CN109705201B - Cotton verticillium wilt resistance related gene GhABC and coding protein and application thereof - Google Patents

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to a cotton verticillium wilt resistance related gene GhABC, and a coding protein and application thereof. The nucleotide sequence of the cotton verticillium wilt resistance related gene GhABC is shown in SEQ ID No.2, and the amino acid sequence of the coded protein is shown in SEQ ID No. 1. After the gene silencing plant is inoculated with the cotton verticillium wilt pathogenic bacterium Vd080 spore suspension, the silencing plant shows more susceptible diseases, and the mechanism research shows that the synthesis of xylem and callose in the silencing plant is reduced, the ROS is reduced, and the down-regulation expression of partial defense genes is realized. The GhABC is proved to be positively correlated with the verticillium wilt resistance of cotton, and the GhABC can be used as a candidate gene for cotton breeding for disease resistance.

Description

Cotton verticillium wilt resistance related gene GhABC and coding protein and application thereof

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to a cotton verticillium wilt resistance related gene GhABC, and a coding protein and application thereof.

Background

Cotton is an important textile raw material and an important economic crop, however, cotton verticillium wilt serving as a worldwide disease causes great harm to the yield of cotton, the disease serving as a soil-borne vascular bundle disease has no ideal prevention and treatment measures, and the selection of disease-resistant varieties through genetic engineering becomes an important technical means for solving the cotton verticillium wilt.

Disclosure of Invention

The invention aims to provide a verticillium wilt resistant gene GhABC of cotton.

It is still another object of the present invention to provide a protein encoded by the above gene.

It is still another object of the present invention to provide a recombinant expression vector containing the above gene.

It is still another object of the present invention to provide a recombinant strain containing the above gene.

It is still another object of the present invention to provide use of the above gene.

The invention also aims to provide application of the protein.

The amino acid sequence of the GhABC protein provided by the invention is shown in SEQ ID No. 1:

the nucleotide sequence of the GhABC gene provided by the invention is shown in SEQ ID No. 2:

ATGGATGGTTTAGAAAGAGTTCGAAGTCGAAATCCCAGCAGAAGAACGGGGCATAGCAGCATAGGGAGGAGCTTAAGTAGGAGTAGTTGGAACATGGAAGATGTGTTTTCAGGTTCCAGAAGAAGTAGCCGTGTGGAAGATGATGAAGAAGCTCTAAAATGGGCTGCTATCGAGAGACTACCCACATATGATCGGCTGAGGACAAGCATCATGCAGTCCTTTGTGGATCATGAAATCATTGGCAACAAGATGGAACATAGAGAGGTTGATGTTAGAAACCTTGACATGAACGACAGACAAAAATTCATCGACATGCTCTTCAAGGTTGCTGAGGAAGATAATGAGAAATTCTTGAAGAAGTTCAGAAACAGGATCGATAAGGTTGGGATTACACTTCCAACAGTAGAAGTTAGATTCAACCATCTGACGATTGAAGCCGACTGCTACGTTGGCAGCAGAGCTCTTCCAACTCTTGTAAACTCTGCTAGAAACCTTGCAGAATCGGCTCTTGGCCTCCTTGGAATCAGTTTTGCCAAGAAAGCAAACCTCACAATTCTTAAAGATGCTTCTGGGATTATTAAACCATCAAGGATGACACTCTTACTAGGCCCACCCTCTTCTGGGAAAACAACCCTTTTGCTGGCATTGGCCGATAAGTTGGACCCAAGCTTAAGGGTTAAAGGAGAAGTCACATACAACGGATATAAACTAAAGGAATTTGTTGCTAGAAAGACATCCGCATATATCAGTCAAAATGATGTTCATGTCGGAGAAATGACAGTGAAAGAAACCTTGGATTTCTCAGCAAGATGTCAGGGTGTTGGGACACGATACGATCTGTTAAGTGAGCTTGCTAGAAGGGAAAAAGATGCAGGGATTTTCCCAGAAGCTGATGTAGACCTTTTCATGAAGGCAACTTCAGTGGAAGGAATTGAAAGCAGCCTTATCACTGATTACACACTCAAAATATTGGGGCTCGACATATGCAAGGATATCATCGTTGGAGACGAGATGCAGCGTGGAATTTCCGGAGGTCAAAAGAAAAGAGTAACAACAGGGGAGATGATTGTTGGTCCCACCAAGACACTATTCATGGATGAAATATCAACGGGTCTTGATAGTTCCACGACATACCAGATAGTGAAGTGCTTGCAGCAGGTTGTGCACCTAACAGAGGGCACAATCTTGATGTCACTATTGCAGCCTGCTCCAGAGACTTACGATCTCTTTGATGATATCATCCTCTTATCTGAGGGTCAAATTGTCTATCAAGGTCCACGAGAACACGTTGTTGAGTTCTTTGAGAGCTGTGGTTTCAAATGTCCCGAGAGGAAAGGAACTGCTGACTTTTTGCAAGAGGTTACCTCAAAGAAGGACCAAGAACAATATTGGGCGGACAAAAGAAAGCCATACAGATACATTACAGTAACTGAATTTGCAAACAGGTTCAAGCACTTCCATGTCGGAATGCAGCTACAGAGTGAGCTAGCTGTGCCTTTCGACAAGTCAAGAGGCCACCGAGCGGCATTGGCCTTCCAGAAATACTCTATGTCCAAAATGGAGCTTCTTAAGGCCTGTTGGGACAAAGAATGGCTATTGATCAAAAGGAATTCTTTTATTTATGTGTTTAAGACGGTCCAAATTATCATCGTGGCATTCATCTCGTCTACTGTCTTTTTGAGAACTGAAATGCACCAGAGGGATTTGAACGATGCGCAACTCTATATTGGCTCACTTCTGTTTGGAATGATCATCAACATGTTCAATGGCTTCGCTGAGCTCTCCCTTATGATTAGTAGGCTTCCAGTGTTCTACAAGCAAAGAGACCTCTTATTCCACCCTGTCTGGACTTTCACTCTGCCCACTTTCTTGCTCCGGGTTCCGATATCTATTTTGGAAACAGTTGCTTGGATGGCTGTAACTTATTACACTGTAGGATATGCACCTGAGGCCAGCAGGTTTTTCAAAAACTTCCTGTTGGTGTTTTCAGTACAACAAATGGCATCTGGTCTATTTCGGCTCATTGCCGGATTATGCAGAACAATGATCATAGCTAACACTGGTGGGGTTCTTACACTTCTCCTCGTGTTCTTGCTGGGAGGTTTCATCATTCCTAAACGTGAAATTCCAAGTTGGTGGGAGTGGGCTCACTGGATTTCACCTTTGACTTACGGTTTCAATGCCTTTACTGTGAATGAAATGTTTGCGTCAAGGTGGATGAATAGACAGGTTTCAAACAGTTCGACTAGCCTGGGGCTACAAGTGCTTGATAGCTTTGATGTCCCAAACGATGAAAACTGGTATTGGATTGGTGCAGGTGCTCTTCTAGGGTTCGCAGTGCTCTTCAACATTCTCTTCACCTTTGCGCTTATATACTTAAGCCCCCTTGGAAAGCCGCAGGCTATAATTTCGGAGGAAACGGTGGAAGAGCTAGAGGCTAATAATGTGGATTCTAATGAAGAACCAAGGTTAATGAGACCAGAATCGAGTAAATATTCATTCTCTGCAGATGCAAGCAATGCAGTAGAAATGGAAATCCGAAGAATGAGCAGTCGAGCTGATTCCCACGGAATGAGCAGGAATGATTCTCAAGTTGATGCAGCCACTGGTGTTGCCCCAAAGAGAGGAATGGTTCTTCCCTTCACTCCTCTAGCAATGTCTTTTGACACTGTCGATTACTACGTTGATATGCCACCTGAAATGAAGGCACAAGGAGTTGGTGAGGATAGGTTACAACTACTTCGGGGAGTAACAGGTGCATTTAGGCCTGGAGTGTTGACTGCATTGATGGGAGTCAGTGGAGCAGGGAAGACAACATTGATGGATGTTCTAGCAGGAAGAAAGACCGGTGGATATATTGAGGGTGATATCAGAATATCCGGATTCCCAAAGAAACAAGAAACCTTTGCAAGAATTTCTGGATACTGTGAACAAACTGATATTCACTCACCACAAGTGACTATCAGAGAATCCTTAATTTACTCAGCATTCCTACGACTTCCAAAAGAAATCAGCAACGAGGAAAAGATGATTTTCGTGGATGAAGTAATGGAACTAGTAGAATTAAGCAATCTCAAGGATGCCATAGTAGGGTTGCCTGGAGTCACAGGGTTGTCAACAGAGCAAAGAAAGAGGTTAACAATTGCAGTAGAGCTTGTTGCTAATCCCTCGATCATTTTCATGGATGAACCGACATCCGGTCTTGATGCGAGGGCAGCAGCCATTGTCATGAGGACTGTCAGAAACACCGTGGACACCGGAAGAACGGTTGTCTGCACCATTCATCAGCCTAGTATTGATATCTTTGAAGCCTTTGATGAATTGCTACTAATGAAGAGAGGAGGTCAGGTGATTTACTCCGGACCATTAGGCCGAAATTCTCATAAGATCATCGAATATTTTGAGGCAATTCCTGGAGTTCCCAAAATTAAGGAAAAGTATAATCCAGCTACATGGATGTTAGAAGTGAGCTCTATAGCAGCTGAAGTTAGGCTCGGAATTGATTTTGCTGAACACTACAAATCATCTTCCTTGTATCAGAGAAACAAGGCGTTAGTAAATGAGTTAAGCACACCACCTCCAGGAGCTAAAGACCTCTATTTTGCCACTCAGTACTCACAAACTACATTGGGTCAATTCAAATCATGCTTTTGGAAACAATGGTGGACTTACTGGAGAAGTCCAGATTATAACCTTGTCAGATACTTCTTCACTTTGGTCACTGCTCTCTTGGTTGGTTCTATTTTCTGGCAGATCGGCACTGACAGGAGTAAAGCATCTGATCTTACAATGATCATCGGTGCAATGTATGCTGCAGTCATATTTGTTGGAATCAATAACTGCTCAACAGTTCAACCAGTCATAGCCATTGAAAGAACAGTGTTCTATCGTGAAAGAGCTGCTGGGATGTACTCTGCATTACCTTATGCCCTTGCGCAGGTGCTTTGTGAAATACCTTACGTATTTGGCCAAACCGTATACTATACACTTATAGTGTATGCCATGGTGGGCTTTCAATGGACAGTGGCAAAGTACTTCTGGTTTTTCTTTGTCAGCTTCTTCACCTTCCTTTACTTTACATACTACGGAATGATGACTGTTTCGATCACACCAAACCATCAAATATCATCTATATTTGCTGCAGCATTCTATTCAGTCTTTAATCTTTTCTCCGGCTTCTTCATTCCAAGACCAAGAATTCCTGGTTGGTGGATCTGGTATTACTGGATTTGCCCGGTTGCATGGACAATTTACGGATTGATTGCGTCACAATATGGAGATCTTGAAGACAAAATTAGTGTACCTGGCGTCTCTCCTGACCCTACTATTAAGTCGTATATTAAAGATCAGTACGGCTATGATTCAGACTTCATGGGGCCAGTTGCTGCAGTTTTGGTTGGCTTTGGAGTATTTTTTGCCACTTTGTTTGCCTACTGCATAAGGACACTCAATTTCCAGACCAGATAA

the invention also provides a recombinant expression vector and a cell containing the coding gene.

The invention provides application of GhABC protein and a coding gene thereof in cotton verticillium wilt resistance.

After the plant material is inoculated with cotton verticillium wilt pathogenic bacteria, the serine (Ser, S) at positions 855, 859 and 863 of the GhABC protein is subjected to phosphorylation modification, and the lysine (Lys, K) at position 440 is subjected to ubiquitination modification.

Furthermore, test results show that the protein is up-regulated in cotton body in a key disease-resistant period after inoculation of pathogenic bacteria, the expression of the GhABC gene is activated under the stress of the pathogenic bacteria, and after treatment of hormones (jasmonic acid, ethylene, salicylic acid and hydrogen peroxide), the expression of the GhABC gene is changed, and the GhABC gene is regulated and controlled by the hormones.

The invention constructs a silencing vector of the gene GhABC, and inhibits the expression of the GhABC in cotton by utilizing a virus-mediated gene silencing technology. After the gene silencing plant is inoculated with a cotton verticillium wilt pathogenic bacterium Vd080 spore suspension, the silencing plant shows more susceptible diseases, and the mechanism research finds that the synthesis of xylem and callose in the silencing plant is reduced, the ROS is reduced, and the down-regulation expression of partial defense genes is realized, so that the GhABC gene is proved to be positively correlated with the cotton verticillium wilt resistance, and the GhABC gene can be used for screening cotton disease resistance breeding.

Drawings

FIG. 1 shows GhABC expression in resistant/susceptible varieties under pathogenic stress;

FIG. 2 shows GhABC expression in disease-resistant varieties after hormone treatment;

FIG. 3 shows GhABC expression in susceptible varieties after hormone treatment;

FIG. 4 shows the albinism and morbidity of the leaves of plants after gene silencing;

FIG. 5 shows the accumulation of xylem in plants following gene silencing;

FIG. 6 shows the accumulation of callose in plant leaves after gene silencing;

FIG. 7 shows the burst of reactive oxygen species in the leaves of plants following gene silencing;

FIG. 8 shows the relative expression levels of the relevant defense genes in plants after pathogen treatment.

Detailed Description

Example 1 acquisition of Cotton Gene GhABC

The method comprises the steps of taking cotton No.2 planted in a disease-resistant variety and Ji cotton No. 11 infected by a disease-susceptible variety as plant materials, inoculating pathogen Vd080 of cotton verticillium wilt, extracting cotton holoprotein, and taking cotton which is not inoculated with pathogen as a blank control. Performing enzymolysis on the extracted total protein with trypsin, and using metal oxide TiO₂The phosphorylation peptide containing serine (S), threonine (T) and tyrosine (Y) is enriched by utilizing the affinity of the phosphate group, the acetylation peptide in the complex sample is specifically enriched by utilizing the motif antibody with high affinity to the acetylation lysine, the ubiquitination peptide in the complex sample is specifically enriched by utilizing the motif antibody with high affinity to the ubiquitination lysine, the different modified enrichments are subjected to a liquid chromatography mass spectrometry (LC-MS/MS) protein quantitative method, the quantitative analysis of large-scale protein modification is realized, and the biological information analysis is carried out according to the quantitative analysis result.

According to the results of the biogenic analysis, the protein GhABC with the amino acid sequence shown as SEQ ID No.1 is obtained by screening. The phosphorylation intensity of the protein GhABC in a disease-resistant control variety is 15.12, and the phosphorylation intensity in a disease-susceptible variety is 9.60; the ubiquitination intensity in the disease-resistant variety is 5.49, and the ubiquitination intensity in the disease-susceptible variety is 1.35.

Example 2 expression of GhABC in Cotton after pathogen and hormone treatment

The experimental materials are No.2 cotton and No. 11 cotton which are disease-resistant varieties of cotton planted in vermiculite sandy soil paper bowls.

After the root injury, Vd080 spore suspension is inoculated, and root RNA is extracted at 12h, 24h, 48h and 72h respectively.

With 0.5mM hydrogen peroxide (H)₂O₂) 0.1mM Salicylic Acid (SA), 0.15mM methyl Jasmonate (JA) and 1mM Ethylene (ET) are sprayed on the leaf surface until the leaf surface drips, and root RNA is extracted in 12h, 24h, 48h and 72h respectively.

The fluorescent quantitative primer is designed as follows: GhABC-F: TTCAGCCTGTTGGTCGTG, GhABC-R: GCGGGATTATTATGTCCTTG. Detecting the expression of GhABC after the treatment of pathogenic bacteria and hormone.

As shown in FIG. 1, in susceptible and disease-resistant varieties, GhABC is inhibited from being expressed in the early stage of pathogenic bacteria inoculation, but is greatly increased in the critical stage of disease resistance. It can be seen that GhABC plays an important role in plant disease resistance.

As shown in FIGS. 2 and 3, after hormone treatment, GhABC of the disease-resistant variety was sensitive to all hormones, but expression was suppressed except for ET, and GhABC of the disease-sensitive variety was sensitive to ET and H₂O₂Sensitive, indicating that the expression of GhABC is probably influenced by ET and H₂O₂Regulation and control of (1).

Example 3 study of GhABC function Using Virus-mediated Gene silencing technology (VIGS)

1. Silencing gene GhABC in cotton

Primer for designing GhABC silencing vector

ABC-VIGS-F:GCTCTAGATCAGAAACACCGTGGACA，

ABC-VIGS-R:GGGGTACCTTAACTCATTTACTAACGCCTT。

Amplifying a silent segment by taking cDNA of cotton plant No.2 in a disease-resistant variety as a template, transforming pYL-156 vector, transforming escherichia coli DH5 alpha competent cells, extracting pYL-156-GhABC plasmid after sequencing verification is correct, transforming agrobacterium GV3101 competence, carrying out colony PCR verification, carrying out amplification culture, taking pYL-156 idle load as a control, taking pYL-156-PDS as a positive control (after the PDS gene is silenced, the whitening phenomenon of leaves) to mix and stand with helper plasmid pYL-192, and injecting cotton plant No.2 cotton leaves by a needle-free injector. Dark culture is carried out for 24h after injection treatment, and the culture is carried out at 22 ℃ under normal illumination. When the positive control shows a albino phenotype, detecting the expression level of GhABC in the silent plant by using fluorescent quantitative PCR, and selecting the plant with better silent effect for further test.

2. Disease resistance study of silenced plants

Selecting plants with good silencing effect, and inoculating 10mL of Verticillium dahliae spore solution (the concentration is 2 multiplied by 10) when one true leaf is initialized⁷CFU/mL), placed in a greenhouse at 25 ℃ for growth under normal light. After inoculation, samples were taken at different time periods, and silent plant RNA was extracted for detecting expression of the defense genes shown in table 1. And (3) detecting the accumulation of callose in xylem and leaf callose of the cotton stem and detecting the outbreak of active oxygen in the cotton leaf when inoculating the strain for 3 d. And detecting the necrosis of leaf cells at 15 d. At 20d, the plants were investigated for disease.

TABLE 1 RT-qPCR primers for Cotton defense-related genes

As shown in FIG. 4, the disease index of the silenced plant is calculated to be 60.12 + -0.99 and the disease rate is 100% after counting the disease condition, while the disease index of the control is 21.92 + -2.68 and the disease rate is 83.74 + -2.56%, and the disease index and the disease rate of the silenced plant and the control are very different.

As shown in fig. 5, after cotton seedling stalks were stained with phloroglucinol and incubated with concentrated sulfuric acid, accumulation of xylem of silenced plants was observed to be significantly lower than that of non-silenced plants under an orthographic microscope. As shown in FIG. 6, cotton leaves were stained with aniline blue, and plants visibly silenced under UV excitation had a lower accumulation of callose than controls. As shown in fig. 7, the active oxygen burst experiments showed less brown precipitate in the leaves of the silenced plants, indicating a weaker active oxygen burst. More cell necrosis was observed in silent plants by staining cotton leaves with aniline blue.

The expression levels of defensive enzyme genes or defensive enzyme metabolism genes, such as phenylalanine ammonia lyase (GhPAL), cinnamic acid-4-hydroxylase (GhC4H1), peroxidase (GhPOD) and polyphenol oxidase (GhPOO), in silenced plants are reduced to different degrees after inoculation with pathogenic bacteria at different time intervals after inoculation with pathogenic bacteria. The expression levels of marker genes GhHSR203J and GhHIN1 of anaphylaxis in cotton are lower than those of a control in the whole detection period, and the fact that the silencing of GhABC causes the reduction of active oxygen is shown. GhPR3 is a marker gene of an Ethylene (ET) signal pathway, and the expression of the gene at the initial stage of pathogen inoculation in a silent plant is obviously lower than that of a control, which indicates that ET plays a positive regulation role in the initial stage of GhABC disease resistance. GhJaZ1 is an identification gene of a JA signal channel, and the expression in a silent plant is higher than that of a control, indicating that GhABC is possibly negatively regulated by JA. GhPR1 and GhNPR1 are identification genes of a salicylic acid signal channel, and the expression quantity in a silent plant is higher than that in a control, which indicates that salicylic acid plays a role in negative regulation in GhABC disease resistance. GhNOA1 is a nitric oxide pathway gene, and the expression level in silent plants is obviously lower than that of a control, indicating that GhABC is related to a nitric oxide pathway. Therefore, the GhABC gene has an important positive regulation function in cotton verticillium wilt resistance, and can be used as an important candidate gene for cotton verticillium wilt resistance breeding.

Sequence listing

<110> Cotton research institute of Chinese academy of agricultural sciences

<120> verticillium wilt resistance related gene GhABC of cotton, and coding protein and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

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<211> 1488

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<213> Cotton (Gossypium spp)

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Gly His Ser Ser Ile Gly Arg Ser Leu Ser Arg Ser Ser Trp Asn Met

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Glu Asp Val Phe Ser Gly Ser Arg Arg Ser Ser Arg Val Glu Asp Asp

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Glu Glu Ala Leu Lys Trp Ala Ala Ile Glu Arg Leu Pro Thr Tyr Asp

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Arg Leu Arg Thr Ser Ile Met Gln Ser Phe Val Asp His Glu Ile Ile

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Gly Asn Lys Met Glu His Arg Glu Val Asp Val Arg Asn Leu Asp Met

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Asn Asp Arg Gln Lys Phe Ile Asp Met Leu Phe Lys Val Ala Glu Glu

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Asp Asn Glu Lys Phe Leu Lys Lys Phe Arg Asn Arg Ile Asp Lys Val

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Gly Ile Thr Leu Pro Thr Val Glu Val Arg Phe Asn His Leu Thr Ile

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Glu Ala Asp Cys Tyr Val Gly Ser Arg Ala Leu Pro Thr Leu Val Asn

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Ser Ala Arg Asn Leu Ala Glu Ser Ala Leu Gly Leu Leu Gly Ile Ser

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Phe Ala Lys Lys Ala Asn Leu Thr Ile Leu Lys Asp Ala Ser Gly Ile

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Ile Lys Pro Ser Arg Met Thr Leu Leu Leu Gly Pro Pro Ser Ser Gly

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Lys Thr Thr Leu Leu Leu Ala Leu Ala Asp Lys Leu Asp Pro Ser Leu

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Arg Val Lys Gly Glu Val Thr Tyr Asn Gly Tyr Lys Leu Lys Glu Phe

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Val Ala Arg Lys Thr Ser Ala Tyr Ile Ser Gln Asn Asp Val His Val

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Gly Glu Met Thr Val Lys Glu Thr Leu Asp Phe Ser Ala Arg Cys Gln

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Gly Val Gly Thr Arg Tyr Asp Leu Leu Ser Glu Leu Ala Arg Arg Glu

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Lys Asp Ala Gly Ile Phe Pro Glu Ala Asp Val Asp Leu Phe Met Lys

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Ala Thr Ser Val Glu Gly Ile Glu Ser Ser Leu Ile Thr Asp Tyr Thr

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Leu Lys Ile Leu Gly Leu Asp Ile Cys Lys Asp Ile Ile Val Gly Asp

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Glu Met Gln Arg Gly Ile Ser Gly Gly Gln Lys Lys Arg Val Thr Thr

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Gly Glu Met Ile Val Gly Pro Thr Lys Thr Leu Phe Met Asp Glu Ile

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Ser Thr Gly Leu Asp Ser Ser Thr Thr Tyr Gln Ile Val Lys Cys Leu

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Gln Gln Val Val His Leu Thr Glu Gly Thr Ile Leu Met Ser Leu Leu

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Gln Pro Ala Pro Glu Thr Tyr Asp Leu Phe Asp Asp Ile Ile Leu Leu

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Ser Glu Gly Gln Ile Val Tyr Gln Gly Pro Arg Glu His Val Val Glu

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Phe Phe Glu Ser Cys Gly Phe Lys Cys Pro Glu Arg Lys Gly Thr Ala

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Asp Phe Leu Gln Glu Val Thr Ser Lys Lys Asp Gln Glu Gln Tyr Trp

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Ala Asp Lys Arg Lys Pro Tyr Arg Tyr Ile Thr Val Thr Glu Phe Ala

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Asn Arg Phe Lys His Phe His Val Gly Met Gln Leu Gln Ser Glu Leu

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Ala Val Pro Phe Asp Lys Ser Arg Gly His Arg Ala Ala Leu Ala Phe

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Gln Lys Tyr Ser Met Ser Lys Met Glu Leu Leu Lys Ala Cys Trp Asp

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Lys Glu Trp Leu Leu Ile Lys Arg Asn Ser Phe Ile Tyr Val Phe Lys

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Thr Val Gln Ile Ile Ile Val Ala Phe Ile Ser Ser Thr Val Phe Leu

