenic bacteria will subsequently produce sporangia and zoospores, which are spread by wind, rain, soil, etc., with multiple reinfestations (Ristaino et al, 1991; Ristain et al, 1992; Ristain0 et al, 1993; Schlub et al, 1983; Springer et al, 1982).
The current prevention and treatment work of the pepper phytophthora blight is mainly based on the traditional chemical agent prevention and treatment. The control method has the advantages that the series of problems of heavy metal exceeding and pesticide residue caused by phytophthora capsici brings great threat to human health and environmental safety.
Disclosure of Invention
The invention aims to solve the technical problem of how to inhibit plant leaf necrosis caused by Bax and/or how to inhibit phytophthora capsici from infecting plants.
The protein is the following protein A1), A2) or A3):
A1) the protein with the amino acid sequence of the sequence 1 in the sequence table or the protein with the amino acid sequence of the sequence 1 in the sequence table;
A2) protein which comprises an amino acid sequence shown in a sequence 1 in a sequence table and is related to leaf cell necrosis and/or phytophthora capsici infection;
A3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of A2), has the same property with the protein shown in A1), and is related to leaf cell necrosis and/or phytophthora capsici infection;
A4) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).
Wherein the A2 is A21 or A22 as follows:
a21, protein with amino acid sequence shown as sequence 3;
a22, the amino acid sequence of which is shown as 16 th to 329 th positions in the sequence 3;
the protein in A3 can be homologous proteins RxLR101pi and RxLR101pp of a21, and the results of homology alignment with a21(RxLR101) are shown in fig. 1.
Wherein the fusion protein in A4 is A41, A42, A43 or A44, wherein:
a41, protein with amino acid sequence shown as sequence 5;
a42, protein with amino acid sequence shown as sequence 9;
a43, protein with amino acid sequence shown as sequence 7;
a44, protein with amino acid sequence shown as 16 th-340 th position in sequence 7;
a45, protein with amino acid sequence shown as sequence 11;
a46, the amino acid sequence of which is shown as 16 th-343 th position in the sequence 11.
The invention also provides a biological material related to the protein, which is any one of the following B1) to B7):
B1) a nucleic acid molecule encoding the protein of any one of claims 1-3;
B2) an expression cassette comprising the nucleic acid molecule of B1);
B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);
B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;
B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;
B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);
B7) a transgenic plant organ containing B1) the nucleic acid molecule or a transgenic plant organ containing B2) the expression cassette.
Wherein, B1) is the nucleic acid molecule with the nucleotide of R as follows B101, B102, B103, B104, B105, B106, B107, B108, B109, B110, B111 and B112:
b101, cDNA molecules or DNA molecules of a
b102, cDNA molecules or DNA molecules of a sequence 10 in a sequence table;
b103, cDNA molecules or DNA molecules of a sequence 4 in a sequence table;
b104, is a cDNA molecule or a DNA molecule at the 46 th to the 987 th site in the sequence 4 in the sequence table;
b105, cDNA molecules or DNA molecules of a sequence 6 in a sequence table;
b106, cDNA molecules or DNA molecules of a
b107, cDNA molecules or DNA molecules of 46 th to 1020 th sites in a
b108, cDNA molecules or DNA molecules of a sequence 12 in a sequence table;
b109, and a cDNA molecule or a DNA molecule at the 46 th to the 1032 th position in the sequence 12 in the sequence table.
A method for inhibiting phytophthora capsici from infecting a plant, comprising introducing into the plant a recombinant vector containing said B101, B105, B106 or B107 or introducing into the plant a recombinant microorganism containing said B101, B105, B106 or B107.
The method for inhibiting phytophthora capsici from infecting plants is applied to regulation and control of phytophthora capsici infection of plants, preparation of products for resisting phytophthora capsici infection of plants, cultivation of plants resisting phytophthora capsici infection or plant breeding.
Any one of the following P1-P9 uses of the protein, or the biomaterial:
the use of P1, the protein, or the biomaterial for modulating plant leaf cell necrosis;
use of P2, the protein, or the biomaterial in the manufacture of a product for reducing necrosis of plant leaves;
use of P3, said protein, or said biological material for growing plants that reduce necrosis of plant leaves;
the use of P4, the protein, or the biological material for the manufacture of a product for reducing plant leaf necrosis;
use of P5, said protein, or said biomaterial for modulating phytophthora capsici infestation in a plant;
the use of P6, the protein, or the biomaterial in the manufacture of a product for increasing the resistance of a plant to phytophthora capsici infestation;
the use of P7, said protein, or said biological material for growing plants resistant to phytophthora capsici infestation;
the application of P8, the protein or the biological material in preparing a product for resisting phytophthora capsici infection of plants;
use of P9, the protein, or the biological material in plant breeding.
