CN111690659B - GhVLN2 gene and application thereof in resisting cotton verticillium wilt - Google Patents

Abstract

One purpose of the invention is to provide a GhVLN2 gene, the base sequence of which is shown as SEQ ID NO.1 or the amino acid sequence of which is shown as SEQ ID NO. 2. Experiments prove that the invention discovers a new gene GhVLN2, the expression of which is reduced after verticillium dahliae is infected by verticillium dahliae; the inhibition expression of GhVLN2 can improve the disease resistance of cotton to verticillium dahliae. The gene and the coded protein thereof have important functions in the process of resisting the verticillium wilt of cotton, and a new target gene is provided for improving the verticillium wilt resistance of cotton by using a gene engineering method.

Description

GhVLN2 gene and application thereof in resisting cotton verticillium wilt

Technical Field

The invention belongs to the technical field of cotton verticillium wilt prevention and control.

Background

Cotton is the most important natural fiber crop in the world and has an important position in the economic development of many countries. Vascular bundle disease, cotton verticillium wilt, caused by the soil filamentous fungus verticillium dahliae Kleb is a major disease threatening cotton production. Since the 90 s, the verticillium wilt of cotton in China is rapidly spread and continuously occurs in hundreds of cotton-planting counties (cities) in China, most of main cotton-producing areas become verticillium wilt serious areas, the quality and the yield of the cotton in China are seriously influenced, and the economic loss is huge. Although cotton breeders at home and abroad screen a batch of verticillium wilt resistant varieties by a conventional breeding method, the breeding of new disease-resistant varieties of cotton needs to be improved urgently due to the restriction of factors such as long breeding period, insufficient germplasm resources, poor broad spectrum and the like. Therefore, the molecular mechanism and genetic basis of cotton and verticillium wilt pathogen interaction are deeply researched, the key genes determining the verticillium wilt resistance are separated and identified, and then the genetically modified cotton variety with stable heredity and verticillium wilt resistance is obtained by utilizing a genetic engineering method.

The plant cytoskeleton consists of a microfilament skeleton and a microtubule skeleton, wherein the microfilament skeleton is a fibrous network structure formed by polymerizing monomer actin (G-actin), and participates in various biological processes including growth and development, cell assembly, organelle movement, biotic and abiotic stress signal response and the like (Day et al, 2011). Recent studies have found that the microfilament scaffold plays an important role in plant defense against pathogenic microorganisms, responding to a variety of microorganisms and elicitors by increasing microfilament numbers and fasciculations (Takemoto and Hardham, 2004; hunty-Ridilla et al, 2013). In vivo, microfilaments are regulated by a variety of actin-binding proteins, including actin-depolymerizing factors ADF, profilin, formin, fimbrin, etc. (Hussey et al, 2006; van Gisbergen and Bezanilla, 2013). Actin-binding proteins have been found to play an important role in host-pathogen interaction. The micro-filament nucleation protein Arp2/3 complex can mediate the motility of bacteria when invading host; capping proteins can block the positive end of the microwire, preventing the growth of the microwire on the bacterial surface (Loisel et al, 1999); the pre-fibrin profile, which promotes the polymerization of microfilaments, and the filamin fimbrin, which promotes bundling of microfilaments, are involved in actin-based movements when bacteria invade the host (Serio et al, 2010). Altered levels of ADF expression can enhance plant non-host resistance to fungal and bacterial diseases (Miklis et al, 2007); arabidopsis ADF4 gene function research finds that AtADF4 is not involved in resisting pathogen invasion, but is involved in plant disease resistance reaction as a component of a plant disease resistance signal pathway (Tian et al, 2009; Henty-Ridilla et al, 2011; 2014; Porter et al, 2012).

Villin proteins belong to the Villin/gelsolin superfamily members, which contain 6 typical highly conserved gelsolin domains and one Villin headpiece domain (Friederich et al, 1999; Klahre et al, 2000). There are 5 villin genes in Arabidopsis, and their translation products are highly conserved (Khurana et al, 2010; Huang et al, 2014). Biochemical activity studies showed that arabidopsis VLN1 only had microwire bunching activity independent of Ca ions, while the other 4 VLNs all had capping, bunching and cleavage activities (Huang et al, 2014). In general, villin genes are involved in polar growth of plants, and gene deletion mutations affect the elongation growth of leaves, roots, root hairs and pollen tubes (Zhang et al, 2010; Zhang et al, 2011 b; Bao et al, 2012; van der Houning et al, 2012; Huang et al, 2014; Wu et al, 2015). There has been no report on the function of villin in plant-pathogen interactions.

Disclosure of Invention

One purpose of the invention is to provide a GhVLN2 gene, the base sequence of which is shown as SEQ ID NO.1 or the amino acid sequence of which is shown as SEQ ID NO. 2.

The second purpose of the invention is to provide the application of the GhVLN2 gene in resisting cotton verticillium wilt.

