CN111748567A

CN111748567A - Barley stripe disease pathogenicity gene pgssk2 and application thereof

Info

Publication number: CN111748567A
Application number: CN202010677653.9A
Authority: CN
Inventors: 孟亚雄; 梁倩倩; 王化俊; 汪军成; 李葆春; 马小乐; 司二静; 杨轲; 边秀秀; 黄志磊
Original assignee: Gansu Agricultural University
Current assignee: Gansu Agricultural University
Priority date: 2020-07-14
Filing date: 2020-07-14
Publication date: 2020-10-09

Abstract

The invention provides a barley stripe disease pathogenicity gene pgssk2 and application thereof, belongs to the field of biotechnology engineering, and particularly provides a novel gene pgssk2 derived from barley stripe pathogen-wheat sclerotinia sclerotiorum. The tolerance of barley stripe disease pathogenic bacteria to prochloraz is enhanced by the pgssk2 gene, and a large quantity of crops resistant to related pesticides and herbicides of the prochloraz are cultivated by transforming the pgssk2 gene mutant, so that the agricultural production is improved.

Description

Barley stripe disease pathogenicity gene pgssk2 and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a barley stripe disease pathogenic gene pgssk2 and application thereof in regulation of hypha growth, osmotic pressure sensitivity, bacteriostatic agent tolerance, cell wall integrity and pathogenicity.

Background

Wheat sclerotinia sclerotiorum (Pyrenophora graminea) is pathogenic bacteria of barley stripe disease, mainly causes barley leaf diseases, and the fungi of Deuteromycotina (Deuteromycotina) of the genus is pathogenic bacteria transmitted through seeds, and is systemically transmitted in infected barley plants, so that the whole infected barley plants can be attacked, and the prevention and the control are very difficult. Pathogenic fungi respond to environmental changes through signal cascade amplification and adjust the expression of each gene, and signal genes involved in the process of invading hosts by the pathogenic fungi mainly comprise: heterotrimeric G protein gene, MAP kinase, and cAMP-dependent protein kinase gene. In eukaryotes, the MAP signaling pathway plays a central role in response to a variety of extracellular signals. Among them, the high osmotic pressure glycerol (HOG) pathway, which responds to osmotic stress, is one of the most characteristic MAP kinase modules in saccharomyces cerevisiae, and activation of the HOG pathway is regulated by two membrane-bound osmotic sensors, SLN1P and SHOLP, which transmit signals through two independent branches of the pathway, which converge in the MEPK-encoded pbs2p gene. Under hyperosmotic stress, sln1 was not activated, accumulation of unphosphorylated Ssk1 activated the downstream MAP kinase cascade by sequential phosphorylation (Ssk2/Ssk22-Pbs2-Hog1), the Hog pathway of the filamentous fungus Neurospora crassa consisted of one osmosensing histidine kinase (OS1), phosphotransferase protein histidine (HPT1), two regulatory proteins (RRG1 and RRG2) and downstream MAPK cascade reactions (OS5 and OS2) with higher homology to Hog1 Saccharomyces cerevisiae SSK2/SSK22, Pbs2, respectively. Until now, the related research of the two-component signal system members of the barley stripe pathogen is blank, and the application mainly researches a pgssk2 gene coded by barley stripe pathogen mapkk kinase and the application thereof in an RNAi mode, and aims to provide technical support for barley disease resistance breeding.

The problems existing in the prior art are as follows: no report of the gene and function of barley stripe disease (wheat type sclerotinia) pgssk2 is found in the prior art.

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 pgssk2, obtains a pgssk2 mutant by an RNAi interference technology, and further provides application of the gene in regulation of hypha growth 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 pgssk2, the full-length sequence of which is shown in a sequence table SEQ ID NO. 1.

2. A barley stripe disease pathogenic gene pgssk2 gene clone and a gene function verification method thereof comprise the following steps:

(1) cloning and sequence analysis of pgssk2 gene;

(2) constructing an interference vector of pgssk2 gene;

(3) preparing protoplast and genetically transforming the vector;

(4) hypha growth experiments;

(6) osmotic pressure sensitive and bacteriostatic tolerance experiments;

(7) cell wall integrity experiments;

(8) determination of pathogenicity of barley stripe disease.