Wherein the plant is a monocotyledon or a dicotyledon.
Wherein the plant is Nicotiana benthamiana or Capsicum annuum.
The invention has the beneficial effects that RxLRnudix (protein A1)) and RxLR101 (protein A2)) in the invention can inhibit PCD induced by Bax, and RxLR101-P.p and RxLR101-P.i (protein A3)) can also inhibit PCD induced by Bax. RxLRnudix (protein A1)) and RxLR101-NLS fused with RxLRnudix-NLS (protein A4)) can more strongly inhibit the infection of phytophthora capsici zoospores. Thereby providing a theoretical basis for designing a new strategy for preventing and treating the pepper phytophthora blight by effectively utilizing the disease resistance mechanism of the plant.
Drawings
FIG. 1 is a diagram showing an alignment of homologous gene sequences;
FIG. 2.RxLR101 truncated mode diagram
FIG. 3. pathogenicity analysis of RxLR effector molecules on Nicotiana benthamiana, FIG. 3a is a functional verification diagram of RxLRnudix, wherein A represents RxLR101, B represents RxLRnudix, and C represents buffer; d represents the empty vector pBIN-GFP2, and E represents INF 1. FIG. 3b is a functional verification diagram of RxLR101-P.i and RxLR101-P.p, wherein A represents unloaded pBIN-
FIG. 4 shows inhibition analysis of RXRR, in 4a, A represents RxLR101, B represents RxLR101-P.p, RxLR101-P.i, C represents buffer, and D represents empty vector pBIN-
FIG. 5 Phytophthora capsici zoospore infestation of RxLR101 and-RxLRnudix in example 3
FIG. 6 qPCR detection of RxLR101 and-RxLRnudix in example 3
FIG. 7 inhibition assay of RxLR101 fusion proteins, wherein A represents RxLR101-NES, RxLRnudix-NES, B represents RxLR101-NLS, RxLRnudix-NLS, CRxLR101, D-unloaded pBIN-GFP2, E represents RxLRnudix, and F represents buffer, respectively. The right panel shows trypan blue staining results.
FIG. 8 shows the results of infection of Phytophthora capsici zoospores with RxLR101-NES/NLS in example 5
FIG. 9 results of RxLR101-NES/NLSqPCR detection in example 5
FIG. 10. results of example 5 infection of RxLRnudix-NES/NLS with Phytophthora capsici zoospores
FIG. 11 shows the results of RxLRnudix-NES/NLSqPCR assay in example 5.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Vectors and strains
The cloning Vector used for the experiments, pEASY-T3 clone Vector, was purchased from Beijing Quanyujin Biotechnology Ltd, the plant expression Vector pBIN-GFP2 (disclosed in "A Virus Essential CRN efficiency of Phytophtora capsaici supress feedbacks Defence and indecs Cell Death in plantarnulus", publicly available from the teaching laboratory of the sinus tract dragon), pYF2-PsNLS-hSpCas9, pYF2.3G-Ribo-sgRNA (disclosed in "A Phytophta capsaici efficiency Targets ACD11 binding clones of primers which regulated ROS-Mediated feedback in Argpsis", available from the teaching laboratory of the sinus tract), the technology pBIIKS + platinum.
Escherichia coli (Escherichia coli) DH 5. alpha. competent cells were purchased from Kyoto Kogyo gold Biotechnology Ltd, and Agrobacterium (Agrobacterium tumefaciens) strain GV3101 was purchased from TransGen Biotechnocroration.
Phytophthora capsici strain SD33(Phytophthora capsici) (Yong Jian Jia, Bao Zhen Feng, Wen Xiu Sun and Xiu Guo Zhuang, J. phytophathol, 157:585-591,2009) is publicly available from Shandong university of agriculture, and is only used for repeating the relevant experiments of the present invention and is not used for other purposes.
Bax Agrobacterium strains (publicly available from professor sinus professor laboratories, Nanjing university of agriculture, sinus tract), disclosed in the literature "functional analysis of 6 RxLR of Phytophthora capsici and 1 CRN effector of Phytophthora sojae").
The biological material can be obtained from Shandong agricultural university by the general university of Phytophthora capsici Jul0201 and Phytophthora capsici Aug0202 (2008), and is only used for repeating the relevant experiments of the invention and can not be used for other purposes. Primers involved in specific examples of the invention are shown in table 1:
table 1 introduction table:
