阅读说明：本技术 一种辣椒疫霉效应蛋白及其编码基因和应用 (Phytophthora capsici effector protein and coding gene and application thereof ) 是由 张修国 李京 朱春原 艾聪聪 于 2019-09-20 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效应蛋白及其编码基因和应用,所述辣椒疫霉效应蛋白具有如序列1所述的氨基酸序列。本发明的辣椒疫霉效应蛋白RxLR98344可以抑制Bax所引起的细胞程序性死亡,但不能抑制INF1、CRN4引起的细胞死亡。(The invention relates to the technical field of biology, in particular to phytophthora capsici effector protein and a coding gene and application thereof, wherein the phytophthora capsici effector protein has an amino acid sequence shown as a sequence 1. The phytophthora capsici effector protein RxLR98344 can inhibit apoptosis caused by Bax, but cannot inhibit cell death caused by INF1 and CRN 4.)

1. A protein, wherein the protein is a1) or a2), wherein:

A1) the amino acid sequence is protein of sequence 1 in a sequence table;

A2) the amino acid sequence is the protein at the 22 th to 185 th positions in the sequence table 1.

2. The protein of claim 1, wherein the protein is derived from Phytophthora capsici.

3. The biomaterial related to the protein of any one of claims 1-2, which is any one of the following B1) to B7):

B1) a nucleic acid molecule encoding the protein of any one of claims 1-2;

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.

4. The related biological material according to claim 3, wherein: B1) the nucleic acid molecule is the nucleotide B11) or B12) as follows:

B11) a cDNA molecule or DNA molecule consisting of the nucleotide of the sequence 2 in the sequence table;

B12) a cDNA molecule or DNA molecule consisting of nucleotides 64 to 555 in a sequence 2 in a sequence table.

5. A method of inhibiting bax-induced plant cell death by expressing the protein of claims 1-2 in a target plant.

6. Use of a protein according to claim 1 or 2, a biological material according to claim 3 or 4 or a method according to claim 5 for inhibiting bax-induced plant cell death, for producing a product for inhibiting bax-induced plant cell death, for growing a plant resistant to bax-induced plant cell death or for plant breeding.

7. The method of claim 6 or the use of claim 6, wherein the plant is a monocot or a dicot.

8. The method of claim 7 or the use of claim 6, wherein the plant is Nicotiana benthamiana or Capsicum annuum.

Technical Field

The invention relates to the technical field of biology, in particular to phytophthora capsici effector protein and a coding gene and application thereof.

Background

Phytophthora capsici (Phytophthora capsicii) is a main pathogenic bacterium causing Phytophthora capsici, and the pathogenic bacterium causing Phytophthora capsici in China belongs to the kingdom of algae (Chromista), Oomycetes (Oomycotes), Peronosporales (Peronosporales), Peronosporaceae (Pythiaceae), Phytophthora (Phytophthora), and Phytophthora capsici (Phytophthora capsicii) (Palim et al, 2012).

Phytophthora capsici is one of the main diseases infecting capsicum, also called black convulsion, and is the main disease in the production and planting process of capsicum (Zhang politician et al, 2006; Zhuzongyuan et al, 1995). The pepper phytophthora blight is distributed and spread all over the world, the occurrence and infection of the pepper phytophthora blight are not limited by geographical environments, and the pepper phytophthora blight has been reported in all continents of Latin America, Europe, Asia and the like (Pengzheng et al, 1991). More than ten national pepper phytophthora blight diseases in the united states, argentina, italy, japan, korea, russia, bulgaria, india, etc. occur universally and cause severe economic losses (zuolin et al, 1999). Since the early Shu of the 20 th century 60 year \32450pepperphytophthora blight is discovered in Jiangsu province, the occurrence of pepper phytophthora blight has been reported in Liaoning province (city), Shanghai province, Jilin province, Shandong province, Xinjiang province, Hunan province, Hubei province, inner Mongolia province, Gansu province, Heilongjiang province, Shaanxi province, Guizhou province, Yunnan province, Guanzhou province and the like, and the disease tends to be aggravated year by year (GauLin et al, 2002). The pepper phytophthora blight is a destructive and traumatic soil-borne disease, and can be attacked in the whole growth and development period of pepper. Phytophthora capsici pathogenic bacteria can seriously damage roots, stems, leaves, flowers and fruits of capsicum, the disease is serious in stem nodes and branch parts, and all the phytophthora capsici pathogenic bacteria are necrotic and broken from the serious disease to the diseased part (Zhang politician, 2006). The pepper phytophthora blight can infect pepper, and can also harm various solanaceous and cucurbitaceous vegetable crops such as watermelon, tomato, eggplant, cucumber, etc.

In long-term plant and pathogen competition, plants have evolved resistance (R) proteins that recognize effectors to combat pathogen infestation. The disease-resistant processes of plants are diverse (Hiller et al, 2004). Disease resistance is often associated with plant perception of signal molecules, i.e., elicitors, produced by avirulent pathogens. These two types of resistance were used to develop studies of potato resistance to late blight race specific or vertical levels of resistance or levels of field resistance. Vertical resistance is based on gene-gene interactions, where pathogen avirulence gene interactions directly or indirectly recognize the major resistance genes (R genes) of plants. The allergic reaction (HR) is turned on and the infected cells undergo programmed cell death together with neighboring cells. This may prevent further growth of biological pathogens. Horizontal or field resistance is referred to as a second type of resistance and is multigenotypic.