3. An application of a barley stripe disease pathogenic gene pgssk2 gene in regulation of hypha growth rate.

4. An application of a barley stripe disease pathogenic gene pgssk2 gene in regulating osmotic pressure and fungal inhibitor sensitivity.

5. The application of a barley stripe disease pathogenic gene pgssk2 gene in participating in cell wall integrity.

6. An application of a barley stripe disease pathogenic gene pgssk2 in regulating and controlling the pathogenicity of barley stripe disease.

7. An application of a barley stripe disease pathogenic gene pgssk2 in improving the resistance of barley to prochloraz.

The application has at least the following beneficial effects:

(1) the application provides a novel gene pgssk2 derived from barley stripe germ, Pyrenophora graminea (Pyrenophora graminea).

(2) The pgssk2 gene strengthens the tolerance of barley stripe disease pathogenic bacteria to prochloraz. Therefore, by transforming pgssk2 gene mutant, a large amount of crops resistant to prochloraz related pesticides and herbicides can be cultivated, and agricultural production is improved.

Drawings

FIG. 1 sequence of the pgssk2 gene

FIG. 2 partial sequence alignment of exophyta dematitidis, Glarea lozoyensis, Paraphaeospora polosa, Borrytis cinerea, Pyrenophora grandinea and Saccharomyces cerevisiae

Wherein, black shading indicates identical amino acids, green or pink shading indicates similar amino acids, the signal acceptor domain is underlined, and missing amino acids are indicated ";

FIG. 3 phylogenetic analysis of pgssk2 Gene

FIG. 4S _ TKC domain of the pgssk2 gene

FIG. 5 schematic diagram of the construction of the interference vector pSilent pgssk2

FIG. 6 interference vector pSilent pgssk2 interference strategy diagram

FIG. 7 restriction enzyme digestion and PCR validation of RNAi vector pSilentssk2 of pgssk2 gene

Wherein, M1: markDL 2000; m2: markDL 10000; 1: HindIII is cut into 150 bp; 2: carrying out enzyme digestion on Kpn I and SphI, wherein the size is 424 bp; 3: xho I enzyme digestion, the size is 998 bp; 4: HindIII and XhoI are cut by enzyme, and the sizes are 424bp and 150 bp; 5: SphI and XhoI are cut by enzyme, and the sizes are 574bp and 424 bp; 6: PCR identification of the pSilent pgssk2 vector.

FIG. 8 PCR validation of interfering strains

Wherein, M: markDL15000+ 2000; 1: PCR analysis and verification of WT strains; 2: verifying hygromycin PCR analysis of the plasmid; 3: PCR analysis verification of delta pgssk2-7 strain; 4: PCR analysis verification of delta pgssk2-21 strain

FIG. 9 SDS-PAGE analysis of PGSSK2 recombinant protein

Wherein, M: protein standard molecular weight; pET-32 a; pET32a-pgssk 2; pET32a-pgssk2 induction; 4. purified PGSSK2 recombinant protein

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

Wherein A and B represent significance (P.ltoreq.0.01) between wild strain (WT) and interfering strain (. DELTA.pgssk 2-7 and. DELTA.pgssk 2-21)

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 chitin content analysis

FIG. 17 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 pgssk2-7 and delta pgssk2-21 strains, and photographed and recorded 3 days after inoculation. (B) Barley seeds were treated with WT, Δ pgssk2-7 and Δ pgssk2-21 strains at 4 ℃ for 30 days before cultivation, and photographed after cultivation for 20 days. (C) Infection rate of barley after inoculation with WT, Δ pgssk2-7 and Δ pgssk2-21 strains. (D) Tendencies of growth of barley leaves infected with WT, Δ pgssk2-7 and Δ pgssk2-21 strains. (E) The transcription level of mRNA of the delta pgssk2 gene in the wild strain WT in the parasitic state and the non-parasitic state is shown by asterisks, and the significant difference (P is less than or equal to 0.05) exists between the parasitic state and the non-parasitic state.

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.

