阅读说明：本技术 一种辣椒疫霉侵染植物相关蛋白及其应用 (Phytophthora capsici infected plant-related protein and application thereof ) 是由 张修国 李京 朱春原 艾聪聪 于 2019-08-21 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中第19-384位的蛋白质；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90％以上的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 of which the amino acid sequence is the sequence 1 in the sequence table or the protein of which the amino acid sequence is the 19 th to 384 th sites in the sequence table 1; 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 more than 90% of 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 following protein A1), A2) or A3):

A1) the protein of which the amino acid sequence is the sequence 1 in the sequence table or the protein of which the amino acid sequence is the 19 th to 384 th sites in the sequence table 1;

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 more than 90% of 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).

2. The protein of claim 1, wherein a2 is a21, a22, a23, a24, a25, or a26 wherein:

a21, protein with amino acid sequence shown as sequence 3;

a22, protein with the amino acid sequence shown as 19 th to 384 th positions in the sequence 3;

a23, protein with amino acid sequence shown as sequence 3;

a24, protein with the amino acid sequence shown as 19 th to 384 th positions in the sequence 3;

a25, protein with amino acid sequence shown as sequence 3;

a26, the amino acid sequence of which is shown as 19 th to 384 th positions in the sequence 3.

3. The protein of claim 1, wherein the fusion protein in A3 is A31, A32, A33 or A34, wherein:

a31, protein with amino acid sequence shown as sequence 9;

a32, the amino acid sequence of which is shown as 19 th to 394 th amino acid sequence in the sequence 9;

a33, protein with amino acid sequence shown as sequence 11;

a34, the amino acid sequence of which is shown as 19 th to 398 th positions 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 B9):

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, B110, B111 and B112:

b101, cDNA molecules or DNA molecules of a sequence 2 in a sequence table;

b102, cDNA molecules or DNA molecules of 55 th to 1152 th sites in a sequence 2 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 of 55 th to 1152 nd site in a 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 55 th to 1152 th sites in a sequence 6 in a sequence table;

b107, cDNA molecules or DNA molecules of a sequence 8 in a sequence table;

b108, cDNA molecules or DNA molecules of 55 th to 1152 th sites in a sequence 8 in a sequence table;

b109, cDNA molecules or DNA molecules of a sequence 10 in a sequence table;

b110, a cDNA molecule or a DNA molecule of 55 th to 1182 th in a sequence 10 in a sequence table;

b111, cDNA molecules or DNA molecules of a sequence 12 in a sequence table;

b112, cDNA molecules or DNA molecules of 55 th to 1194 th in a sequence 10 in a sequence table.

6. 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.

7. Use according to claim 6, characterized in that: the plant of claim 6, which is a monocotyledonous plant or a dicotyledonous plant.

8. 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 areas, 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 successively produce sporangia and zoospores, which are spread by wind, rain, soil, etc., with multiple reinfestations (Ristaino et al, 1991; Ristain et al, 1992; Ristain0et 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 inhibiting phytophthora capsici from infecting plants and/or providing phytophthora capsici from infecting plant-related proteins.

The invention provides a protein, which is the following protein A1), A2) or A3):

A1) the amino acid sequence is protein of sequence 1 in a 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 more than 90% of 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).

Wherein the A2 is A21, A22, A23, A24, A25 or A26 as follows, wherein:

a21, protein with amino acid sequence shown as sequence 3;

a22, protein with the amino acid sequence shown as 19 th to 384 th positions in the sequence 3;

a23, protein with amino acid sequence shown as sequence 3;

a24, protein with the amino acid sequence shown as 19 th to 384 th positions in the sequence 3;

a25, protein with amino acid sequence shown as sequence 3;

a26, the amino acid sequence of which is shown as 19 th to 384 th positions in the sequence 3.

