CN108130320A - Verticillium dahliae pathogenesis related protein and its encoding gene, application and mutant - Google Patents

Abstract

The invention relates to the field of pathogenic mechanism of Verticillium dahliae of cotton, in particular to pathogenicity-related protein of Verticillium dahliae of cotton and its coding gene, application and mutant. The nucleotide sequence of the gene is shown in SEQ ID No.2. The present invention screens and obtains the low pathogenicity mutant T1027, the growth rate of the mutant has no change compared with the wild type, but the amount of sporulation and the secretion of crude toxin are significantly reduced, and the gene inserted by T-DNA is identified as glucanase . Cotton Verticillium dahliae mutant T1027 is an ideal strain for studying the molecular pathogenic mechanism of cotton Verticillium dahliae. The expression and modification of its genes and proteins can be used as target sites for the design and screening of antifungal agents, and it has good application potential.

Description

Cotton Verticillium dahliae pathogenicity-related proteins and their coding genes, applications and mutants

technical field

The invention relates to the field of pathogenic mechanism of Verticillium dahliae of cotton, in particular to pathogenicity-related protein of Verticillium dahliae of cotton and its coding gene, application and mutant.

Background technique

Cotton verticillium wilt (Verticillium Wilt) is the primary disease affecting the high quality and high yield of cotton (Gossypium hirsutum L.) in major cotton producing areas in the world.

The main reasons for the outbreak of Verticillium wilt in cotton are as follows: continuous cropping in the field leads to the continuous accumulation of pathogenic bacteria in the soil; Lag, variety resistance loss quickly. In addition, the pathogenic mechanism of the pathogen is complex, and the interaction mechanism between the pathogen and the host is still unclear, which is also the basis and difficulty of research in this field. To reveal its pathogenic mechanism in detail, it is necessary to isolate and identify pathogenic related genes. At present, there are few studies on the pathogenic molecular mechanism of Verticillium dahliae to cotton at home and abroad, but the identification of pathogenicity-related genes is the basic work for in-depth study of its molecular pathogenic mechanism and the interaction between pathogenic bacteria and cotton plants.

Contents of the invention

The present invention aims to develop a shape-stable mutant of Verticillium dahliae of cotton with low pathogenicity, evaluate its application potential in the functional verification of key pathogenic genes and the study of molecular pathogenic mechanism, and complete the present invention.

The purpose of the present invention is to provide a pathogenicity related protein of Verticillium dahliae of cotton.

Another object of the present invention is to provide pathogenicity-related genes of Verticillium dahliae in cotton.

Another object of the present invention is to provide the application of the above-mentioned Verticillium dahliae pathogenicity-related protein.

Another object of the present invention is to provide the application of the above-mentioned pathogenicity-related genes of Verticillium dahliae in cotton.

Another object of the present invention is to provide a method for reducing the pathogenicity of Verticillium dahliae in cotton.

Another object of the present invention is to provide a mutant of Verticillium dahliae of cotton with reduced pathogenicity.

Another object of the present invention is to provide the application of the above-mentioned Verticillium dahliae mutants.

According to the pathogenicity-related protein of Verticillium dahliae of cotton of the present invention, its amino acid sequence is shown in SEQ ID No.1

MKTAILSGLSLLAVAMAQQVGTEEPEVHPKITWKRCTGTNGSNCATVNGEIVIDANWRWAHNVGGYENCFDGNDWTGLCTGAEDCAKNCAVEGANYGATYGVSTSGDALTLKFVTQHNFGTNIGSRTYLMENESKYQMFTLNNNELAFDVDLSTIPCGMNSALYLVPMKPDGGLSDEPNNKAGAKYGTGYCDAQCARDLKFINGKGNIVGWEPSPTDDNAGVGAMGSCCAEIDIWESNRESFAFTPHACKNNSYHVCTDTTCGGTYSEDRYGGGCDANGCDYNPYRLGNTEFYGQGKTVDTRSKFTVISRFRDNVSEQVFIQNGNVIIPPKPTLAGLTQFNAAITPEFCTAYPTIFGDRNRHDEIGGHTQLNAAYRLPMVLVLSVWADHFANMLWLDSIYPPERRGEPGVARGRCPETGRTPADVIRNHPNASVTWSNIRFGPIGSTHNTSGAVSPPVTPPSSTTRPVTPPTTTTRAATTTTRVAPPTTTTASTGGPTAPKWGQCGGQGWTGPTVCAAGSTCQAQNQWYSQCL

The pathogenicity-related gene of Verticillium dahliae of cotton according to the present invention encodes the above-mentioned pathogenicity-related protein of Verticillium dahliae of cotton.