Example 1

The embodiment provides a barley stripe disease pathogenicity gene pgssk2, wherein the full-length sequence of the gene is shown in a sequence table SEQ ID NO. 1. The DNA sequence of pgssk2 is 4906bp long and the sequence contains a 52bp intron located at the position from nucleotide 3619bp to 3671 bp. The sequence of the pgssk2 gene is shown in FIG. 1. PGSSK2 encodes a 1384 amino acid protein and the 1384 amino acid sequence of PGSSK2 was analyzed for homology to Botrytis cinerea, Exophiala dematitidis, Glarea lozoyensis, Paraphaeospora sporogenes and Saccharomyces cerevisiae: 51.52%, 51.33%, 51.27%, 67.92% and 26.78% (FIG. 2). Phylogenetic analysis results showed that p.graminea, p.sporolos and e.dermatitidis were clustered together (fig. 3). The CDART (conserved domain technical regenerative tool) program predicted the conserved S _ TCK signal acceptor domain (protein serine/threonine kinase catalytic domain, mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (1077-1349aa), MAPK kinase (1084-1363 aa)) in PGSSK2 (FIG. 4).

Example 2

The embodiment provides a method for cloning a barley stripe disease pathogenic gene pgssk2 gene and verifying the gene function thereof, which comprises the following steps:

(1) the cloning and sequence analysis of pgssk2 gene includes homologous alignment analysis of ssk2(Genbank ID: NP 013106) sequence and barley stripe disease QWC genome sequencing result, the homologous sequence is named as pgssk2, the pgssk2 gene sequence is cloned in a homologous mode, a primer pair pgssk2-F1/pgssk2-R1 (table 1) is used as a primer to amplify a target fragment, a product obtained by PCR amplification is connected with pMD19-T vector (Takara Biotech) after being purified, and the product is sent to a company for sequencing. Domain prediction of the PGSSK2 protein was performed using SMART software (http:// SMART. embl-heidelberg. de /) and cdart (conserved domain architecture recovery reagent) programs.

(2) RNAi vector construction of pgssk2 Gene includes construction of RNA interference vector of pgssk2 gene by hairpin strategy using fungal interference vector pSilent-1(6.9kilobases [ kb ]), and amplification of target fragments pgssk2-1(741bp) and pgssk2-2(741bp) using PCR primer pair pgssk2-XhoI/pgssk 2-HindIII and PCR primer pair pgssk2-KpnI/pgssk2-SphI (Table 1), respectively, using cDNA of barley streak disease WT as template. Construction of pSilent pgssk2 vector Using two-step strategy, restriction enzyme XhoI and Hind III are used to cleave the pgssk2-1 and the vector pSilent-1 respectively, both fragments are recovered separately, T4 ligase is used to join the pgssk2-1 fragment and the vector pSilent-1, and the pgssk2-1(741bp) fragment is inserted into the XhoI/Hind III site of the vector pSilent-1 to obtain the vector pSilent pgssk 2-1. The two fragments were recovered by digesting the pgssk2-2 and the vector pSilent pgssk2-1 with restriction enzymes KpnI and SphI respectively, and the vector pSilent pgssk2 was obtained by ligating the pgssk2-2 fragment with pSilent pgssk2-1, pgssk2-2 fragment (424bp) by T4 ligase and inserting the KpnI/SphI site of the vector pSilent pgssk2-1, and the XhoI/HindIII site and the KpnI/SphI site of the vector pSilent 2 interference vector pSilent 2 (FIG. 5) was obtained by inserting the reverse complement sequences of pgssk2-1 (821 and 5bp of psssk 2) and Ppgssk 2-2 (1245 and 821bp of psssk 2) into the vector pSilent pgssk 3626.

(3) Protoplast preparation and genetic transformation of the interference vector comprises suspending 100. mu.L of freshly prepared protoplast of the WT strain (1X 108cells/mL) in STC buffer in a 1.5mL centrifuge tube, mixing 1-10. mu.g of plasmid pSilentpgssk2 DNA, placing in freshly prepared crushed ice 20minutes, adding 100. mu.L of LPTC buffer to the suspended suspension centrifuge tube, gently mixing, placing the mixture in freshly prepared crushed ice 20min, then adding 800. mu.L of PTC buffer, and placing the mixture at room temperature for 15 min. The protoplast transformation mixture (500. mu.L/plate) was spread on a plate containing 15mL of regeneration medium using a glass spreader, the plate was incubated at 25 ℃ for 48 hours, a layer of 10mL of PDA medium containing 70. mu.g/mL hygromycin (hygromycin B) was spread on the plate, and incubation was continued at 25 ℃ for 2-3 days until a regeneration colony grew out. Regenerated single colonies were transferred to PDA plates with hygromycin concentration of 100. mu.g/mL. And continuously transferring the culture medium into 3 generations, and carrying out subsequent PCR detection on colonies with stable growth conditions. The PCR, RT-PCR and western blot verification of the transformant in the step (4) comprises the steps of extracting RNA, carrying out PCR amplification verification on the transgenic strain by using hygromycin specific primers and Hyg-1/Hyg-2, and simultaneously detecting the expression of pgssk2 in the transgenic strain by using real-time fluorescent quantitative PCR (qRT-PCR).

QRT-PCR was performed on a 7300Real-Time PCR system using SYBR premixed Ex Taq (Perfect Real Time, Takara, Japan). RNA of wild strain and interference strain is extracted, cDNA is reverse transcribed, and cDNA of each transformant is diluted by 1:5 to carry out transgene expression analysis. The real-time PCR mixture consists of 12.5. mu.L TB Green Advantage qPCRPremix (2X) (Takara Bio, Mountain View, CA), 0.5. mu.L forward primer, 0.5. mu.L reverse primer, 1. mu.L diluted cDNA, and beta-actin gene of barley streak pathogen as reference gene, and the target gene is pgssk 2. The experiment was repeated in triplicate and the primers used are shown in Table 1.

The PGSSK2 gene is detected by a western blot method, the method refers to 2.1.6, the PGSSK2 gene is connected with an expression vector pET32a, PGSSK2 protein is induced and expressed, a Ni-NTA His Resin column is purified, protein liquid is collected and analyzed by SDS-PAGE electrophoresis, a protein immune mouse obtains a primary antibody, serum of mouse anti-recombinant PGSSK2 is taken as the primary antibody, goat anti-mouse IgG (Bioss, China) is taken as a secondary antibody, and the membrane is detected by immune combined chemiluminescence (bio-star, Hercules, CA) according to the instructions of a manufacturer.

TABLE 1 primers used in the cloning and functional Studies of the pgssk2 Gene

(1) RNAi vector construction of barley stripe germ pgssk2 gene

According to the design of constructing an inverted repeat RNAi vector pSilent pgssk2 with pgssk2 as a target (FIG. 6), constructing a pSilent pgssk2 vector through enzyme digestion and connection, carrying out enzyme digestion analysis according to restriction sites on the pSilent pgssk2 vector, and generating a specific band (shown in FIG. 7, an arrow below a lane 1) at the position of about 150bp by HindIII enzyme digestion, wherein the size of the specific band is consistent with the size of an intron of the vector between two HindIII enzyme digestion sites; cleaved with KpnI and SphI, the size of the fragment was about 424bp (FIG. 7, arrow below lane 2), which is consistent with the size of the interference arm fragment; the fragment was digested with Xho I, 998bp (FIG. 7, arrow below lane 3), which was consistent with the sum of the two interfering arms plus the size of the vector intron; HindIII and XhoI are cut by enzyme, the sizes are 424bp and 150bp, and the bands just conform to the sizes of the interference arms and the vector intron; the size of the fragment was 574bp and 424bp (FIG. 7, arrow below lane 4) using SphI and XhoI, and was consistent with the size of the interference arm plus intron (574bp) and one interference arm (150 bp).

(2) Verification of interfering strains

An interference vector pSilent pgssk2 is transformed on a protoplast regeneration system of barley stripe pathogen to obtain 42 transformants, 2 pgssk2 interference mutants are randomly selected to carry out RT-PCR and western blot verification. 3 mutants all had the expected hygromycin fragment of 1026bp as detected by PCR with hygromycin specific primer Hyg-1/Hyg-2 (Table 1) (FIG. 8); the fluorescent quantitative PCR result shows that the transcription level of pgssk2 gene is remarkably reduced in RNAi interference strains delta pgssk2-7 and delta pgssk2-21 by taking an untransformed wild strain as a control (WT) (P is less than or equal to 0.01), and the reduction values of the relative expression levels of the delta pgssk2-7 mutants and the delta pgssk2-21 mutants are respectively as follows: 43.4% and 44%, and the expression level decreases by Δ pgssk2-7 and Δ pgssk2-21, which are significantly different from the expression level decrease of the wild strain (P.ltoreq.0.01). And further carrying out western blot verification. The result of PGSLN protein purification is shown in FIG. 9, and a fragment of about 154KD is obtained; the expression results are shown in FIG. 10, and consistent with the results of fluorescence quantification, the western blot verified that the bands of Δ pgssk2-7 and Δ pgssk2-21 are weak, and these results confirm that the gene interference strain of pgssk2 has been obtained in this study.

(3) Relationship between pgssk2 and vegetative growth of barley streak germ hyphae

Interference of pgssk2 gene in barley stripe disease WT has little effect on radial growth of barley stripe pathogen, and the growth rates of pgssk2 interfering strains (. DELTA.pgssk 2-7 and. DELTA.pgssk 2-21) on PDA, CM, BM, V8 and MM media were: Δ pgssk 2-7: 10.9, 9.5, 9.0, 9.1 and 8.7 mm/d; Δ pgssk 2-21: 10.9, 9.3, 9.0, 9.0 and 8.7 mm/d; WT: no significant differences (P.ltoreq.0.01) were found between 10.4, 9.5, 9.2, 8.9 and 9.3mm/d (FIG. 11; Table 2), Δ pgssk2-7 and Δ pgssk2-21 and the wild strain WT.

The above results indicate that pgssk2 is not associated with the growth and development of hyphae of barley streak pathogen.

TABLE 2 growth rates of hyphae on different media

Example 3

The embodiment provides an application of a barley stripe disease pathogenic gene pgssk2 in regulating osmotic pressure and a fungal inhibitor, and the application comprises the following steps:

(1) RNAi vector construction of barley stripe germ pgssk2 gene

(2) Verification of interfering strains

(3) Correlation of pgssk2 Gene with osmotic pressure and fungal inhibitors

To examine the role played by the pgssk2 gene in adapting to osmotic pressure, wild and interfering strains (. DELTA.pgssk 2-7 and. DELTA.pgssk 2-21) were grown in the following types of media: ionic stress (Na +), oxidative stress (H2O2), heavy metal stress (CoCl2), osmotic stress (sorbitol) and fungistatic stress (the benzimidazole fungistatic agents carbendazim, the sterol demethylation inhibitor tebuconazole and prochloraz, the phenylpyrrole fludioxonil and the dicarboximide fungistatic agent iprodione).

The radial growth rates of the wild strain WT and the interference strains delta pgssk2-7 and delta pgssk2-21 under sorbitol stress were 8.7, 7.7 and 7.9mm/d, the radial growth rates were 69.2%, 49.2% and 52.3%, and the growth rates of the interference strains (delta pgssk2-7 and delta pgssk2-21) were significantly reduced (P.ltoreq.0.05). There were no significant differences under oxidative stress (H2O2), heavy metal stress (CoCl2), ionic stress (Na +) and glycerol stress (fig. 12, table 3). The above results indicate that the pgssk2 gene is associated with osmotic pressure (Sorbitol) stress.

TABLE 3 growth rates of wild and interfering strains on different stress media

Fungal inhibitor susceptibility experiments showed that the tolerance of the interfering strains Δ pgssk2-7 and Δ pgssk2-21 to fenamido was significantly higher than that of the wild-type strain WT (P.ltoreq.0.05) (FIG. 13; Table 4), and that the growth rates of WT, Δ pgssk2-7 and Δ pgssk2-21 were: 4.5, 6.4 and 6.7%. Compared with wild strains, the sensitivity of the interference strains delta pgssk2-7 and delta pgssk2-21 to tebuconazole is obviously increased, and the relative growth rates of the delta pgssk2-7, the delta pgssk2-21 and WT are as follows: 8.9, 9.2 and 11.3%. The above results indicate that pgssk2 gene enhances the tolerance of barley stripe disease pathogen to prochloraz and the sensitivity to tebuconazole.

TABLE 4 growth rates of wild and interfering strains on different bacteriostatic media

Example 4

The embodiment provides an application of a barley stripe disease pathogenic gene pgssk2 in participating in cell wall integrity, and the application verification comprises the following steps:

(1) RNAi vector construction of barley stripe germ pgssk2 gene

(2) Verification of interfering strains

(3) Correlation of pgssk2 Gene with cell wall integrity

To examine the cell wall integrity of wild and interfering strains. Several cell wall inhibitor CR (100. mu.g/mL), SDS (0.01%) and CFW (200. mu.g/mL) plates (FIG. 14) were inoculated, where CFW inhibits the fungal cell wall assembly of chitin and beta-1, 4-glucan by binding to chitin of the hyphal cell wall. CR prevents the normal assembly of cell wall components by binding to beta-1, 3-glucan in the cell wall; SDS reduces cell membrane stability and damage to the cell wall increases fungal sensitivity. The relative growth rate of the hyphae of the interfering strain on the SDS plate is obviously lower than that of the WT, and the growth rates on the SDS plate are respectively as follows: Δ pgssk2-7, 56.0%; Δ pgssk2-21, 55%; WT, 63.2% (P.ltoreq.0.05) (FIG. 15).

Further chitin content was determined and found to be significantly lower than WT for the interfering strains with Δ pgssk2-7 and Δ pgssk2-21 (0.85% vs. 0.82% vs. 0.99%; P ≦ 0.05) (FIG. 16). These results indicate that pgssk2 is not involved in cell wall chitin synthesis regulation.

Example 5

The embodiment provides an application of a barley stripe disease pathogenic gene pgssk2 in regulation and control of barley stripe disease pathogenicity, and the application verification comprises the following steps:

(1) RNAi vector construction of barley stripe germ pgssk2 gene

(2) Verification of interfering strains

(3) Correlation of pgssk2 gene and pathogenicity of barley stripe pathogen

To evaluate the pathogenicity of pgssk2, the study inoculated leaf barley with bacteria from both the wild and the interfering strains. Both leaf blades of barley inoculated with WT and the Δ pgssk2-7 and Δ pgssk2-21 strains exhibited symptoms of infection, with the appearance of tan lesions (FIG. 17-A). Barley growth trends were observed 20 days after sowing barley seeds inoculated with the wild strain and the interfering strain (FIG. 17-D). Barley plants infected with both WT and Δ pgssk2-7 and Δ pgssk2-21 strains grew weakly and showed streaky leaf surfaces (FIG. 17-B; transcription level of pgssk2 was significantly decreased in WT parasitism (P.ltoreq.0.01) and relative expression of pgssk2 was increased in parasitism (FIG. 17-E) compared to non-parasitism, which indicates that pgssk2 was not involved in pathogenesis.

The foregoing is directed to embodiments of the present application and it is noted that numerous modifications and adaptations may be made by those skilled in the art without departing from the principles of the present application and are intended to be within the scope of the present application.

<110> university of agriculture in Gansu province

<120> barley stripe disease pathogenicity gene pgssk2 and application thereof

<160>1

<210>1

<211>4906

<212>DNA

<213> barley striped bacteria-Pyrenophora graminea (Pyrenophora graminea)

<400>1

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181 atgagggttt gggttcagag tgttttctgg gtgagtgtcg gggggtctcg ggtaaggagt

241 cgggattgga ttcgaagcgg atatgtgtgt ttttgcgtgg tgagcgacgg ggggttattg

301 gaggttgttg tgcctcctcc tctacttcat catgtgtcga tggaggtgag ggctcggctt

361 gcagggggtt gtcgaataaa gccgcgccgc gcatataccg gtaaatctcg tattggtatt

421 cttcgctcgc gtcgccttgg aagagtgcgg gatccttgtc tagatccagg taagcgacat

481 cgacgacgtc gtcttgtccg tcgtcgccag atgtggtgct cgagatgtgt gataggtcga

541 tatcatcctg ggatagcgag ggcacgatgt cggcgcggga tagcgtgtaa tcaatgactg

601 tggtttccag gcccgtgaag cggaatttgc ggcgtaaagg gtcgcggatg aggggctgca

661 atacgtctcc gtctgtgcgt gaagaccgca cgcagatgtt gccgagatgc aggtcgcgat

721 gctcgaatcg acatgcttct tctgctttgc ctagacttat tgctacaccc caaaagacgt

781 cccagacttc ccatatggaa ctcaaaccgc cgttctccat catcttttcg cagtcagtac

841 cggcgtcttg catctcaacg acggcccaga gttggttttc atcgtagctg gcttttttgc

901 tgggatctgg gaactcgctt ttcttgccgc gaggacgaga cttgttccat tccttccagg

961 cgtcgttgaa ggacgttgaa ggtctaccct ggacaatagt gaggtcacgg aaaacggtga

1021 agccaggtat gtgagtcagg ttctggagga gcctaacctc tgaaactaca tcctcaacat

1081 gcgatttcca ttgatcctcc tcctctcgtt gagccatctc cttttccatc tgcgcttcgc

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1201 gggccacgat cttgagcacc gattcttctg tgacaccatt cgcggaagat gtagcagata