The effector in the cytoplasm is a modular protein that constitutes two major functional domains, consisting of an N-terminal region with a highly conserved sequence motif (signal peptide) and an RxLR-dEER motif (arginine, any amino acid, leucine, arginine) that can facilitate secretion and transfer into plant cells. The other is the C-terminal domain carrying the RxLR domain transport effector. The RxLR region plays a role in secretion and targeting, and the C-terminal domain carries effector activity and functions inside plant cells. The RxLR effector Avr3a secreted by Phytophthora infestans is capable of inhibiting allergic cell death of host cells. The main function of secreted effector proteins is therefore to inhibit the cell signal transduction pathway that mediates the plant defense response (Win et al, 2007; Bhattacharjee et al, 2006; Nunes et al, 2007).

The RxLR region plays a role in secretion and targeting and the C-terminal region carries effector activity and is manipulated within plant cells. CRN is a modular protein with effector activity encoded by the C-terminal region.

The RxLR effector has a modular structure, comprising a signal peptide, followed by a relatively conserved RxLR or RxLR variant (RxLR variant, e.g. KxLR or RxLK) motif, which is functionally secreted into the host cell, while the remaining C-terminal domain carries effector activity. In addition, nearly half of the RxLR effectors contain one or more repeat modules consisting of adjacent W, Y and L motifs. In particular, most RxLR proteins have extensive sequence divergence and lack similarity to other known proteins (Guo et al, 2009). An intrinsically disordered protein region is a flexible fragment that lacks stable secondary or tertiary structure under physiological conditions. The presence of flexible fragments within proteins is common in nature and eukaryotic proteomes have been shown to have a higher degree of disordered protein appearance than prokaryotic proteomes. Disordered proteins have their respective biological functions, which are usually associated with signal transduction, recognition and transcriptional regulation (Luofluoring et al, 1994). To date, several classes of RxLR effector crystal structures have been resolved, including ATR1 and ATR13 from h. However, the prior art is not complete in the research of effector proteins in phytophthora capsici, and the pathogenic mechanism and the influence on the cell death mechanism are not clear.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a phytophthora capsici effector protein RxLR98344 and a coding gene and application thereof.

The invention provides a protein, which is A1) or A2), wherein:

A1) the amino acid sequence is protein of sequence 1 in a sequence table;

A2) the amino acid sequence is the protein at the 22 th to 185 th positions in the sequence table 1.

Wherein the protein is derived from phytophthora capsici.

The invention also provides a biological material related to any one of the proteins, which is any one of the following B1) to B7):

B1) a nucleic acid molecule encoding the protein;

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.

Further, B1) the nucleic acid molecule is the following nucleotide B11) or B12), wherein:

B11) a cDNA molecule or DNA molecule consisting of the nucleotide of the sequence 2 in the sequence table;

B12) a cDNA molecule or DNA molecule consisting of nucleotides 64 to 555 in a sequence 2 in a sequence table.

The invention also provides a method for inhibiting cell death of a plant caused by bax, wherein the protein is expressed in a target plant.

In the method, the method for expressing the protein in the target plant is to introduce a nucleic acid molecule encoding the protein into the target plant.

Further, a method for expressing the protein in a target plant is to transfect a recombinant bacterium containing a nucleic acid molecule encoding the protein into the target plant.

The application of the protein, the biological material or the method in inhibiting the plant cell death caused by bax, preparing a product for inhibiting the plant cell death caused by bax, cultivating a plant resistant to the plant cell death caused by bax or plant breeding is also within the protection scope of the invention.

Further, the target plant or plant is a monocotyledon or dicotyledon.

Further, the target plant or plant is Nicotiana benthamiana or Capsicum annuum.

The phytophthora capsici effector protein RxLR98344 of the invention cannot cause cell death of the nicotiana benthamiana leaves, but can inhibit apoptosis caused by Bax.

Drawings

FIG. 1 shows PCR amplification of RxLR98344 gene;

FIG. 2 PCR amplification of recombinant plasmid by colony PCR.

FIG. 3 is a diagram of the verification of the pathogenicity function of RxLR98344 gene in Nicotiana benthamiana;

FIG. 4 is a photograph of Bunshi tobacco leaves expressing the RxLR98344 effector and Bax genes in sequence.

Detailed Description

The present invention is further illustrated by the following examples, which are intended to be illustrative only and not limiting.

List of experimental materials

1. Bacterial species and plasmids

Phytophthora capsici pathogenic strain SD33 (Phytophthora capsici) RxLR effector molecule screening and functional study of RxLR23 "documents disclosed, and available from the key laboratory of vegetable pest biology province of the university of shandong agriculture, college of plant protection, and agriculture); 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"). Escherichia coli DH5 alpha, BL21(DE3), BL21(DE3) PlysS, Rosetta (DE3), Agrobacterium GV3101 competence and pEASY-T3clone Vector were purchased from TransGen Biotech corporation (from building 4 of Toyol International scientific park, Guangzhou, North road 1, Yongzhuantai, Haizi, Beijing); expression vector pET28a (available from Beijing Solebao technologies, Inc.), pBIN-GFP2 vector (disclosed in "A viral Essential CRN Effect of photophthora capsilici Suppopres Host Defence and industries Cell Death in Plant Nucleus" publicly available from the Sphacelotheca sinuosa, publicly available from the Spodoptera sinuosa).

2. Test plant

The present study used seeds of nicotiana benthamiana (Phytophthora capsici) RxLR effector molecule screening and functional study of RxLR23, published in the literature, publicly available from the major laboratories of vegetable pest biology province of the plant protection institute of university of shandong agriculture. The Nicotiana benthamiana is suitable for growing in a constant temperature greenhouse at the temperature of 22 ℃, under the conditions of 16h of illumination and 8h of darkness.