CN109705201B - Cotton verticillium wilt related gene GhABC and its encoded protein and application - 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 related gene GhABC and its encoded protein and application

technical field

The invention belongs to the field of agricultural biotechnology, and particularly relates to a cotton verticillium wilt resistance-related gene GhABC and its encoded protein and applications.

Background technique

Cotton is an important textile raw material and an important economic crop. However, as a worldwide disease, cotton verticillium wilt causes huge harm to cotton production. As a soil-borne vascular disease, there is no ideal It has become an important technical means to solve cotton Verticillium wilt through genetic engineering breeding of disease-resistant varieties.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a cotton verticillium wilt resistance phase gene GhABC.

Another object of the present invention is to provide the encoded protein of the above-mentioned gene.

Another object of the present invention is to provide a recombinant expression vector containing the above-mentioned gene.

Another object of the present invention is to provide a recombinant strain containing the above-mentioned gene.

Still another object of the present invention is to provide applications of the above-mentioned genes.

Another object of the present invention is to provide the application of the above-mentioned protein.

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

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

ATGGATGGTTTAGAAAGAGTTCGAAGTCGAAATCCCAGCAGAAGAACGGGGCATAGCAGCATAGGGAGGAGCTTAAGTAGGAGTAGTTGGAACATGGAAGATGTGTTTTCAGGTTCCAGAAGAAGTAGCCGTGTGGAAGATGATGAAGAAGCTCTAAAATGGGCTGCTATCGAGAGACTACCCACATATGATCGGCTGAGGACAAGCATCATGCAGTCCTTTGTGGATCATGAAATCATTGGCAACAAGATGGAACATAGAGAGGTTGATGTTAGAAACCTTGACATGAACGACAGACAAAAATTCATCGACATGCTCTTCAAGGTTGCTGAGGAAGATAATGAGAAATTCTTGAAGAAGTTCAGAAACAGGATCGATAAGGTTGGGATTACACTTCCAACAGTAGAAGTTAGATTCAACCATCTGACGATTGAAGCCGACTGCTACGTTGGCAGCAGAGCTCTTCCAACTCTTGTAAACTCTGCTAGAAACCTTGCAGAATCGGCTCTTGGCCTCCTTGGAATCAGTTTTGCCAAGAAAGCAAACCTCACAATTCTTAAAGATGCTTCTGGGATTATTAAACCATCAAGGATGACACTCTTACTAGGCCCACCCTCTTCTGGGAAAACAACCCTTTTGCTGGCATTGGCCGATAAGTTGGACCCAAGCTTAAGGGTTAAAGGAGAAGTCACATACAACGGATATAAACTAAAGGAATTTGTTGCTAGAAAGACATCCGCATATATCAGTCAAAATGATGTTCATGTCGGAGAAATGACAGTGAAAGAAACCTTGGATTTCTCAGCAAGATGTCAGGGTGTTGGGACACGATACGATCTGTTAAGTGAGCTTGCTAGAAGGGAAAAAGATGCAGGGATTTTCCCAGAAGCTGATGTAGACCTTTTCATGAAGGCAACTTCAGTGGAAGGAATTGAAAGCAGCCTTATCACTGATTACACACTCAAAATATTGGGGCTCGACATATGCAAGGATATCATCG TTGGAGACGAGATGCAGCGTGGAATTTCCGGAGGTCAAAAGAAAAGAGTAACAACAGGGGAGATGATTGTTGGTCCCACCAAGACACTATTCATGGATGAAATATCAACGGGTCTTGATAGTTCCACGACATACCAGATAGTGAAGTGCTTGCAGCAGGTTGTGCACCTAACAGAGGGCACAATCTTGATGTCACTATTGCAGCCTGCTCCAGAGACTTACGATCTCTTTGATGATATCATCCTCTTATCTGAGGGTCAAATTGTCTATCAAGGTCCACGAGAACACGTTGTTGAGTTCTTTGAGAGCTGTGGTTTCAAATGTCCCGAGAGGAAAGGAACTGCTGACTTTTTGCAAGAGGTTACCTCAAAGAAGGACCAAGAACAATATTGGGCGGACAAAAGAAAGCCATACAGATACATTACAGTAACTGAATTTGCAAACAGGTTCAAGCACTTCCATGTCGGAATGCAGCTACAGAGTGAGCTAGCTGTGCCTTTCGACAAGTCAAGAGGCCACCGAGCGGCATTGGCCTTCCAGAAATACTCTATGTCCAAAATGGAGCTTCTTAAGGCCTGTTGGGACAAAGAATGGCTATTGATCAAAAGGAATTCTTTTATTTATGTGTTTAAGACGGTCCAAATTATCATCGTGGCATTCATCTCGTCTACTGTCTTTTTGAGAACTGAAATGCACCAGAGGGATTTGAACGATGCGCAACTCTATATTGGCTCACTTCTGTTTGGAATGATCATCAACATGTTCAATGGCTTCGCTGAGCTCTCCCTTATGATTAGTAGGCTTCCAGTGTTCTACAAGCAAAGAGACCTCTTATTCCACCCTGTCTGGACTTTCACTCTGCCCACTTTCTTGCTCCGGGTTCCGATATCTATTTTGGAAACAGTTGCTTGGATGGCTGTAACTTATTACACTGTAGGATATGCACCTGAGGCCAGCAGGTTTTTCAAAAACTTCCTGTTGGTGTTTTCAGTACAACAAAT GGCATCTGGTCTATTTCGGCTCATTGCCGGATTATGCAGAACAATGATCATAGCTAACACTGGTGGGGTTCTTACACTTCTCCTCGTGTTCTTGCTGGGAGGTTTCATCATTCCTAAACGTGAAATTCCAAGTTGGTGGGAGTGGGCTCACTGGATTTCACCTTTGACTTACGGTTTCAATGCCTTTACTGTGAATGAAATGTTTGCGTCAAGGTGGATGAATAGACAGGTTTCAAACAGTTCGACTAGCCTGGGGCTACAAGTGCTTGATAGCTTTGATGTCCCAAACGATGAAAACTGGTATTGGATTGGTGCAGGTGCTCTTCTAGGGTTCGCAGTGCTCTTCAACATTCTCTTCACCTTTGCGCTTATATACTTAAGCCCCCTTGGAAAGCCGCAGGCTATAATTTCGGAGGAAACGGTGGAAGAGCTAGAGGCTAATAATGTGGATTCTAATGAAGAACCAAGGTTAATGAGACCAGAATCGAGTAAATATTCATTCTCTGCAGATGCAAGCAATGCAGTAGAAATGGAAATCCGAAGAATGAGCAGTCGAGCTGATTCCCACGGAATGAGCAGGAATGATTCTCAAGTTGATGCAGCCACTGGTGTTGCCCCAAAGAGAGGAATGGTTCTTCCCTTCACTCCTCTAGCAATGTCTTTTGACACTGTCGATTACTACGTTGATATGCCACCTGAAATGAAGGCACAAGGAGTTGGTGAGGATAGGTTACAACTACTTCGGGGAGTAACAGGTGCATTTAGGCCTGGAGTGTTGACTGCATTGATGGGAGTCAGTGGAGCAGGGAAGACAACATTGATGGATGTTCTAGCAGGAAGAAAGACCGGTGGATATATTGAGGGTGATATCAGAATATCCGGATTCCCAAAGAAACAAGAAACCTTTGCAAGAATTTCTGGATACTGTGAACAAACTGATATTCACTCACCACAAGTGACTATCAGAGAATCCTTAATTTACTCAGCATTCCTACGACTT CCAAAAGAAATCAGCAACGAGGAAAAGATGATTTTCGTGGATGAAGTAATGGAACTAGTAGAATTAAGCAATCTCAAGGATGCCATAGTAGGGTTGCCTGGAGTCACAGGGTTGTCAACAGAGCAAAGAAAGAGGTTAACAATTGCAGTAGAGCTTGTTGCTAATCCCTCGATCATTTTCATGGATGAACCGACATCCGGTCTTGATGCGAGGGCAGCAGCCATTGTCATGAGGACTGTCAGAAACACCGTGGACACCGGAAGAACGGTTGTCTGCACCATTCATCAGCCTAGTATTGATATCTTTGAAGCCTTTGATGAATTGCTACTAATGAAGAGAGGAGGTCAGGTGATTTACTCCGGACCATTAGGCCGAAATTCTCATAAGATCATCGAATATTTTGAGGCAATTCCTGGAGTTCCCAAAATTAAGGAAAAGTATAATCCAGCTACATGGATGTTAGAAGTGAGCTCTATAGCAGCTGAAGTTAGGCTCGGAATTGATTTTGCTGAACACTACAAATCATCTTCCTTGTATCAGAGAAACAAGGCGTTAGTAAATGAGTTAAGCACACCACCTCCAGGAGCTAAAGACCTCTATTTTGCCACTCAGTACTCACAAACTACATTGGGTCAATTCAAATCATGCTTTTGGAAACAATGGTGGACTTACTGGAGAAGTCCAGATTATAACCTTGTCAGATACTTCTTCACTTTGGTCACTGCTCTCTTGGTTGGTTCTATTTTCTGGCAGATCGGCACTGACAGGAGTAAAGCATCTGATCTTACAATGATCATCGGTGCAATGTATGCTGCAGTCATATTTGTTGGAATCAATAACTGCTCAACAGTTCAACCAGTCATAGCCATTGAAAGAACAGTGTTCTATCGTGAAAGAGCTGCTGGGATGTACTCTGCATTACCTTATGCCCTTGCGCAGGTGCTTTGTGAAATACCTTACGTATTTGGCCAAACCGTATACTATACACTTATAGTGTATG CCATGGTGGGCTTTCAATGGACAGTGGCAAAGTACTTCTGGTTTTTCTTTGTCAGCTTCTTCACCTTCCTTTACTTTACATACTACGGAATGATGACTGTTTCGATCACACCAAACCATCAAATATCATCTATATTTGCTGCAGCATTCTATTCAGTCTTTAATCTTTTCTCCGGCTTCTTCATTCCAAGACCAAGAATTCCTGGTTGGTGGATCTGGTATTACTGGATTTGCCCGGTTGCATGGACAATTTACGGATTGATTGCGTCACAATATGGAGATCTTGAAGACAAAATTAGTGTACCTGGCGTCTCTCCTGACCCTACTATTAAGTCGTATATTAAAGATCAGTACGGCTATGATTCAGACTTCATGGGGCCAGTTGCTGCAGTTTTGGTTGGCTTTGGAGTATTTTTTGCCACTTTGTTTGCCTACTGCATAAGGACACTCAATTTCCAGACCAGATAA