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Arg Thr Glu Met His Gln Arg Asp Leu Asn Asp Ala Gln Leu Tyr Ile

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Gly Ser Leu Leu Phe Gly Met Ile Ile Asn Met Phe Asn Gly Phe Ala

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Glu Leu Ser Leu Met Ile Ser Arg Leu Pro Val Phe Tyr Lys Gln Arg

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Asp Leu Leu Phe His Pro Val Trp Thr Phe Thr Leu Pro Thr Phe Leu

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Leu Arg Val Pro Ile Ser Ile Leu Glu Thr Val Ala Trp Met Ala Val

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Thr Tyr Tyr Thr Val Gly Tyr Ala Pro Glu Ala Ser Arg Phe Phe Lys

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Asn Phe Leu Leu Val Phe Ser Val Gln Gln Met Ala Ser Gly Leu Phe

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Arg Leu Ile Ala Gly Leu Cys Arg Thr Met Ile Ile Ala Asn Thr Gly

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Gly Val Leu Thr Leu Leu Leu Val Phe Leu Leu Gly Gly Phe Ile Ile

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Pro Lys Arg Glu Ile Pro Ser Trp Trp Glu Trp Ala His Trp Ile Ser

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Pro Leu Thr Tyr Gly Phe Asn Ala Phe Thr Val Asn Glu Met Phe Ala

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Ser Arg Trp Met Asn Arg Gln Val Ser Asn Ser Ser Thr Ser Leu Gly

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Leu Gln Val Leu Asp Ser Phe Asp Val Pro Asn Asp Glu Asn Trp Tyr

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Trp Ile Gly Ala Gly Ala Leu Leu Gly Phe Ala Val Leu Phe Asn Ile

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Leu Phe Thr Phe Ala Leu Ile Tyr Leu Ser Pro Leu Gly Lys Pro Gln

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Ala Ile Ile Ser Glu Glu Thr Val Glu Glu Leu Glu Ala Asn Asn Val

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Asp Ser Asn Glu Glu Pro Arg Leu Met Arg Pro Glu Ser Ser Lys Tyr

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Ser Phe Ser Ala Asp Ala Ser Asn Ala Val Glu Met Glu Ile Arg Arg

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Met Ser Ser Arg Ala Asp Ser His Gly Met Ser Arg Asn Asp Ser Gln

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Val Asp Ala Ala Thr Gly Val Ala Pro Lys Arg Gly Met Val Leu Pro

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Phe Thr Pro Leu Ala Met Ser Phe Asp Thr Val Asp Tyr Tyr Val Asp

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Met Pro Pro Glu Met Lys Ala Gln Gly Val Gly Glu Asp Arg Leu Gln

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Leu Leu Arg Gly Val Thr Gly Ala Phe Arg Pro Gly Val Leu Thr Ala

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

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Ala Gly Arg Lys Thr Gly Gly Tyr Ile Glu Gly Asp Ile Arg Ile Ser

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Gly Phe Pro Lys Lys Gln Glu Thr Phe Ala Arg Ile Ser Gly Tyr Cys

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Glu Gln Thr Asp Ile His Ser Pro Gln Val Thr Ile Arg Glu Ser Leu

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Ile Tyr Ser Ala Phe Leu Arg Leu Pro Lys Glu Ile Ser Asn Glu Glu

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Lys Met Ile Phe Val Asp Glu Val Met Glu Leu Val Glu Leu Ser Asn

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Leu Lys Asp Ala Ile Val Gly Leu Pro Gly Val Thr Gly Leu Ser Thr

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Glu Gln Arg Lys Arg Leu Thr Ile Ala Val Glu Leu Val Ala Asn Pro

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Ser Ile Ile Phe Met Asp Glu Pro Thr Ser Gly Leu Asp Ala Arg Ala

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Ala Ala Ile Val Met Arg Thr Val Arg Asn Thr Val Asp Thr Gly Arg

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Thr Val Val Cys Thr Ile His Gln Pro Ser Ile Asp Ile Phe Glu Ala

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Phe Asp Glu Leu Leu Leu Met Lys Arg Gly Gly Gln Val Ile Tyr Ser

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Gly Pro Leu Gly Arg Asn Ser His Lys Ile Ile Glu Tyr Phe Glu Ala

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Ile Pro Gly Val Pro Lys Ile Lys Glu Lys Tyr Asn Pro Ala Thr Trp

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Met Leu Glu Val Ser Ser Ile Ala Ala Glu Val Arg Leu Gly Ile Asp

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Phe Ala Glu His Tyr Lys Ser Ser Ser Leu Tyr Gln Arg Asn Lys Ala

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Leu Val Asn Glu Leu Ser Thr Pro Pro Pro Gly Ala Lys Asp Leu Tyr

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

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Phe Trp Lys Gln Trp Trp Thr Tyr Trp Arg Ser Pro Asp Tyr Asn Leu

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Val Arg Tyr Phe Phe Thr Leu Val Thr Ala Leu Leu Val Gly Ser Ile

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Phe Trp Gln Ile Gly Thr Asp Arg Ser Lys Ala Ser Asp Leu Thr Met

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Ile Ile Gly Ala Met Tyr Ala Ala Val Ile Phe Val Gly Ile Asn Asn