A21 is obtained by replacing GAL of the amino acids at the 34 th to the 36 th positions in the sequence 1 with DTD, T of the amino acid at the 41 th position with A, T of the amino acid at the 46 th position with N, K of the amino acid at the 93 rd position with R, M of the amino acid at the 320 th position with P, and keeping the other amino acid sequences in the sequence 1 unchanged;

a23 is obtained by replacing 34 th to 36 th amino acids of the sequence 1 from GAL to DTD, replacing 41 th amino acid from T to A, replacing 46 th amino acid from T to N, and replacing 93 rd amino acid from K to R, and keeping other amino acid sequences of the sequence 1 unchanged;

a25 is the substitution of GAL to DTD for the amino acids at positions 34-36 in the sequence 1, the substitution of T to A for the amino acid at position 41, the substitution of T to N for the amino acid at position 46, and the substitution of K to R for the amino acid at position 93; the amino acid sequence of SEQ ID No. 1 was maintained by replacing the amino acid at position 221 with K and the amino acid at position 284 with Q and R.

Wherein the fusion protein in A3 is A31, A32, A33 or A34, wherein:

a31, protein with amino acid sequence shown as sequence 9;

a32, the amino acid sequence of which is shown as 19 th to 394 th amino acid sequence in the sequence 9;

a33, protein with amino acid sequence shown as sequence 11;

a34, the amino acid sequence of which is shown as 19 th to 398 th positions in the sequence 11.

The invention also provides a biological material related to the protein, which is any one of the following B1) to B9):

B1) a nucleic acid molecule encoding the protein of claim 1;

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 sequence 2 in a sequence table;

b102, cDNA molecules or DNA molecules of 55 th to 1152 th sites in a sequence 2 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 of 55 th to 1152 nd site in a 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 55 th to 1152 th sites in a sequence 6 in a sequence table;

b107, cDNA molecules or DNA molecules of a sequence 8 in a sequence table;

b108, cDNA molecules or DNA molecules of 55 th to 1152 th sites in a sequence 8 in a sequence table;

b109, cDNA molecules or DNA molecules of a sequence 10 in a sequence table;

b110, a cDNA molecule or a DNA molecule of 55 th to 1182 th in a sequence 10 in a sequence table;

b111, cDNA molecules or DNA molecules of a sequence 12 in a sequence table;

b112, cDNA molecules or DNA molecules of 55 th to 1194 th in a sequence 10 in a sequence table.

Any of the following applications of the protein, the biomaterial, P1-P9 should also be within the scope of the present invention:

use of P1, the protein of claim 1, or the biomaterial of claim 2 or 3 for modulating leaf cell necrosis in a plant;

use of P2, a protein according to claim 1, or a biomaterial according to claim 2 or 3 in the manufacture of a product for reducing necrosis of plant leaves;

use of P3, the protein of claim 1, or the biomaterial of claim 2 or 3 for growing plants that reduce leaf necrosis;

use of P4, the protein of claim 1, or the biomaterial of claim 2 or 3 for the manufacture of a product for reducing leaf necrosis in plants;

use of P5, a protein according to claim 1, or a biomaterial according to claim 2 or 3 for modulating phytophthora capsici infestation in plants;

use of P6, a protein according to claim 1, or a biomaterial according to claim 2 or 3 for the manufacture of a product for increasing the resistance of plants to phytophthora capsici infestation;

use of P7, a protein according to claim 1, or a biomaterial according to claim 2 or 3 for growing plants resistant to phytophthora capsici infestation;

use of P8, a protein according to claim 1, or a biomaterial according to claim 2 or 3 for the preparation of a product for combating phytophthora capsici infestation in plants;

use of P9, the protein of claim 1, or the biological material of claim 2 or 3 in plant breeding.

Wherein the plant is a monocotyledon or a dicotyledon.

The plant of claim 4, wherein said plant is Nicotiana benthamiana or Capsicum annuum.

The invention has the advantages that the RxLR23 can not only cause slight cell necrosis on the Shinikin, but also cause relatively obvious necrosis on the hot pepper, and the subsequent experimental results show that the RxLR23 starts to have the symptom of necrosis about 1-3 days after the Shinikin and the hot pepper are instantaneously expressed, and about 3-5 days can form grey-brown necrotic spots on the hot pepper leaves, and the necrotic spots are not enlarged and belong to relatively obvious cell death reaction. The homologous genes of RxLR23, RxLR3-1, RxLR6-2 and RxLR8-2 can also cause the necrosis of the leaves of Nicotiana benthamiana and capsicum, but the functions of the homologous genes are different from those of RxLR23 due to the change of individual amino acid sites; the RxLR23 homologous gene is changed in the front-segment amino acid sequence, so that the subcellular localization of the homologous gene is changed to a certain extent; the early expression level of the effector RxLR23 in plants is higher. The function of causing cell necrosis by RxLR23 is mainly concentrated in cell nucleus, the difference of RxLR23 nuclear localization has influence on the infection of phytophthora capsici, and the difference of the corresponding fusion protein RxLR23NLS and RxLR23NES has certain influence on the infection of the phytophthora capsici, for example, the fusion protein RxLR23NLS can inhibit the infection of the phytophthora capsici. 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 the results of PCR electrophoresis, wherein M: marker 2000; lanes 1-2: RxLR 23; lanes 3-4: RxLR 3-1; lanes 5-6: RxLR 6-2; lanes 7-8: RxLR 8-2;