The present invention also provides recombinant expression vectors and cells comprising the above-mentioned encoding genes.

The invention provides the application of GhABC protein and its encoding gene in cotton resistance to Verticillium wilt.

The GhABC protein provided by the present invention undergoes phosphorylation modification at the 855th, 859th and 863th positions of serine (Ser, S) after the plant material is inoculated with cotton Verticillium wilt pathogen, and the 440th lysine (Lys, K) Ubiquitination has occurred.

Further, the experimental results showed that after inoculation with pathogenic bacteria, the expression of this protein was up-regulated in cotton during the critical period of disease resistance, indicating that the expression of GhABC gene was activated under the stress of pathogenic bacteria. and hydrogen peroxide), the expression of GhABC gene was altered, indicating that the GhABC gene is regulated by hormones.

The invention constructs a gene GhABC silencing vector, and uses the virus-mediated gene silencing technology to inhibit the expression of GhABC in cotton. After the gene-silenced plants were inoculated with Vd080 spore suspension of the cotton verticillium wilt pathogen, the silenced plants were more susceptible to the disease. The mechanism study found that the synthesis of xylem and callose in the silenced plants decreased, ROS decreased, and the expression of some defense genes was down-regulated, which proved that It was concluded that GhABC gene was positively correlated with cotton resistance to Verticillium wilt, and GhABC gene could be used for the screening of cotton disease resistance breeding.

