阅读说明：本技术 来源于帚枝霉属真菌的激发子蛋白在防治木薯细菌性萎蔫病中的应用 (Application of elicitor protein derived from mycosphaerella sp in prevention and treatment of bacterial wilt of cassava ) 是由 李超萍 杨扬 时涛 王国芬 蔡吉苗 李博勋 黄贵修 于 2020-06-24 设计创作，主要内容包括：本发明开了一种来源于帚枝霉属真菌的激发子蛋白在防治木薯细菌性萎蔫病的应用。所述应用是用来源于帚枝霉属真菌的激发子蛋白诱导木薯产生对细菌性萎蔫病的抗病性或者提木薯的抗病性中的应用。该激发子蛋白是具有下述氨基酸残基序列之一的蛋白质：1)序列表中的SEQ ID №.1的氨基酸残基序列；2)将序列表中的SEQ ID №.1氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有激发子蛋白功能的由SEQ ID №：1衍生的蛋白质。实验证明,该蛋白激发子SbES可以诱导木薯产生或者提高植物对靶标病原菌的抗性,降低发病率及病情指数,为提高植物抗病性提供了新的途径,在农业生产上具有广阔的应用前景。(The invention discloses application of elicitor protein derived from mycosphaerella sp. The application is the application of elicitor protein derived from the mycosphaerella fungus to induce the cassava to generate disease resistance to bacterial wilt or to improve the disease resistance of the cassava. The elicitor protein is a protein having one of the following amino acid residue sequences: 1) the SEQ ID No. 1 in a sequence table; 2) the SEQ ID No. 1 amino acid residue sequence in the sequence table is substituted and/or lost and/or added by one or more amino acid residues, and the amino acid residue sequence has the function of stimulating the protoprotein and is composed of the amino acid sequence shown in SEQ ID No. 1: 1, or a derivative thereof. Experiments prove that the protein elicitor SbES can induce cassava to generate or improve the resistance of plants to target pathogenic bacteria, reduce the morbidity and disease index, provide a new way for improving the disease resistance of the plants, and has wide application prospect in agricultural production.)

1. An application of elicitor protein from cladosporium endophyticus or a coding gene thereof in inducing cassava to generate disease resistance or improving the disease resistance of the cassava, wherein the elicitor protein from the cladosporium endophyticus is the protein shown in 1) or 2) or 3) as follows:

1) protein consisting of an amino acid residue sequence of SEQ ID No. 1 in a sequence table;

2) protein consisting of 106 th and 387 th amino acid sequences of SEQ ID No. 1 in a sequence table;

3) the amino acid residue sequence described in 1) or 2) is substituted and/or deleted and/or added by one or more amino acid residues, and has the function of elicitor protein, and the protein has the sequence shown in SEQ ID No: 1, or a derivative thereof.

2. The use according to claim 1, wherein the improvement of the disease resistance of cassava is an activity of inducing cassava to develop resistance to bacterial wilt disease caused by Xanthomonas carpet (Xanthomonas axonopodis).

3. An application of elicitor protein from fungus of the genus Scopulariopsis or a coding gene thereof in inducing cassava to generate defense reaction to diseases; the protein is a protein shown in the following 1) or 2) or 3):

1) protein consisting of an amino acid residue sequence of SEQ ID No. 1 in a sequence table;

2) protein consisting of 106 th and 387 th amino acid sequences of SEQ ID No. 1 in a sequence table 1;

3) the SEQ ID No. 1 amino acid residue sequence in the sequence table is substituted and/or lost and/or added by one or more amino acid residues, and the amino acid residue sequence has the function of stimulating the protoprotein and is composed of the amino acid sequence shown in SEQ ID No. 1: 1-derived protein;

the disease is caused by Xanthomonas axonopodis (Xanthomonas axonopodis).