Further, the application method comprises the following steps: taking an ORF 3 'end sequence (5'-GGGGTACCGGTGTAGTATCTGAAACCAG-3') and a 3' untranslated region sequence (5'-GCTCGAGCTTTACTTTCTGCCCATG-3') of GhVLN2 as primers, carrying out enzyme digestion on an amplified fragment by Kpn I and Xho I, connecting an obtained enzyme digestion product with a pTRV2 vector (between nucleotide sites 1682 and 1696) subjected to the same enzyme digestion, and obtaining a recombinant vector pTRV2-GhVLN 2; the recombinant vector pTRV2-GhVLN2 is introduced into Agrobacterium GV3101 to obtain recombinant bacterium pTRV2-GhVLN2/GV 3101.

The invention reduces the expression level of GhVLN2 by using a virus-mediated gene silencing method, and improves the disease resistance of cotton to verticillium wilt.

The invention separates and identifies a gene GhVLN2 responding to greensickness infection from cotton variety upland cotton, identifies the function of the gene in the greensickness resistance process, and finds that the inhibition expression of GhVLN2 can improve the greensickness resistance of the cotton.

Experiments prove that the invention discovers a new gene GhVLN2, the expression of which is reduced after verticillium dahliae is infected by verticillium dahliae; the inhibition expression of GhVLN2 can improve the disease resistance of cotton to verticillium dahliae. The gene and the coded protein thereof have important functions in the process of resisting the verticillium wilt of cotton, and a new target gene is provided for improving the verticillium wilt resistance of cotton by using a gene engineering method.

Drawings

FIG. 1 is an expression analysis diagram of GhVLN 2.

Wherein, 0, verticillium dahliae is not inoculated; 3-48hpi, 3-48 hours after the verticillium dahliae is inoculated.

FIG. 2 is a graph of expression level analysis of GhVLN2 in control (TRV:00) and silencing GhVLN2(TRV: GhVLN2) cotton.

FIG. 3 is a graph of disease resistance analysis of cotton expressing GhVLN 2.

Wherein A is a phenotype chart of control (TRV:00) and silencing GhVLN2(TRV: GhVLN2) cotton inoculated with verticillium dahliae for 2 weeks; b is disease index statistics. Represents significant difference, P < 0.05.

Detailed Description

Example 1

Cloning and expression level analysis of cotton GhVLN2 gene

Cloning of GhVLN2 gene of cotton

A cDNA encoding a gene of a gesolin domain-containing protein family is isolated from roots of cotton (Gossypium hirsutum cv TM-1), the gene is named GhVLN2, the nucleotide base of the gene is shown as SEQ ID NO.1, the Open Reading Frame (ORF) of the gene is 313 th to 3153 th nucleotides from the 5' end of a sequence 1 of a sequence table, the protein encoded by the gene is named GhVLN2, and the amino acid sequence of the protein is shown as SEQ ID NO.2 and consists of 946 amino acid residues.

Second, expression analysis of GhVLN2 Gene

RNA of cotton infected by verticillium dahliae at different time is extracted, cDNA is obtained through reverse transcription, the 5 ' untranslated region sequence of GhVLN2 is used as a primer (upstream primer: 5'-TGCTTAGCTCTTTCTCGTCCC-3'; downstream primer: 5'-ACAACTGGTTTCACAAGCGA-3'), cotton histone3 gene is used as an internal reference, and the primer of the internal reference is used as an upstream primer: 5'-GCCAAGCGTGTCACAATTATGC-3'; downstream primer: 5'-ACATCACATTGAACCTACCACTACC-3'), so that the relative expression quantity of the GhVLN2 gene is obtained. The result is shown in figure 1, compared with a non-infected control, the expression level of GhVLN2 after cotton is infected by verticillium dahliae is obviously reduced, and the expression level reaches the lowest peak after the cotton is infected by the verticillium dahliae 24 hours, which indicates that the gene participates in the immune response process of the cotton to the verticillium dahliae (figure 1).

Example 2

Construction and function identification of GhVLN2 plant expression vector

1. Construction of recombinant plant expression vectors

Construction of pTRV2-GhVLN2 plant expression vector

The 3 'end sequence (5'-GGGGTACCGGTGTAGTATCTGAAACCAG-3') and 3' untranslated region sequence (5'-GCTCGAGCTTTACTTTCTGCCCATG-3') of ORF of GhVLN2 are used as primers, the amplified fragment is cut by Kpn I and Xho I, and the obtained cut product is connected with pTRV2 vector (between 1682 and 1696 nucleotide sites) which is cut by the same enzyme, so as to obtain the recombinant vector pTRV2-GhVLN 2.

The recombinant vector is characterized in that nucleotides 2881 to 3285 in an amino acid sequence shown as SEQ ID NO.2 are inserted into pTRV2 vector.