According to the pathogenicity-related gene of Verticillium dahliae dahliae of the present invention, the nucleotide sequence of said gene is shown in SEQ ID No.2

ATGAAGACTGCGATTCTCAGCGGCCTCTCCCTGCTGGCCGTGGCCATGGCCCAGCAGGTCGGCACCGAGGAGCCCGAGGTTCACCCCAAGATCACCTGGAAGCGCTGCACCGGCACCAACGGCAGCAACTGTGCTACCGTCAACGGTGAGATCGTCATCGACGCCAACTGGCGCTGGGCCCACAACGTCGGCGGCTACGAGAACTGCTTCGACGGCAACGACTGGACCGGTCTCTGCACCGGCGCCGAGGACTGCGCCAAGAACTGCGCCGTCGAGGGTGCCAACTACGGCGCCACCTACGGTGTCTCGACCAGCGGCGATGCCCTCACCCTGAAGTTCGTCACCCAGCACAACTTTGGCACCAACATTGGCTCGCGCACCTACCTCATGGAGAACGAGTCCAAGTACCAGATGTTCACCCTGAACAACAACGAGCTGGCCTTCGACGTCGACCTGTCGACCATCCCCTGCGGCATGAACTCGGCCCTCTATCTCGTCCCCATGAAGCCCGATGGTGGTCTCTCTGACGAGCCCAACAACAAGGCTGGTGCCAAGTACGGTACCGGTTACTGTGACGCCCAGTGCGCCCGTGACCTGAAGTTCATCAACGGCAAGGGCAACATTGTCGGCTGGGAGCCCTCGCCCACCGACGACAACGCCGGTGTCGGTGCCATGGGATCTTGCTGCGCCGAGATCGACATCTGGGAGTCCAACCGCGAGTCGTTCGCCTTCACCCCCCACGCCTGCAAGAACAACAGCTACCACGTCTGCACCGACACCACCTGCGGCGGTACCTACTCCGAGGACCGCTACGGCGGTGGCTGCGACGCCAACGGCTGCGACTACAACCCTTACCGCCTCGGCAACACCGAGTTCTACGGCCAGGGCAAGACGGTTGACACACGCTCCAAGTTCACCGTCATCTCCCGCTTCCGTGACAACGTCTCCGAGCAGGTCTTCATCCAGAACGGCAACGTCATCATTCCCCCCAAGCCCACTC TCGCCGGCCTGACCCAGTTCAACGCCGCCATCACCCCCGAGTTCTGCACGGCCTACCCCACCATCTTCGGTGACCGCAACCGCCACGACGAGATTGGCGGACACACTCAGCTCAACGCTGCCTACCGCCTGCCCATGGTCCTCGTCCTGTCTGTCTGGGCCGACCACTTCGCCAACATGCTCTGGCTCGACTCCATCTACCCCCCCGAGCGCCGTGGCGAGCCCGGTGTCGCCCGTGGCCGCTGCCCCGAGACCGGCCGCACCCCCGCCGATGTCATCCGCAACCACCCCAACGCCTCCGTCACCTGGTCCAACATCCGCTTCGGCCCCATCGGCTCCACCCACAACACCTCCGGCGCCGTCAGCCCCCCGGTCACCCCTCCCAGCTCGACCACTCGCCCCGTCACGCCCCCTACCACCACCACCCGCGCCGCCACCACCACCACCCGCGTTGCTCCTCCCACCACCACCACCGCCTCCACCGGCGGCCCTACCGCTCCCAAGTGGGGCCAGTGCGGTGGCCAGGGCTGGACCGGCCCTACCGTCTGCGCCGCCGGCTCTACCTGCCAGGCCCAGAACCAGTGGTACTCTCAG TGCCTGTAA

The present invention also provides a vector comprising the above-mentioned pathogenicity-related gene of Verticillium dahliae of cotton.