4. An application of elicitor protein from fungus of the genus Scopulariopsis or a coding gene thereof in preparing a medicament for preventing cassava diseases;

the protein is a protein shown in the following 1) or 2) or 3):

1) protein consisting of an amino acid residue sequence of SEQ ID No. 1 in a sequence table;

2) protein consisting of 106 th and 387 th amino acid sequences of SEQ ID No. 1 in a sequence table 1;

3) the SEQ ID No. 1 amino acid residue sequence in the sequence table is substituted and/or lost and/or added by one or more amino acid residues, and the amino acid residue sequence has the function of stimulating the protoprotein and is composed of the amino acid sequence shown in SEQ ID No. 1: 1, or a derivative thereof.

5. Use according to any one of claims 1 to 4, characterized in that: the cDNA nucleotide sequence of the coding gene is shown as the following 1), 2) or 3):

1) SEQ ID No: 2;

2) SEQ ID No: 2 at the 5' end of the sequence at positions 316-1164;

3) SEQ ID No: 4;

4) a nucleotide sequence that can hybridize under stringent conditions with the DNA sequence described in 1) or 2) or 3);

5) a nucleotide sequence which has more than 90 percent of homology with the nucleotide sequence 1) or 2) or 3) and codes protein with the function of an exciton protein.

6. Use according to any one of claims 1 to 4, characterized in that: the nucleotide sequence of the genome gene of the coding gene is shown as the following 1), 2) or 3):

1) SEQ ID No: 3;

2) a nucleotide sequence that can hybridize with the DNA sequence of 1) under strict conditions;

3) and SEQ ID No: 3 has more than 90 percent of homology and the coded protein has the nucleotide sequence of the exciton protein function.

7. The use according to claim 4, wherein the disease is a bacterial wilt disease caused by Xanthomonas carpi (Xanthomonas axonopodis).

8. A method for improving the disease resistance of cassava is to use the following proteins to control plant diseases:

the protein is a protein shown in the following 1) or 2) or 3):

1) protein consisting of an amino acid residue sequence of SEQ ID No. 1 in a sequence table;

2) protein consisting of 106 th and 387 th amino acid sequences of SEQ ID No. 1 in a sequence table;

3) the amino acid residue sequence described in 1) or 2) is substituted and/or deleted and/or added by one or more amino acid residues, and has the function of elicitor protein, and the protein has the sequence shown in SEQ ID No: 1-derived protein;

the plant diseases are cassava bacterial wilt diseases caused by Xanthomonas axonopodis (Xanthomonas axonopodis);

the way of using the protein to control cassava diseases is to spray the protein on cassava or to inject the protein on cassava tissues.

9. The method of claim 8, wherein: the protein is obtained by the following method: transferring the recombinant vector for expressing the protein into pichia pastoris to express to obtain the protein; the recombinant vector is obtained by inserting a DNA fragment shown in a sequence between EcoRI and KpnI enzyme recognition sites of pRICZA to express a protein consisting of the 106 th and 387 th amino acid sequences of SEQ ID No. 1 in a sequence table.

10. The method of claim 9, wherein: the pichia is pichia X-33.

Technical Field

The invention belongs to the field of molecular biology, and relates to an elicitor protein derived from a mycosphaerella and an application of a coding gene of the elicitor protein in prevention and treatment of bacterial wilt of cassava.

Background

The protein elicitors are some proteins secreted by bacteria and fungi, and mainly comprise allergic protein (Harpin), Cryptogein (Cryptogein), Activator (Activator) and the like. Can stimulate plants to obtain resistance by inducing the expression of plant defense related genes and activating defense signal pathways in plants such as salicylic acid and the like, and enhance the autoimmune capacity of the plants to resist the infection of pathogenic microorganisms. Until now, many new elicitors of protein types have been discovered, but there is no report on elicitor proteins and their coding genes that can induce plants to generate disease resistance in M.virescens.