1261 gtcggtatac ctctgaaaaa gacgcttccg caatctttgt gatttcgaaa tgcggttcta

1321 gttcgcctaa ccagtcttgg aagcccacaa tcctctttct gtcactgaga gccagtagcg

1381 gagacgcata ggcagagtaa acggtttcag gctcaggtgt tggttccggg gtaggtaact

1441 gtggtcttcg ttgcttcgat ctaggacgta tgggtgtctc ctcatttctt gtgacctcgc

1501 cagctgtggg gataggggta gggacaagtc gcttcttctc cttcttcttt ttcgaaccgg

1561 actggacctg gccatgagtt tcgttcaaag cgttgatgtt ccgatcgctc agaactttgc

1621 gcggtttcct tggcttcttt agcatggcgg ggtcttcttc taccggggcc ctgatgtcca

1681 aagctcgcat gctttcctcc agagcatcgt agattggaat cgaattctct tttgcggtat

1741 ctggatcggt cgcggacgat tccagtggtc tagattgttt ctttgtgatg cgcacttttg

1801 gcgaattcct tggttgctcc atttgtaaac cctcaaaccc cttctccaac gccgacatgt

1861 catcatcctt ctgcttctgt tgtgctgttg actctgctgc tgtcaagagc tgatgccgcc

1921 aatcatttgt tgtgttgcta gccacatttt ggctccccac gactttgtgc gcaacatcct

1981 tctctggcga cagaaacttt gtataggcag gccgcccccc agttctcttc ttcccgtata

2041 caatttttgg tggcatgtca ggcggctgag cgaatagtat ggctgttgac gttaggtcgg

2101 gcgtctggct ggacctgcca gggtcgcttt gcacgggtcc acgtgggaaa ctctaagcga

2161 ggacaaaccg ttgacccggc aacagacagg gttctgacac atgcgtatcg atagtcacaa

2221 gcagagaagc agagaagtac aatctcacac gattaacatt acaatcaaaa gctactttgt

2281 tctccggcgc cagcctcatc agtcgcaata tcaaaacaaa gcctctcgcc ccgagacgat

2341 catattgtgt acggtaaaac gggtgtccag cctccctttt ctaaagccaa gaaagatatt

2401 acataggaaa ggtgtgtagc aaggtttgtc tgaaagacaa gtgtatggcg agtgcgtcca

2461 tgaccaatga gaaaacgcca agtgcaacaa taagtagacc cagaatattg acgaaactaa

2521 agaaaaagac ccatggaaaa catgagggcc gtgcaaaatg gtgttatgtc aatttacaac

2581 cgaataactt ttgatatttg gtaggcgagg aaaatcgtgc aacgcatagc agggaagaaa

2641 gcaacaggca gacttgtgac tccgtacaac cgaagaggcg tgtgagtgtg aaagctgagc

2701 ttactcatct ccgcttccgc tttcaattgg cggtgtctcg gcctcttcct gcagcaccgg

2761 aggcgggacg agactcttgc gcttattttt cctttcagct tccgcatcct tgttcgtacc

2821 gtttgtatcc ggtggagcgg ccgcgagtgt tgggctgggg gtaactggtt tcttgtttgg

2881 ggcaatactc gcatagctac cggagagctt cgatgtggtg tcattgttct cagacttgtc

2941 ggcaaaatct ttccacttgc gccaaccgtc gaaatcgatc agggcttgca tgcatcccca

3001 ttctttgact ttgcgctcga gccagacgaa gttgacgggc tgcagtgtgt tagtgaaagc

3061 taatggaaaa gaagtctatc cttcttacct ttgtcaagaa atcgttgcag ccagccgcca

3121 atgcctcatg cctatcactt tgcagcgaac tggcagtgag ggcgacaata atgacagggc

3181 tcttgaacat gcccttgtca ccaagccggt cgtctgcaac tagctcatct ttgcccttgg

3241 catcctggcc gagcctgtta ttgggcgctt cgctgcttcc actgctgaag acgccaatgc

3301 tgttgacacg ctccaatctc ctaatctcct ttgtcgcctc gagaccgttc atgacgggca

3361 gctgaatgtc catgagtacc aagtggaaac caccagcctt ccacttggtc acggcctcct

3421 ttccattcat tgcgcgctgc cagcggactt tgagacgctg cataaaagct cccaagactc

3481 gcaggttgat aatgttatct tccacaatga gcacgttgat aggtggcact gacccatcga

3541 gcaatccagc aggcaagctg ggtgcctcga gtggagcctt cttccgcttc ttcttcggct

3601 gaggcgacga gatacgcggg gtcggcggag ttgactcatg gagggttggt tggtgtgtga

3661 tgaggtcggc agtagcggga ggtgtgggta tcgggttatt cgatgcagcc gagttcttcg

