阅读说明：本技术 大麦条纹病致病性基因pgssk1及其应用 (Barley stripe disease pathogenicity gene pgssk1 and application thereof ) 是由 王化俊 梁倩倩 汪军成 姚立蓉 孟亚雄 马小乐 李葆春 司二静 杨轲 边秀秀 陈 于 2020-07-14 设计创作，主要内容包括：本发明提供大麦条纹病致病性基因pgssk1及其应用,属于生物技术工程领域,具体提供了一个来源于大麦条纹病菌-麦类核腔菌的新基因pgssk1。pgssk1基因可以调控大麦条纹病菌丝的生长及发育,通过pgssk1基因干扰突变体,培育大批量抗大麦条纹病病菌的突变体植株,可高效解决大麦条纹病病害的侵袭,实现了简单、易行、高效培育抗大麦条纹病植株的目的。新基因pgssk1基因加强了大麦条纹病菌对戊唑醇的敏感性。(The invention provides a barley stripe disease pathogenicity gene pgssk1 and application thereof, belongs to the field of biotechnology engineering, and particularly provides a novel gene pgssk1 derived from barley stripe pathogen-wheat sclerotinia sclerotiorum. The pgssk1 gene can regulate the growth and development of barley stripe disease hypha, and the pgssk1 gene interference mutant can be used for cultivating a large batch of barley stripe disease germ-resistant mutant plants, so that the attack of barley stripe disease can be efficiently solved, and the aim of simply, easily and efficiently cultivating barley stripe disease-resistant plants is fulfilled. The sensitivity of barley stripe germ to tebuconazole is enhanced by the new gene pgssk 1.)

1. The barley stripe disease pathogenicity gene pgssk1 is characterized in that the full-length sequence of pgssk1 is shown as SEQ ID NO:1 is shown.

2. The method for gene cloning and gene function verification of the barley stripe disease pathogenicity gene pgssk1 according to claim 1, comprising the following steps:

(1) cloning and sequence analysis of pgssk1 gene; (2) constructing an interference vector of pgssk1 gene; (3) preparing protoplast and genetically transforming the vector; (4) hypha growth experiments; (5) osmotic pressure sensitive and bacteriostatic tolerance experiments; (6) cell wall integrity experiments; (7) determination of pathogenicity of barley stripe disease.

3. The application of a barley stripe disease pathogenic gene pgssk1 gene in regulation of hypha growth rate is characterized in that the full-length sequence of pgssk1 is shown as SEQ ID NO:1 is shown.

4. The application of a barley stripe disease pathogenic gene pgssk1 gene in regulation of osmotic pressure and fungal inhibitor sensitivity is characterized in that the full-length sequence of pgssk1 is shown as SEQ ID NO:1 is shown.

5. The application of a barley stripe disease causing gene pgssk1 in participating in cell wall integrity is characterized in that the full-length sequence of pgssk1 is shown as SEQ ID NO:1 is shown.

6. The application of a barley stripe disease pathogenic gene pgssk1 in regulation and control of barley stripe disease pathogenicity is characterized in that the full-length sequence of pgssk1 is shown as SEQ ID NO:1 is shown.

Technical Field

The invention belongs to the field of biotechnology engineering, and particularly relates to a barley stripe disease new gene pgssk1 and application thereof in regulation of hypha growth, osmotic pressure sensitivity, bacteriostatic agent tolerance, cell wall integrity and pathogenicity.

Background

The Barley stripe disease (Barley leaf stripe) has serious harm to Barley, and is the main cause of yield reduction of Barley. The disease is a systemic fungal disease caused by seed-borne bacteria, while some signal proteins in different filamentous fungi have different functions, and one of the signal proteins is a reflection regulator protein RR in a yeast two-component signal pathway. In the corn microsporum, RR signal proteins ChSSK1 and ChSKN7 both contribute to the osmotic adaptation and sensitivity to the phenylpyrrole fungicide fludioxonil. In bread mildew rubra, RR signaling protein has multiple functions, RRG-1 controls vegetative cell integrity, hyperosmotic sensitivity, fungicide resistance, and RRG-2 is only involved in oxidative stress. In rice blast, RR signaling proteins are involved in pathogenicity in addition to increased resistance to osmotic stress and fluorobipyrimidine fungicides. However, the function of RR protein in barley stripe disease-causing bacteria has not been studied to date. The method mainly researches a pgssk1 gene for coding RR protein and functions of the gene in barley stripe germs in an RNAi mode, and aims to lay a foundation for disease-resistant breeding of the barley stripe germs.

Disclosure of Invention

In view of the defects of the prior art, the invention provides a barley stripe disease (wheat type nuclear cavity bacterium) pathogenic gene pgssk1, obtains a pgssk1 mutant by an RNAi interference technology, and further provides functions of the gene in the aspects of regulating and controlling hypha growth and differentiation, osmotic stress, bacteriostatic agent tolerance, cell wall integrity, pathogenicity and the like.

In order to achieve the purpose, the invention adopts the following technical scheme:

1. a barley stripe disease pathogenicity gene pgssk1, the full-length sequence of which is shown in a sequence table SEQ ID NO. 1.

2. A barley stripe disease pathogenic gene pgssk1 gene clone and a gene function verification method thereof comprise the following steps:

(1) cloning and sequence analysis of pgssk1 gene;

(2) constructing an interference vector of pgssk1 gene;

(3) preparing protoplast and genetically transforming the vector;

(4) hypha growth experiments;

(5) osmotic pressure sensitive and bacteriostatic tolerance experiments;

(6) cell wall integrity experiments;

(7) determination of pathogenicity of barley stripe disease.