2. Acquisition and identification of cotton capable of inhibiting expression of GhVLN2

The recombinant vector pTRV2-GhVLN2 is introduced into Agrobacterium GV3101 to obtain recombinant bacterium pTRV2-GhVLN2/GV 3101.

The recombinant bacteria are injected into the cotyledon of New Youngao No. 7 of upland cotton by a microinjection method. Two weeks later, empty vector control and pTRV2-GhVLN2 cotton root RNA were extracted and the resulting cDNA was reverse transcribed as template.

RT-QPCR was performed using GhVLN2 gene primer (forward primer: 5'-TGCTTAGCTCTTTCTCGTCCC-3'; reverse primer: 5'-ACAACTGGTTTCACAAGCGA-3') and Ghhistone3 as primers for internal reference (forward primer: 5'-GCCAAGCGTGTCACAATTATGC-3'; reverse primer: 5'-ACATCACATTGAACCTACCACTACC-3').

The Ghhistone3 gene is used as an internal reference, and the relative expression quantity of GhVLN2 in wild-type and transgenic cotton roots is calculated.

The results show that the relative expression level of GhVLN2 in the three lines is much lower than that of the wild type, indicating that cotton expressing GhVLN2 is inhibited (FIG. 2).

3. Phenotypic analysis of cotton inhibiting expression of GhVLN2

The control and transgenic cotton are inoculated with verticillium dahliae, and after 2 weeks, most leaves of the control plants are observed to be yellowed, wilted, dried and shed, while the disease of cotton inhibiting the expression of GhVLN2 is obviously reduced. Statistical results show that the disease index of cotton plants expressing GhVLN2 is obviously lower than that of a control group, namely, the resistance of cotton to verticillium wilt can be enhanced by reducing the expression of GhVLN2 (figure 3).

Table: index statistics of cotton disease for control (TRV:00) and silent GhVLN2(TRV: GhVLN 2).

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> GhVLN2 gene and application thereof in resisting cotton verticillium wilt

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Gln Lys Pro Gly Thr Glu Ile Trp Arg Ile Glu Asn Phe Gln Pro Val

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Pro Leu Pro Lys Ser Asp Tyr Gly Lys Phe Tyr Leu Gly Asp Ser Tyr

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Phe Lys Ser Asn Phe Asp Ser Trp Pro Ala Gly Ser Ala Ala Pro Gly

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Arg Val Ala Glu Phe Leu Lys Pro Gly Val Ala Leu Lys His Ala Lys

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Glu Gly Lys Glu Ser Ser Ala Phe Trp Phe Ala Leu Gly Gly Lys Leu

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Leu Phe Thr Phe Ser Leu Asn Lys Gly Lys Phe Glu Val Glu Glu Val

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Tyr Asn Phe Ser Gln Asp Asp Leu Leu Thr Glu Asp Ile Leu Ile Leu

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Asp Thr His Ala Glu Val Phe Val Trp Val Gly Gln Cys Val Glu Pro

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Lys Glu Lys Gln Asn Ala Phe Glu Ile Gly Gln Lys Tyr Ile Asp Met

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Ala Ala Ser Leu Glu Gly Leu Ser Pro His Val Pro Leu Tyr Lys Val

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Thr Glu Gly Asn Glu Pro Cys Phe Phe Thr Thr Phe Phe Ser Trp Asp

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Ser Thr Gln Ala Thr Val Gln Gly Asn Ser Phe Gln Lys Lys Val Ala

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Leu Leu Phe Gly Ala Ser His Ala Val Glu Ala Gln Asp Arg Ser Asn

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

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Leu Ser Ser Val Leu Thr Ala Glu Lys Lys Lys Gln Ser Pro Asp Ala

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Ser Pro Ile Lys Ser Thr Ser Ser Thr Pro Ala Val Thr Ser Pro Pro

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Gly Ser Thr Gln Ser Thr Phe Ser Tyr Glu Gln Leu Lys Ala Lys Ser

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Ser Asp Glu Glu Phe Gln Ala Val Phe Gly Met Glu Lys Glu Ala Phe

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Claims (2)

1. A kind ofGhVLN2The application of the gene in resisting cotton verticillium wilt is characterized in that the nucleic acid sequence of the gene is shown as SEQ ID NO.1 or the amino acid sequence is shown as SEQ ID NO. 2.

2. Use according to claim 1, characterized in that the method is: to be provided withGhVLN2The 3 '-end sequence GGGGTACCGGTGTAGTATCTGAAACCAG and the 3' -untranslated region sequence GCTCGAGCTTTACTTTCTGCCCATG of ORF of (5) are primers for amplifying a fragmentKpnI andXhoi, enzyme digestion is carried out, and an obtained enzyme digestion product is connected with a pTRV2 vector subjected to the same enzyme digestion to obtain a recombinant vector pTRV2-GhVLN 2;

the recombinant vector pTRV2-GhVLN2 is introduced into Agrobacterium GV3101 to obtain recombinant bacterium pTRV2-GhVLN2/GV 3101.

Images