The invention also provides the application of mutating the above gene to reduce the pathogenicity of Verticillium dahliae in cotton.

The present invention also provides Verticillium dahliae in which the above-mentioned gene is mutated.

The method for reducing the pathogenicity of Verticillium dahliae of cotton according to the present invention comprises the step of mutating the pathogenicity related protein of Verticillium dahliae of cotton or the gene related to pathogenicity of Verticillium dahliae of cotton.

According to a specific embodiment of the present invention, starting from the Verticillium dahliae T-DNA insertion mutant library, through biological shape measurement and pathogenicity measurement, compared with the wild type, the amount of sporulation and crude toxin are significantly reduced, The T-DNA insertion mutant T1027 with significantly weakened pathogenicity to cotton has a preservation number of CCTCC M 2017655. The test confirmed that the mutant strain is a T-DNA single insertion mutant.

According to the specific embodiment of the present invention, with the help of TAIL-PCR technology and the genome database of VdLs.17, the flanking sequence of the T-DNA insertion was obtained, and the alignment result showed that it was glucanase, and it was successfully cloned from the wild-type strain Vd080 The pathogenicity-related gene GLU was obtained.

In the present invention, the T-DNA insertion mutant library of the strong pathogenic sclerotia type strain Vd080 of Verticillium dahliae dahliae is used as a platform, and the pathogenicity of the mutant is measured by using a bottomless paper bowl quantitative dipping method, and its biology is analyzed. Characteristics, the low pathogenicity mutant T1027 was screened, the growth rate of the mutant was not changed compared with the wild type, but the sporulation production and crude toxin secretion were significantly reduced, and the gene inserted by T-DNA was identified as glucanase . Cotton Verticillium dahliae mutant T1027 is an ideal strain for studying the molecular pathogenic mechanism of cotton Verticillium dahliae. The expression and modification of its genes and proteins can be used as target sites for the design and screening of antifungal agents, and it has good application potential.

Description of drawings

Fig. 1 is the electrophoresis diagram of hygromycin gene in positive mutants detected by PCR, wherein M: DL2000 DNA maker; swimming lanes 1-25: positive mutants; swimming lane 26: Vd080.

Figure 2 shows the detection of the copy number of T-DNA insertion by Southern hybridization, in which lane 6 is T1027.

Figure 3 shows the colony morphology of wild-type Vd080 and mutant T1027 on PDA medium.

Figure 4 Determination of the pathogenicity of wild-type Vd080 and mutant T1027 on cotton.

Verticillium dahliae T1027 (Verticillium dahliae), which was preserved in the China Center for Type Culture Collection (CCTCC) (address: Wuhan University, Wuhan, China, postcode 430072) on November 03, 2017, and the preservation number is CCTCC No: M2017655.

Detailed ways

Embodiment 1 Construction of Verticillium dahliae T-DNA insertion mutant library of cotton

Cotton Verticillium dahliae Vd080 isolated from a severely diseased field in Xinji, Hebei, as the starting strain, will carry

For the Agrobacterium pCTHyg binary vector, the Agrobacterium-mediated transformation (ATMT) method was used to construct a T-DNA insertion mutant library with a capacity of 3000 carrying hygromycin (HPH) resistance selection markers. Whether there is a hygromycin label is an important basis for screening positive transformants. Use hygromycin-specific primers Hyg-F/Hyg-R (Hyg-F: 5′-GCCAAGCTTGCATGCCTGCAGGTC-3′; Hyg-R: 5′-GCGCTCGAG

TCCTCTAGAAAAGAA GGATTAC-3′) Amplified to obtain a specific band of 895bp is a positive transformant (Figure 1). The Southern hybridization technique was used to detect the copy number of T-DNA insertion in the mutant (Figure 2).

The acquisition of embodiment 2 low pathogenicity mutant T1027 bacterial strain

The pathogenicity of cotton Verticillium dahliae mutants was determined by using the upland cotton susceptible variety Jimian 11 as the identification host, and using the method of quantitative inoculation in vermiculite sandy soil with bottomless paper pots. After two rounds of primary screening and re-screening, the mutant T1027 with significantly reduced pathogenicity was obtained. Compared with the wild-type relative disease index of 50, the relative disease index of T1027 was only 8.7, and the pathogenicity was greatly reduced. See Figure 3 for comparison.

Example 3 Mutant T1027 biological shape analysis

3.1 Observation of colony morphology and determination of growth rate

Take the wild-type strain Vd080 as a control, drop 5 μL of mutant spore suspension with a concentration of 1×107/mL in the center of the PDA medium, and repeat 5 times for each strain, and culture at a constant temperature of 25°C for 10 days, and record the number of colonies growth form. And at 5d, 7d, 9d and 11d, adopt the cross method to measure the colony growth diameter respectively, and calculate the growth rate.

The results showed that compared with the wild-type Vd080, the sclerotium production of the mutant T1027 decreased sharply, and the synthesis of melanin also decreased significantly. But the growth rate of the two tends to be the same, both are 4.5mm/d.

3.2 Determination of spore production and crude toxin content

Taking the wild-type strain Vd080 as a control, drop 5 μL of the mutant spore suspension in 40 mL of the Chasch medium, 25 ° C, 150 rpm shaking culture for 6 days, and measure the amount of sporulation by means of a hemocytometer.

After continuing to shake and cultivate for 19 days, the bacterial solution was taken at 13000 rpm and centrifuged for 30 minutes. Take the supernatant, according to the Coomassie Brilliant Blue G-250 method, use bovine serum albumin as the standard protein to make a standard curve, and use a microplate reader (Synergy HT, American BioTek Company) to measure the protein concentration of Verticillium dahliae toxin as the crude toxin content.

The results showed that due to the insertion of T-DNA, the sporulation yield of T1027 was reduced from 4.2×10 ⁷ cells/ml of the wild type to 1.3×10 ⁷ cells/ml, which was as high as 67%. The secretion of crude toxin was also greatly reduced, the wild type was 58.7ml/L, while T1027 was only 39% of the wild type, 22.5ml/L.

Identification and cloning of T-DNA inserted genes in embodiment 4 mutant T1027

According to the inner DNA sequences of the T-DNA left arm (LB) and right arm (RB) of the binary vector pCTHyg (used to build the ATMT library), the 3′ and 5′ walking primers R- SP1, R-SP2, R-SP3 and L-SP1, L-SP2 and L-SP3 (Table 1), degenerate primers AP1, AP2, AP3 and AP4 provided by the Genome Walking kit, walking primers and random primers Pairs are used as templates to amplify the flanking sequences of the left and right arms of T-DNA. The primer sequences used are shown in Table 1, and the reaction procedures and reaction systems used were carried out according to the method of the Genome Walking kit. After the reaction, the reaction products of the first, second and third rounds were detected on 1% agarose gel electrophoresis. The specific products of the third round of TAIL-PCR at the 3' end and 5' end were recovered, cloned and sequenced. The sequencing results are first compared with T-DNA pairwise. If there are repeated sequences, the genome database of Verticillium dahliae VdLs.17 (http://www.broadinstitute.org/annotation /genome/verticillium_dahliae/MultiDownloads.html) for comparative analysis, and to extract the basic information of disease-related genes.

With the help of TAIL-PCR technology and comparison with the genome database of VdLs.17, it was found that the mutant T1027 T-DNA insertion flank sequence gene code is VDAG_00752, located in Verticillium dahliae and functionally annotated as glucanase. Design specific primer T1027 (ATGAAGACTGCGATTCTCAG/TTACAGGCACTGAGA GTACC) to clone the gene from wild-type Vd080. It is known that the gene contains 1602 base pairs without introns. The nucleotide sequence of the gene is shown in SEQ ID No.2, encoding 533 amino acids, and its amino acid sequence is shown in SEQ ID No.1.

Table 1 List of specific primers for TAIL-PCR

sequence listing

<110> Cotton Research Institute, Chinese Academy of Agricultural Sciences

<120> Cotton Verticillium dahliae pathogenicity related protein and its coding gene, application and mutant

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 533

<212> PRT

<213> Verticillium dahliae T1027

<400> 1

Met Lys Thr Ala Ile Leu Ser Gly Leu Ser Leu Leu Ala Val Ala Met

1 5 10 15

Ala Gln Gln Val Gly Thr Glu Glu Pro Glu Val His Pro Lys Ile Thr

20 25 30

Trp Lys Arg Cys Thr Gly Thr Asn Gly Ser Asn Cys Ala Thr Val Asn

35 40 45

Gly Glu Ile Val Ile Asp Ala Asn Trp Arg Trp Ala His Asn Val Gly

50 55 60

Gly Tyr Glu Asn Cys Phe Asp Gly Asn Asp Trp Thr Gly Leu Cys Thr

65 70 75 80

Gly Ala Glu Asp Cys Ala Lys Asn Cys Ala Val Glu Gly Ala Asn Tyr

85 90 95

Gly Ala Thr Tyr Gly Val Ser Thr Ser Gly Asp Ala Leu Thr Leu Lys

100 105 110

Phe Val Thr Gln His Asn Phe Gly Thr Asn Ile Gly Ser Arg Thr Tyr

115 120 125

Leu Met Glu Asn Glu Ser Lys Tyr Gln Met Phe Thr Leu Asn Asn Asn

130 135 140

Glu Leu Ala Phe Asp Val Asp Leu Ser Thr Ile Pro Cys Gly Met Asn

145 150 155 160

Ser Ala Leu Tyr Leu Val Pro Met Lys Pro Asp Gly Gly Leu Ser Asp

165 170 175

Glu Pro Asn Asn Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp

180 185 190

Ala Gln Cys Ala Arg Asp Leu Lys Phe Ile Asn Gly Lys Gly Asn Ile

195 200 205

Val Gly Trp Glu Pro Ser Pro Thr Asp Asp Asn Ala Gly Val Gly Ala

210 215 220

Met Gly Ser Cys Cys Ala Glu Ile Asp Ile Trp Glu Ser Asn Arg Glu

225 230 235 240

Ser Phe Ala Phe Thr Pro His Ala Cys Lys Asn Asn Ser Tyr His Val

245 250 255

Cys Thr Asp Thr Thr Cys Gly Gly Thr Tyr Ser Glu Asp Arg Tyr Gly

260 265 270

Gly Gly Cys Asp Ala Asn Gly Cys Asp Tyr Asn Pro Tyr Arg Leu Gly

275 280 285

Asn Thr Glu Phe Tyr Gly Gln Gly Lys Thr Val Asp Thr Arg Ser Lys

290 295 300

Phe Thr Val Ile Ser Arg Phe Arg Asp Asn Val Ser Glu Gln Val Phe

305 310 315 320

Ile Gln Asn Gly Asn Val Ile Ile Pro Pro Pro Lys Pro Thr Leu Ala Gly

325 330 335

Leu Thr Gln Phe Asn Ala Ala Ile Thr Pro Glu Phe Cys Thr Ala Tyr

340 345 350

Pro Thr Ile Phe Gly Asp Arg Asn Arg His Asp Glu Ile Gly Gly His

355 360 365

Thr Gln Leu Asn Ala Ala Tyr Arg Leu Pro Met Val Leu Val Leu Ser

370 375 380

Val Trp Ala Asp His Phe Ala Asn Met Leu Trp Leu Asp Ser Ile Tyr

385 390 395 400

Pro Pro Glu Arg Arg Gly Glu Pro Gly Val Ala Arg Gly Arg Cys Pro

405 410 415

Glu Thr Gly Arg Thr Pro Ala Asp Val Ile Arg Asn His Pro Asn Ala

420 425 430

Ser Val Thr Trp Ser Asn Ile Arg Phe Gly Pro Ile Gly Ser Thr His

435 440 445

Asn Thr Ser Gly Ala Val Ser Pro Pro Val Thr Pro Pro Ser Ser Ser Thr

450 455 460

Thr Arg Pro Val Thr Pro Pro Thr Thr Thr Thr Thr Arg Ala Ala Thr Thr

465 470 475 480

Thr Thr Arg Val Ala Pro Pro Thr Thr Thr Thr Thr Ala Ser Thr Gly Gly

485 490 495

Pro Thr Ala Pro Lys Trp Gly Gln Cys Gly Gly Gln Gly Trp Thr Gly

500 505 510

Pro Thr Val Cys Ala Ala Gly Ser Thr Cys Gln Ala Gln Asn Gln Trp

515 520 525

Tyr Ser Gln Cys Leu

530

<210> 2

<211> 1602

<212>DNA

<213> Verticillium dahliae T1027

<400> 2

atgaagactg cgattctcag cggcctctcc ctgctggccg tggccatggc ccagcaggtc 60

ggcaccgagg agcccgaggt tcaccccaag atcacctgga agcgctgcac cggcaccaac 120

ggcagcaact gtgctaccgt caacggtgag atcgtcatcg acgccaactg gcgctgggcc 180

cacaacgtcg gcggctacga gaactgcttc gacggcaacg actggaccgg tctctgcacc 240

ggcgccgagg actgcgccaa gaactgcgcc gtcgagggtg ccaactacgg cgccacctac 300

ggtgtctcga ccagcggcga tgccctcacc ctgaagttcg tcacccagca caactttggc 360

accaacattg gctcgcgcac ctacctcatg gagaacgagt ccaagtacca gatgttcacc 420

ctgaacaaca acgagctggc cttcgacgtc gacctgtcga ccatcccctg cggcatgaac 480

tcggccctct atctcgtccc catgaagccc gatggtggtc tctctgacga gcccaacaac 540

aaggctggtg ccaagtacgg taccggttac tgtgacgccc agtgcgcccg tgacctgaag 600

ttcatcaacg gcaagggcaa cattgtcggc tgggagccct cgcccaccga cgacaacgcc 660

ggtgtcggtg ccatgggatc ttgctgcgcc gagatcgaca tctgggagtc caaccgcgag 720

tcgttcgcct tcacccccca cgcctgcaag aacaacagct accacgtctg caccgacacc 780

acctgcggcg gtacctactc cgaggaccgc tacggcggtg gctgcgacgc caacggctgc 840

gactacaacc cttaccgcct cggcaacacc gagttctacg gccagggcaa gacggttgac 900

acacgctcca agttcaccgt catctcccgc ttccgtgaca acgtctccga gcaggtcttc 960

atccagaacg gcaacgtcat cattcccccc aagccactc tcgccggcct gacccagttc 1020

aacgccgcca tcacccccga gttctgcacg gcctacccca ccatcttcgg tgaccgcaac 1080

cgccacgacg agattggcgg acacactcag ctcaacgctg cctaccgcct gcccatggtc 1140

ctcgtcctgt ctgtctgggc cgaccacttc gccaacatgc tctggctcga ctccatctac 1200

ccccccgagc gccgtggcga gcccggtgtc gcccgtggcc gctgccccga gaccggccgc 1260

accccccgccg atgtcatccg caaccacccc aacgcctccg tcacctggtc caacatccgc 1320

ttcggcccca tcggctccac ccacaacacc tccggcgccg tcagcccccc ggtcacccct 1380

cccagctcga ccactcgccc cgtcacgccc ctaccacca ccacccgcgc cgccaccacc 1440

accacccgcg ttgctcctcc caccaccacc accgcctcca ccggcggccc taccgctccc 1500

aagtggggcc agtgcggtgg ccagggctgg accggcccta ccgtctgcgc cgccggctct 1560

acctgccagg cccagaacca gtggtactct cagtgcctgt aa 1602

Claims (9)

1. verticillium dahliae pathogenesis related protein, which is characterized in that its amino acid sequence is as shown in SEQ ID No.1.

2. verticillium dahliae pathogenic related gene, which is characterized in that encode verticillium dahliae described in claim 1 and cause a disease GAP-associated protein GAP.

3. verticillium dahliae pathogenic related gene according to claim 2, which is characterized in that the nucleotide of the gene Sequence is as shown in SEQ ID No.2.

4. include the carrier of the verticillium dahliae pathogenic related gene described in claim 2.

5. gene described in claim 2 is mutated to reduce the application of Pathogenicity of Verticillium dahliae of Cotton.

6. the verticillium dahliae that gene described in claim 2 is mutated.

A kind of 7. method for reducing verticillium dahliae pathogenicity, which is characterized in that the method includes mutation claim 1 institutes The step of stating verticillium dahliae pathogenic related gene described in verticillium dahliae pathogenesis related protein or claim 2.

8. verticillium dahliae T-DNA insertion mutation body bacterial strains, which is characterized in that its deposit number is：CCTCC M 2017655。

9. verticillium dahliae T-DNA insertion mutation body bacterial strains are in Pathogenicity of Verticillium dahliae of Cotton research side described in claim 8 The application in face.