3. An application of a barley stripe disease pathogenic gene pgssk1 gene in regulation of hypha growth rate.

4. An application of a barley stripe disease pathogenic gene pgssk1 gene in regulating osmotic pressure and fungal inhibitor sensitivity.

5. The application of a barley stripe disease pathogenic gene pgssk1 gene in participating in cell wall integrity.

6. An application of a barley stripe disease pathogenic gene pgssk1 in regulating and controlling the pathogenicity of barley stripe disease.

The application has at least the following beneficial effects:

(1) the application provides a novel gene pgssk1 derived from barley stripe germ, Pyrenophora graminea (Pyrenophora graminea).

(2) The pgssk1 gene can regulate the growth and development of barley stripe disease hypha, and the pgssk1 gene interference mutant can be used for cultivating a large batch of barley stripe disease germ-resistant mutant plants, so that the attack of barley stripe disease can be efficiently solved, and the aim of simply, easily and efficiently cultivating barley stripe disease-resistant plants is fulfilled.

(3) The sensitivity of barley stripe germ to tebuconazole is enhanced by the new gene pgssk 1.

Drawings

FIG. 1 sequence of the pgssk1 gene

FIG. 2 sequence alignment of Alternaria alternata, Stemphyllium lycopersici, Diplodia seriata, Botrytis cinerea, Zymospora tritici, Pyrenophora graminea and Saccharomyces

Wherein, black shading indicates identical amino acids, green or pink shading indicates similar amino acids, the signal acceptor domain is underlined, the missing amino acids are indicated with ".", the phosphorylation receptor sites phosphorylated by histidine kinase homologues are indicated with "#";

FIG. 3 phylogenetic analysis of pgssk1 Gene

FIG. 4 REC Domain of pgssk1 Gene

FIG. 5 is a schematic diagram of the construction of the interference vector pSilentpgssk1

FIG. 6 shows a schematic diagram of interference strategy of interference vector pSilentpgssk1

FIG. 7 restriction enzyme digestion and PCR validation of RNAi vector pSilentssk1 of pgssk1 gene

Wherein, M1: markDL 2000; m2: markDL 10000; 1: HindIII is cut into 150 bp; 2: kpn I and Bgl II are cut with 741 bp; 3: xho I is cut by enzyme, and the size is 1632 bp; 4: HindIII and XhoI are cut by enzyme, and the sizes are 741bp and 150 bp; 5: BglII and Xho I are cut by enzyme, and the sizes are 891bp and 741 bp; 6: PCR identification of the pSilentpgssk1 vector.

FIG. 8 PCR validation of interfering strains

Wherein, Markers: markDL 10000; 1: PCR analysis and verification of WT strains; 2: verifying hygromycin PCR analysis of the plasmid; 3: PCR analysis verification of delta pgssk1-8 strain; 4: the PCR analysis of the delta pgssk1-24 strain verifies.

FIG. 9 SDS-PAGE analysis of PGSSK1 recombinant protein

Wherein, M: protein standard molecular weight; pET-32 a; pET32a-pgssk 1; pET32a-pgssk1 induction; 4. purified PGSSK1 recombinant protein

FIG. 10 RT-PCR and Westernblot validation of interfering strains

Wherein, indicates significance (P.ltoreq.0.01) between wild strain (WT) and interfering strain (. DELTA.pgssk 1-8 and. DELTA.pgssk 1-24);

FIG. 11 growth of wild-type and interfering strains on different media

FIG. 12 growth of wild-type and interfering strains on different osmotic media

FIG. 13 growth of wild and interfering strains on different bacteriostatic media

FIG. 14 growth of wild and interfering strains on different cell wall inhibitor media

FIG. 15 growth rates of wild and interfering strains on different cell wall inhibitor media

FIG. 16 protoplast release analysis of wild strain WT (A, B) and interference strain Δ pgssk1(C, D)

FIG. 17 protoplast yield analysis

FIG. 18 chitin content analysis

FIG. 19 pathogenicity detection of wild and interfering strains on barley leaves

Wherein (A) two-week-old leaf of barley was inoculated with 5mm of WT, delta pgssk1-8 and delta pgssk1-24 strains, and photographed and recorded 3 days after inoculation. (B) Barley seeds were treated with WT, Δ pgpbs1, Δ pgssk1-8, and Δ pgssk1-24 strains at 4 ℃ for 20 days before cultivation, and photographed after 14 days of cultivation. (C) Infection rates of barley after inoculation with WT, Δ pgssk1-8 and Δ pgssk1-24 strains, asterisks indicate significant differences (P.ltoreq.0.05) between wild and interfering strains. (D) The growth tendency of barley leaves infected with WT, Δ pgssk1-8 and Δ pgssk1-24 strains. (E) The transcription level of mRNA of the Δ pgssk1-8 gene in the wild strain WT is marked by asterisks, which indicate that the difference between the parasitic state and the non-parasitic state is significant (P.ltoreq.0.05).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention are described in detail below with reference to the accompanying drawings. Examples of these preferred embodiments are illustrated in the accompanying drawings. The embodiments of the invention shown in the drawings and described in accordance with the drawings are exemplary only, and the invention is not limited to these embodiments.

It should be noted that, in order to avoid obscuring the technical solution of the present invention with unnecessary details, only the structures and/or processing steps closely related to the solution according to the present invention are shown in the drawings, and other details that are not relevant are omitted.