3721 cgtcagatgc cgtggagtca tggcgcggac ggttgggagg acgggcggac gtggcagagg

3781 aggggatgcc gccgccaagg gtagtgggcg aggcagtaag ggtcctgggg tatttcggcc

3841 gatgtgcatg gagtggccca cgccttgtgc ctccgggtga aggcagggca ggcagctgtc

3901 cggtgttgag cacagcgata gagtgcgaag cgtgggggag gggcatgcgg gtgtcgtgct

3961 gctgctggct cgtacccatg ccagctgggg agggcgcgat aacgggcatg gggggaaaat

4021 actcggtttg ttgatggagg ccatcctcgt ggtgcgcata gtatagtggg acgcgcggcg

4081 atggttttgg cgtccttcgt tggggcacgt cgatgatgag agcctctgcg acagtctgac

4141 caccaggaaa gtagaggtcg agtgtcctgc atatgtcctc ctcaggcccc agggtgcgct

4201 ccgctccacg ttgggcgtcg cgcggcgcaa tgcgaagagt gacatcgggc gagtcgaagc

4261 tccgccccag ggagtttgcg tactttcgga gaatcatatc acgcacatcg tcgaccaggt

4321 cgtcctcgcg gatctgcacg agcgtggccg aagcattggg ccgcttgacc cagatcttgc

4381 ggtgcaacat ttgggcgcta ggctggactg tcggactcag tagagcaggt ggcaacgcag

4441 gcgcaggcga aattgaaggt gatggcagtg gtagtggcag cggcagtggc agtggtgatg

4501 gggacgtgga tgtcgatgcc gaaggcgtag cagatgcgtc gatcgtgtcg ctgcctggag

4561 ccaacagcga gagcgacgac gtctgttgct ggggactgct atattcgaga acagcgggaa

4621 caggcgtagg ctcctccagt gtcagctctg gacatacaag aggctcgtcg gcagtgctgg

4681 tgggtatatg cccgttgggt gctgtattct cggcggcttc gggcgcgccc gcctcggcca

4741 gcttctgcaa cgagacgctc ttgcgtagcg agctgccgac ccgggcttgt ggatgtctct

4801 cgctgcccgc agaggatgtc aaggagcgcg tcgctgtcga ggcggagctg cctcttctga

4861 gcagcttgga acggaccttg tgcagccgcg acttgatgtt gctcat

Claims

1. The barley stripe disease pathogenicity gene pgssk2 is characterized in that the full-length sequence of pgssk2 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 pgssk2 according to claim 1, comprising the following steps: (1) cloning and sequence analysis of pgssk2 gene; (2) constructing a knockout vector and a complementation vector of the pgssk2 gene; (3) preparing protoplast and genetically transforming the vector; (4) hypha growth experiments; (6) osmotic pressure sensitive and bacteriostatic tolerance experiments; (7) cell wall integrity experiments.

3. The application of a barley stripe disease pathogenic gene pgssk2 gene in regulation of hypha growth rate is characterized in that the full-length sequence of pgsln is shown as SEQ ID NO:1 is shown.

4. The application of a barley stripe disease pathogenic gene pgssk2 gene in regulation of osmotic pressure and fungal inhibitor sensitivity is characterized in that the full-length sequence of pgsln is shown as SEQ ID NO:1 is shown.

5. The application of a barley stripe disease pathogenic gene pgssk2 gene in participating in cell wall integrity is characterized in that the full-length sequence of pgsln is shown as SEQ ID NO:1 is shown.

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

7. The application of a barley stripe disease causing gene pgssk2 in improving barley anti-prochloraz effect is characterized in that the full-length sequence of pgsln is shown as SEQ ID NO:1 is shown.