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Cys Ser Thr Val Gln Pro Val Ile Ala Ile Glu Arg Thr Val Phe Tyr

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Arg Glu Arg Ala Ala Gly Met Tyr Ser Ala Leu Pro Tyr Ala Leu Ala

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Gln Val Leu Cys Glu Ile Pro Tyr Val Phe Gly Gln Thr Val Tyr Tyr

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Thr Leu Ile Val Tyr Ala Met Val Gly Phe Gln Trp Thr Val Ala Lys

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Tyr Phe Trp Phe Phe Phe Val Ser Phe Phe Thr Phe Leu Tyr Phe Thr

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Tyr Tyr Gly Met Met Thr Val Ser Ile Thr Pro Asn His Gln Ile Ser

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Ser Ile Phe Ala Ala Ala Phe Tyr Ser Val Phe Asn Leu Phe Ser Gly

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Phe Phe Ile Pro Arg Pro Arg Ile Pro Gly Trp Trp Ile Trp Tyr Tyr

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Trp Ile Cys Pro Val Ala Trp Thr Ile Tyr Gly Leu Ile Ala Ser Gln

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Tyr Gly Asp Leu Glu Asp Lys Ile Ser Val Pro Gly Val Ser Pro Asp

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Pro Thr Ile Lys Ser Tyr Ile Lys Asp Gln Tyr Gly Tyr Asp Ser Asp

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Phe Met Gly Pro Val Ala Ala Val Leu Val Gly Phe Gly Val Phe Phe

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Ala Thr Leu Phe Ala Tyr Cys Ile Arg Thr Leu Asn Phe Gln Thr Arg

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<212> DNA

<213> Cotton (Gossypium spp)

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atggatggtt tagaaagagt tcgaagtcga aatcccagca gaagaacggg gcatagcagc 60