Description of drawings

Figure 1 shows the expression of GhABC in resistant/susceptible varieties under pathogen stress;

Figure 2 shows the expression of GhABC in disease-resistant varieties after hormone treatment;

Figure 3 shows the expression of GhABC in susceptible varieties after hormone treatment;

Fig. 4 shows the albino phenomenon and disease condition of plant leaves after gene silencing;

Figure 5 shows the accumulation of plant xylem after gene silencing;

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

Figure 7 shows the outbreak of reactive oxygen species in plant leaves after gene silencing;

Figure 8 shows the relative expression levels of related defense genes in plants after pathogen treatment.

Detailed ways

Example 1 Obtaining cotton gene GhABC

The resistant varieties of Zhimian No. 2 and susceptible varieties of Jimian No. 11 were used as plant materials. After inoculation with Vd080, the pathogen of cotton verticillium wilt, the whole protein of cotton was extracted, and the cotton without pathogenic bacteria was used as the blank control. The extracted whole protein was enzymatically hydrolyzed by trypsin, and the phosphorylation of serine (S), threonine (T) and tyrosine (Y) was achieved by using the affinity of metal oxide _TiO2 for phosphate groups. Peptide enrichment, using motif antibodies with high affinity for acetylated lysine, specifically enriching acetylated peptide fragments in complex samples, using motif antibodies with high affinity for ubiquitinated lysine, Specific enrichment of ubiquitinated peptides in complex samples, and the enrichment of different modifications is carried out by liquid chromatography-mass spectrometry (LC-MS/MS) protein quantification method to achieve quantitative analysis of large-scale protein modifications. The results were analyzed by bioinformatics.

According to the results of bioinformatics analysis, the protein GhABC whose amino acid sequence is shown in SEQ ID No. 1 was obtained by screening. The phosphorylation intensity of protein GhABC in the resistant control varieties was 15.12, and the phosphorylation intensity in the susceptible varieties was 9.60; the ubiquitination intensity in the resistant varieties was 5.49, and the ubiquitination intensity in the susceptible varieties was 5.49. is 1.35.

Example 2 Expression of GhABC in cotton treated with pathogenic bacteria and hormones

The resistant cotton variety Zhongzhimian 2 and the susceptible variety Jimian 11 were planted in vermiculite sand paper pots as experimental materials.

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

With 0.5 mM hydrogen peroxide (H ₂ O ₂ ), 0.1 mM salicylic acid (SA), 0.15 mM methyl jasmonate (JA) and 1 mM ethylene (ET) sprayed until the leaves drip, respectively. Root RNA was extracted at 12h, 24h, 48h and 72h.

The fluorescent quantitative primers were designed as: GhABC-F:TTCAGCCTGTTGGTCGTG, GhABC-R:GCGGGATTATTATGTCCTTG. The expression of GhABC was detected after treatment with pathogenic bacteria and hormones.

As shown in Figure 1, in susceptible and resistant varieties, the expression of GhABC was inhibited at the initial stage of inoculation with pathogenic bacteria, but its expression was greatly increased during the critical period of disease resistance. It can be seen that GhABC plays an important role in plant disease resistance.

As shown in Figures 2 and 3, after hormone treatment, GhABC of disease-resistant varieties was sensitive to all hormones, but the expression of other varieties except ET was inhibited, and GhABC of susceptible varieties was sensitive to ET and H ₂ O ₂ , indicating the expression of GhABC Possibly regulated by ET and H ₂ O ₂ .

Example 3. Using virus-mediated gene silencing (VIGS) to study the function of GhABC

1. Silencing the gene GhABC in cotton

Design primers for GhABC silencing vector

ABC-VIGS-F:GCTCTAGATCAGAAACACCGTGGACA,

ABC-VIGS-R:GGGGTACCTTAACTCATTTACTAACGCCTT.

Using the cDNA of the disease-resistant variety Zhongzhimian 2 as the template to amplify the silent fragment and transform it into the pYL-156 vector, and transform it into Escherichia coli DH5α competent cells. After sequencing and verification, the pYL-156-GhABC plasmid was extracted and transformed into Agrobacterium GV3101 is competent, after the colony PCR verification is correct, the culture is expanded, with pYL-156 empty as a control, pYL-156-PDS as a positive control (after the PDS gene is silenced, the leaves show whitening phenomenon), and the helper plasmid pYL- 192 After mixing and standing, use a needleless syringe to inject the leaves of No. After the injection treatment, the cells were incubated in the dark for 24 hours, and then incubated at 22°C under normal light. When the positive control showed an albino phenotype, the expression of GhABC in the silenced plants was detected by fluorescence quantitative PCR, and the plants with better silencing effect were selected for further experiments.