Disclosure of Invention

The laboratory separates the high-efficiency biocontrol strain from brachiaria blades, and the endophytic fungus Sarocladium brachiariae HND5 belongs to the genus Scopulariopsis. When the HND5 strain was subjected to whole genome sequencing and analyzed by bioinformatics, a gene encoding an exo-elicitor was found and identified from its genome, and the elicitor protein encoded by the gene was named SbES.

Based on the above, the invention aims to provide an elicitor protein from a fungus belonging to the genus Scopulariopsis and a coding gene thereof, and application of the elicitor protein to biological control of cassava, in particular application of the elicitor protein and the coding gene thereof to induction of disease resistance of cassava or improvement of disease resistance of cassava, or application to induction of defense reaction of rubber, cassava, bananas and cucumbers against diseases and insect pests. The elicitor protein is derived from a protein of a Sarocladium brachiariae (endophytic fungi) strain HND5, named SbES, and is 1) or 2) or 3) below:

1) protein consisting of an amino acid residue sequence of SEQ ID No. 1 in a sequence table;

2) protein consisting of 106 th and 387 th amino acid sequences of SEQ ID No. 1 in a sequence table;

3) the SEQ ID No. 1 amino acid residue sequence in the sequence table is substituted and/or lost and/or added by one or more amino acid residues, and the amino acid residue sequence has the function of stimulating the protoprotein and is composed of the amino acid sequence shown in SEQ ID No. 1: 1, or a derivative thereof.

SEQ ID No. 1 in the sequence table is composed of 387 amino acid residues.

In order to facilitate the purification of SbES encoded by SEQ ID No. 1, a tag as shown in Table 1 can be attached to the amino-terminal or carboxyl-terminal of the protein consisting of the amino acid sequence shown in SEQ ID No. 1 in the sequence Listing.

TABLE 1 sequences of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The SbES can be synthesized artificially, or can be obtained by synthesizing a coding gene and then performing biological expression. The gene encoding SbES can be obtained by deleting one or more amino acid residues from the DNA sequence represented by the 1 st to 1164 th nucleotides from the 5 ' end of sequence 2 in the sequence listing, and/or by performing missense mutation of one or more base pairs, and/or by connecting the coding sequence of the tag shown in table 1 above to the 5 ' end and/or 3 ' end thereof.

The sequence of the coding gene of the promoter protein (SbES) of the M.virens endophytic fungus Sarocladium brachiariae HND5 is as follows, and the cDNA of the promoter protein of the M.virens endophytic fungus Sarocladium brachiariae HND5 is the DNA molecule of 1) or 2) or 3) as follows:

1) DNA molecule shown in sequence 2 in the sequence table;

2) a DNA molecule which is hybridized with the DNA sequence defined in 1) under strict conditions and codes the elicitor protein of the endophytic fungus Sarocladium brachiariae HND5 of the Scopulariopsis;

3) the nucleotide sequence has more than 90 percent of homology with the nucleotide sequence of the sequence 2 in the sequence table, and the coded protein has the functions of improving the plant resistance and inducing the plant defense reaction activity.

The total length of the sequence 2 in the sequence table is 1164 nucleotides, which codes a protein with the length of 387 amino acids (the sequence 1 in the sequence table) and the molecular weight of about 39kD, namely, the protein is the promoter protein (SbES) of the endophytic fungus Sarocladium brachiariae HND5, 1-45 bases are SbES protein signal peptide coding sequences, 46-315 bases are propeptide coding sequences, and 316-1164 bases are secreted mature polypeptide coding sequences.

The nucleotide sequence of the genome gene of the promoter protein (SbES) of the endophytic fungus Sarocladium brachiariae HND5 of the Scopulariopsis is shown as 1), 2) or 3) as follows:

1) SEQ ID No: 3;

2) a nucleotide sequence that can hybridize with the DNA sequence of 1) under strict conditions;

3) and SEQ ID No: 3 has more than 90 percent of homology and the coded protein has the functions of improving the plant resistance and inducing the plant defense reaction activity.