atagggagga gcttaagtag gagtagttgg aacatggaag atgtgttttc aggttccaga 120

agaagtagcc gtgtggaaga tgatgaagaa gctctaaaat gggctgctat cgagagacta 180

cccacatatg atcggctgag gacaagcatc atgcagtcct ttgtggatca tgaaatcatt 240

ggcaacaaga tggaacatag agaggttgat gttagaaacc ttgacatgaa cgacagacaa 300

aaattcatcg acatgctctt caaggttgct gaggaagata atgagaaatt cttgaagaag 360

ttcagaaaca ggatcgataa ggttgggatt acacttccaa cagtagaagt tagattcaac 420

catctgacga ttgaagccga ctgctacgtt ggcagcagag ctcttccaac tcttgtaaac 480

tctgctagaa accttgcaga atcggctctt ggcctccttg gaatcagttt tgccaagaaa 540

gcaaacctca caattcttaa agatgcttct gggattatta aaccatcaag gatgacactc 600

ttactaggcc caccctcttc tgggaaaaca acccttttgc tggcattggc cgataagttg 660

gacccaagct taagggttaa aggagaagtc acatacaacg gatataaact aaaggaattt 720

gttgctagaa agacatccgc atatatcagt caaaatgatg ttcatgtcgg agaaatgaca 780

gtgaaagaaa ccttggattt ctcagcaaga tgtcagggtg ttgggacacg atacgatctg 840

ttaagtgagc ttgctagaag ggaaaaagat gcagggattt tcccagaagc tgatgtagac 900

cttttcatga aggcaacttc agtggaagga attgaaagca gccttatcac tgattacaca 960

ctcaaaatat tggggctcga catatgcaag gatatcatcg ttggagacga gatgcagcgt 1020

ggaatttccg gaggtcaaaa gaaaagagta acaacagggg agatgattgt tggtcccacc 1080

aagacactat tcatggatga aatatcaacg ggtcttgata gttccacgac ataccagata 1140

gtgaagtgct tgcagcaggt tgtgcaccta acagagggca caatcttgat gtcactattg 1200

cagcctgctc cagagactta cgatctcttt gatgatatca tcctcttatc tgagggtcaa 1260

attgtctatc aaggtccacg agaacacgtt gttgagttct ttgagagctg tggtttcaaa 1320

tgtcccgaga ggaaaggaac tgctgacttt ttgcaagagg ttacctcaaa gaaggaccaa 1380

gaacaatatt gggcggacaa aagaaagcca tacagataca ttacagtaac tgaatttgca 1440

aacaggttca agcacttcca tgtcggaatg cagctacaga gtgagctagc tgtgcctttc 1500

gacaagtcaa gaggccaccg agcggcattg gccttccaga aatactctat gtccaaaatg 1560

gagcttctta aggcctgttg ggacaaagaa tggctattga tcaaaaggaa ttcttttatt 1620

tatgtgttta agacggtcca aattatcatc gtggcattca tctcgtctac tgtctttttg 1680

agaactgaaa tgcaccagag ggatttgaac gatgcgcaac tctatattgg ctcacttctg 1740

tttggaatga tcatcaacat gttcaatggc ttcgctgagc tctcccttat gattagtagg 1800

cttccagtgt tctacaagca aagagacctc ttattccacc ctgtctggac tttcactctg 1860

cccactttct tgctccgggt tccgatatct attttggaaa cagttgcttg gatggctgta 1920

acttattaca ctgtaggata tgcacctgag gccagcaggt ttttcaaaaa cttcctgttg 1980

gtgttttcag tacaacaaat ggcatctggt ctatttcggc tcattgccgg attatgcaga 2040

acaatgatca tagctaacac tggtggggtt cttacacttc tcctcgtgtt cttgctggga 2100

ggtttcatca ttcctaaacg tgaaattcca agttggtggg agtgggctca ctggatttca 2160

cctttgactt acggtttcaa tgcctttact gtgaatgaaa tgtttgcgtc aaggtggatg 2220

aatagacagg tttcaaacag ttcgactagc ctggggctac aagtgcttga tagctttgat 2280

gtcccaaacg atgaaaactg gtattggatt ggtgcaggtg ctcttctagg gttcgcagtg 2340

ctcttcaaca ttctcttcac ctttgcgctt atatacttaa gcccccttgg aaagccgcag 2400

gctataattt cggaggaaac ggtggaagag ctagaggcta ataatgtgga ttctaatgaa 2460

gaaccaaggt taatgagacc agaatcgagt aaatattcat tctctgcaga tgcaagcaat 2520

gcagtagaaa tggaaatccg aagaatgagc agtcgagctg attcccacgg aatgagcagg 2580

aatgattctc aagttgatgc agccactggt gttgccccaa agagaggaat ggttcttccc 2640

ttcactcctc tagcaatgtc ttttgacact gtcgattact acgttgatat gccacctgaa 2700

atgaaggcac aaggagttgg tgaggatagg ttacaactac ttcggggagt aacaggtgca 2760

tttaggcctg gagtgttgac tgcattgatg ggagtcagtg gagcagggaa gacaacattg 2820

atggatgttc tagcaggaag aaagaccggt ggatatattg agggtgatat cagaatatcc 2880

ggattcccaa agaaacaaga aacctttgca agaatttctg gatactgtga acaaactgat 2940

attcactcac cacaagtgac tatcagagaa tccttaattt actcagcatt cctacgactt 3000

ccaaaagaaa tcagcaacga ggaaaagatg attttcgtgg atgaagtaat ggaactagta 3060

gaattaagca atctcaagga tgccatagta gggttgcctg gagtcacagg gttgtcaaca 3120

gagcaaagaa agaggttaac aattgcagta gagcttgttg ctaatccctc gatcattttc 3180

atggatgaac cgacatccgg tcttgatgcg agggcagcag ccattgtcat gaggactgtc 3240

agaaacaccg tggacaccgg aagaacggtt gtctgcacca ttcatcagcc tagtattgat 3300

atctttgaag cctttgatga attgctacta atgaagagag gaggtcaggt gatttactcc 3360

ggaccattag gccgaaattc tcataagatc atcgaatatt ttgaggcaat tcctggagtt 3420

cccaaaatta aggaaaagta taatccagct acatggatgt tagaagtgag ctctatagca 3480

gctgaagtta ggctcggaat tgattttgct gaacactaca aatcatcttc cttgtatcag 3540

agaaacaagg cgttagtaaa tgagttaagc acaccacctc caggagctaa agacctctat 3600

tttgccactc agtactcaca aactacattg ggtcaattca aatcatgctt ttggaaacaa 3660

tggtggactt actggagaag tccagattat aaccttgtca gatacttctt cactttggtc 3720

actgctctct tggttggttc tattttctgg cagatcggca ctgacaggag taaagcatct 3780

gatcttacaa tgatcatcgg tgcaatgtat gctgcagtca tatttgttgg aatcaataac 3840

tgctcaacag ttcaaccagt catagccatt gaaagaacag tgttctatcg tgaaagagct 3900

gctgggatgt actctgcatt accttatgcc cttgcgcagg tgctttgtga aataccttac 3960

gtatttggcc aaaccgtata ctatacactt atagtgtatg ccatggtggg ctttcaatgg 4020

acagtggcaa agtacttctg gtttttcttt gtcagcttct tcaccttcct ttactttaca 4080

tactacggaa tgatgactgt ttcgatcaca ccaaaccatc aaatatcatc tatatttgct 4140

gcagcattct attcagtctt taatcttttc tccggcttct tcattccaag accaagaatt 4200

cctggttggt ggatctggta ttactggatt tgcccggttg catggacaat ttacggattg 4260

attgcgtcac aatatggaga tcttgaagac aaaattagtg tacctggcgt ctctcctgac 4320

cctactatta agtcgtatat taaagatcag tacggctatg attcagactt catggggcca 4380

gttgctgcag ttttggttgg ctttggagta ttttttgcca ctttgtttgc ctactgcata 4440

aggacactca atttccagac cagataa 4467

Claims (2)

1. The application of the silencing vector of the cotton gene GhABC in the aspect of negative regulation of cotton verticillium wilt resistance is characterized in that the cotton gene GhABC encodes a protein with an amino acid sequence shown in SEQ ID No. 1.

2. The use of claim 1, wherein the cotton gene GhABC has the nucleotide sequence shown in SEQ ID No. 2.

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