2. Study on disease resistance of silent plants

Plants with better silencing effect were selected, when a true leaf first appeared, inoculated with 10 mL of Verticillium dahliae spore solution (concentration of 2×10 ⁷ CFU/mL), and placed in a 25°C greenhouse for normal light growth. Samples were taken at different time periods after inoculation, and RNA of silenced plants was extracted to detect the expression of defense genes shown in Table 1. At the 3rd day of inoculation, the accumulation of xylem and leaf callose in cotton stems was detected, and the outbreak of reactive oxygen species in cotton leaves was detected. The necrosis of leaf cells was detected at 15d. On the 20th day, the disease condition of the plants was investigated.

Table 1 RT-qPCR primers of cotton defense-related genes

As shown in Figure 4, the disease index of the silent plants was calculated to be 60.12±0.99, and the diseased plant rate was 100%, while the disease index of the control was 21.92±2.68, and the diseased plant rate was 83.74±2.56%. There were extremely significant differences in disease index and diseased plant rate between controls.

As shown in Figure 5, cotton seedling stalks were stained with phloroglucinol and incubated with concentrated sulfuric acid, and the xylem accumulation of silenced plants was observed to be significantly lower than that of non-silenced plants under a normal-view microscope. As shown in Figure 6, when cotton leaves were stained with aniline blue, the accumulation of callose in the silent plants was lower than that in the control under UV excitation light. As shown in Figure 7, ROS burst experiments showed less brown precipitation in the leaves of silenced plants, indicating weaker ROS bursts. Using aniline blue to stain cotton leaves, more cell necrosis can be observed in silenced plants.

Silence defense enzyme genes or defense enzyme metabolism genes in plants, such as phenylalanine ammonia lyase (GhPAL), cinnamic acid-4-hydroxylase (GhC4H1), peroxidase, at different time periods after inoculation. The expression levels of (GhPOD) and polyphenol oxidase (GhPPO) in silenced plants decreased to varying degrees after inoculation with pathogenic bacteria. The expression levels of GhHSR203J and GhHIN1, the marker genes of allergic response in cotton, were lower than those of the control throughout the detection period, indicating that the silencing of GhABC resulted in the reduction of reactive oxygen species. GhPR3 is a marker gene of ethylene (ET) signaling pathway, and its expression in silenced plants at the initial stage of inoculation with pathogenic bacteria was significantly lower than that in control, indicating that ET plays a positive regulatory role in the early stage of GhABC disease resistance. GhJaZ1 is a marker gene of JA signaling pathway, and its expression in silenced plants is higher than that in control, indicating that GhABC may be negatively regulated by JA. GhPR1 and GhNPR1 are marker genes of salicylic acid signaling pathway, and their expression levels in silenced plants were higher than those in controls, indicating that salicylic acid plays a negative role in the regulation of GhABC's disease resistance. GhNOA1 is a nitric oxide pathway gene, and the expression level in silenced plants was significantly lower than that in the control, indicating that GhABC is related to nitric oxide pathway. Therefore, the GhABC gene of the present invention has an important positive regulatory effect in cotton resistance to verticillium wilt, and it can be used as an important candidate gene for cotton resistance to verticillium wilt breeding.

sequence listing

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

<120> Cotton verticillium wilt resistance related gene GhABC and its encoded protein and application

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<170> SIPOSequenceListing 1.0

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<213> Cotton (Gossypium spp)

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

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

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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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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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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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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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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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<213> Cotton (Gossypium spp)

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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 gattatagg 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 cotton gene GhABC in negatively regulating the resistance of cotton verticillium wilt, wherein the cotton gene GhABC encodes a protein whose amino acid sequence is shown in SEQ ID No.1. 2 . The application according to claim 1 , wherein the nucleotide sequence of the cotton gene GhABC is shown in SEQID No.2. 3 .

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