The full length of the sequence 3 in the sequence table is 1375 nucleotides, the nucleotides 1-276 from the 5' end of the sequence 3 are the first exon, the nucleotide 277-341 is the first intron, the nucleotide 342-527 is the second exon, the nucleotide 528-595 is the second intron, the nucleotide 596-1102 is the third exon, the nucleotide 1103-1179 is the third intron, and the nucleotide 1180-1375 is the fourth exon, encoding the protein shown in the sequence 1 in the sequence table.

The stringent conditions can be hybridization and membrane washing at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS.

The coding gene can also be codon optimized nucleotide, and if the coding gene can be a sequence 4 in a sequence table, the sequence can express the exciton protein in pichia pastoris, and the expression quantity is increased and the expression activity is high through codon optimization.

In the application, the induced defense reaction is micro-HR reaction, the accumulation of hydrogen peroxide in leaves, the accumulation of callose in leaves and/or the expression of plant disease resistance gene.

The application of the protein or the coding gene thereof in preparing the medicine for preventing the cassava diseases and insect pests also belongs to the protection scope of the invention.

In the application, the diseases and insect pests are diseases, and the diseases and insect pests are preferably diseases caused by Xanthomonas multocida (Xanthomonas axanopodis).

The application of the elicitor protein and the coding gene thereof as the elicitor protein in stimulating plant defense reaction, anaphylactic reaction and/or stimulating plant disease-resistant gene expression and/or in defending against the invasion of plant diseases and insect pests as protein pesticides belongs to the protection range of the invention, and the plants are rubber, cassava, banana and cucumber.

The invention also provides a method for improving the disease resistance of plants, which uses the following elicitor proteins to control plant diseases:

the exciton protein is a protein shown in the following 1) or 2) or 3):

1) protein consisting of an amino acid residue sequence of SEQ ID No. 1 in a sequence table;

2) protein consisting of 106 th and 387 th amino acid sequences of SEQ ID No. 1 in a sequence table;

3) the amino acid residue sequence described in 1) or 2) is substituted and/or deleted and/or added by one or more amino acid residues, and has the function of elicitor protein, and the protein has the sequence shown in SEQ ID No: 1-derived protein;

the plant disease is cassava disease preferably caused by Xanthomonas axonopodis (Xanthomonas axonopodis);

the elicitor proteins are used to improve disease resistance in plants by spraying the protein onto rubber, tapioca, banana or cucumber or by injection onto plant tissues.

The exciton protein can be obtained by in vitro synthesis, and the specific method is that a recombinant expression vector capable of expressing the exciton protein is transferred into yeast to obtain a transgenic recombinant bacterium for expressing the exciton protein; the yeast is preferably Pichia pastoris X-33 strain.

The recombinant expression vector for expressing the exciton protein is obtained by inserting the nucleotide fragment shown in the sequence 4 in the sequence table into a starting vector, and the starting vector is preferably pRICZA.

The elicitor protein has the activity of inducing cassava to generate resistance to bacterial wilt disease caused by Xanthomonas carpi (Xanthomonas axolopodis), can reduce the morbidity and disease index of the bacterial wilt disease of cassava, and is the elicitor protein with the activity of improving plant resistance and inducing plant defense reaction. It can induce plant defense reaction, and can activate cascade signal system in plant body by combining with receptor on plant cell surface or subcellular surface and induce defense gene expression to play a role in disease-resistant signal path.

Drawings

FIG. 1 is an SDS-PAGE protein gel image of His-SbES fusion proteins in example 2.

FIG. 2 shows cassava diseases caused by SbES protein in example 3 against Xanthomonas axonopodis (Xanthomonas axonopodis). Wherein A is a pathogenic bacteria incidence graph inoculated after blank control spray treatment, B is an pathogenic bacteria incidence graph inoculated after SbES protein treatment spray treatment, and C is an pathogenic bacteria incidence graph inoculated after the Tailing treatment spray treatment.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.