FIG. 2 is a diagram showing the result of PCR electrophoresis, wherein M: marker 2000; lanes 1-2: RxLR 23; lanes 3-4: RxLR 3-1; lanes 5-6: RxLR 6-2; lanes 7-8: RxLR 8-2; lanes 9-10: RxLR23: NES;

FIG. 3 is an analysis graph of expression pattern of RxLR 23;

FIG. 4 is a protein expression diagram of Western blot detection of effector genes;

FIG. 5 pathogenic function analysis of RxLR effector on Nicotiana benthamiana, wherein panel a: 1: RxLR3-1, 2: RxLR23, 3: INF1, 4: no-load GFP, 5: MgCl₂(ii) a And (b) figure: 1: RxLR6-2, 2: RxLR23, 3: INF1, 4: no-load GFP, 5: MgCl₂(ii) a And (c) figure: 1: RxLR8-2, 2: INF1, 3: no-load GFP, 4: MgCl₂；

FIG. 6 statistics of necrotic area of effector-inoculated B.benthamiana leaves;

FIG. 7 is a graph of the pathogenic function of RxLR effector on Capsicum annuum, wherein graph a: 1: RxLR23, 2: INF1, 3: pBIN-GFP, 4: MgCl₂(ii) a And (b) figure: 1: RxLR3-1, 2: RxLR23, 3: INF1, 4: no-load GFP, 5: MgCl₂(ii) a And (c) figure: 1: RxLR6-2, 2: RxLR23, 3: INF1, 4: GFP; 5: MgCl₂(ii) a FIG. d: 1: RxLR8-2, 2: RxLR23, 3: INF1, 4: pBIN-GFP, 5: MgCl₂；

FIG. 8 statistics of necrotic area of effector-inoculated pepper leaves;

FIG. 9. Effect of Effector on Phytophthora capsici infection status;

FIG. 10 statistics of the infected area of effector zoospores

FIG. 11 fluorescent quantitation of effector versus Phytophthora capsici infection q-pcr;

FIG. 12 study of the pathogenic function of RxLR effector on pepper, wherein panel e: 1: RxLR NLS, 2: RxLR23, 3: INF1, 4: pBIN-GFP, 5: MgCl₂. FIG. f：1：RxLR:NES，2：RxLR23，3： INF1，4：pBIN-GFP，5：MgCl₂；

FIG. 13 statistics of necrotic area of effector-inoculated pepper leaves;

FIG. 14 Western blot detection of protein expression of effector genes;

FIG. 15. Effect of Effector on Phytophthora capsici infection;

FIG. 16 area statistics of Phytophthora capsici infestation with effector factors;

FIG. 17 fluorescent quantitation of effector versus Phytophthora capsici infection q-pcr.

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 PlantNucluus", 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 cloning of ligands which regulated ROS-Mediated Defense Response ArgRNA", available from the teaching laboratory of the sinus tract), the experimental Vector pEASY-T3 clone Vector, was purchased from Beijing university Ginkyun Biotech Ltd, the plant expression Vector pBIN-GFP2 (available from the teaching laboratory of the sinus tract Posth).

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 SD33) (Yong Jian Jia, Bao Zhen Feng, Wen XiuuSun and Xiu Guo Zhuang, J.Phytopathol,157:585 591,2009) was publicly available from Shandong university of agriculture, and was used only for repeating the relevant experiments of the present invention and was not used for other purposes.

The biological material can be obtained from Shandong agricultural university by the public, wherein the biological material is only used for repeating relevant experiments of the invention and cannot be used for other purposes.

Primers involved in specific examples of the invention are shown in table 1:

table 1 introduction table: