Cotton ARM protein, coding gene and application of cotton ARM protein in improvement of verticillium wilt resistance of plants

Abstract

The invention provides a cotton ARM protein, a coding gene and application of the cotton ARM protein to improvement of verticillium wilt resistance of plants, and belongs to the technical field of biological agriculture. The invention provides a cotton ARM (Advanced RISC Machines) protein. The amino acid sequence of the cotton ARM protein is shown as SEQ ID NO: 1. The cotton ARM protein provided by the invention improves the resistance of plants to verticillium wilt through negative regulation. The embodiment of the invention shows that after the GhARM gene is knocked out, the verticillium wilt resistance of the cotton is remarkably improved compared with that of a wild type, and other agronomic characters of the cotton are not influenced by knockout of the GhARM gene. The cotton ARM protein enriches a cotton gene resource library, and has important practical application significance on breeding of disease-resistant plant strains.

Description

Technical Field

[0001] The present invention relates to the technical field of biological agriculture, and particularly relates to a cotton ARM protein, a coding gene (GhARM) thereof, and their application in improving the resistance of plants to Verticillium wilt. Background Art

[0002] Verticillium wilt is a soil-borne fungal disease caused by Verticillium dahliae, with a wide host range and great harm. Verticillium dahliae can survive in the soil in the form of microsclerotia for many years. When sensing a host, Verticillium dahliae germinates hyphae and infects from the roots of plants. The infection process from bottom to top increases the difficulty of preventing and controlling Verticillium wilt. Cotton (Gossypium hirsutum L.) is an important economic crop globally. Cotton Verticillium wilt caused by Verticillium dahliae is one of the most serious diseases in cotton production and is known as the "cancer" of cotton.

[0003] Currently, the available control methods include chemical control, biological control, and agricultural control. Among them, using biotechnology means such as transgenic technology to breed and plant resistant varieties is an economical and effective control method. However, due to the complex genetic mechanism of resistance to Verticillium wilt and the few reported genes related to resistance to Verticillium wilt, the shortage of disease-resistant resources and the slow progress of disease-resistant breeding. Summary of the Invention

[0004] In view of this, the present invention provides a cotton ARM protein, which improves the resistance of cotton to Verticillium wilt through negative regulation.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a cotton ARM protein, the amino acid sequence of which is as shown in SEQ ID NO: 1.

[0007] The present invention provides a coding gene of the cotton ARM protein.

[0008] The present invention provides an application of the cotton ARM protein or the coding gene as a target in at least one of the following works:

[0009] Creating plant varieties with resistance to Verticillium wilt and / or yield advantages, identifying or assisting in identifying cotton varieties resistant to Verticillium wilt, regulating the resistance of plants to Verticillium wilt, and increasing plant yield.

[0010] Preferably, the cotton ARM protein or the coding gene improves the resistance of plants to Verticillium wilt through negative regulation.

[0011] The present invention provides a reagent for interfering with the expression of the cotton ARM protein or the coding gene, including an sgRNA targeting the cotton ARM protein or a gene derivative product containing the sgRNA;

[0012] The nucleotide sequence of the sgRNA is as shown in SEQ ID NO: 3.

[0013] Preferably, the gene derivative product containing the sgRNA includes at least one of the following: expression cassette, recombinant vector, and recombinant bacterium.

[0014] The present invention provides an application of the reagent in at least one of the following operations:

[0015] Creating plant varieties resistant to Verticillium wilt and / or with yield advantages, improving the resistance of plants to Verticillium wilt, and increasing the yield of plants.

[0016] The present invention provides a method for improving the resistance of plants to Verticillium wilt, which is to knockout or inhibit the expression of the cotton ARM protein or the coding gene.

[0017] The present invention provides a method for identifying the resistance of plants to Verticillium wilt, which is to detect the expression level of the cotton ARM protein or the coding gene in plants, and judge the resistance of plants to Verticillium wilt according to the level of the expression: the expression level of the cotton ARM protein or the coding gene is positively correlated with the resistance of plants to Verticillium wilt.

[0018] Preferably, the reagent for detecting the expression level of the coding gene includes the forward primer shown in SEQ ID NO: 4 and the reverse primer shown in SEQ ID NO: 5.

[0019] The present invention has the following advantages compared with the prior art:

[0020] The present invention provides a cotton ARM protein, and the amino acid sequence is as shown in SEQ ID NO: 1. The cotton ARM protein has three tandemly arranged Armadillo (ARM) repeat domains, and improves the resistance of plants to Verticillium wilt through negative regulation. In the examples of the present invention, 290 cotton samples were detected, and the results showed that the plants with low expression level of the cotton ARM protein had strong disease resistance, while the plants with high expression level of the cotton ARM protein had weak disease resistance. In the examples of the present invention, the function of the cotton ARM protein was further verified by knocking out the GhARM gene. The results showed that after knocking out GhARM, the resistance of cotton to Verticillium wilt was significantly improved compared with the wild-type line Jin668, and knocking out GhARM did not affect other agronomic traits of cotton. It can be seen that the cotton ARM protein is of great significance for cultivating disease-resistant plant varieties with excellent comprehensive traits.

[0021] The present invention provides a method for improving the resistance of plants to Verticillium wilt by knocking out or inhibiting the expression of the cotton ARM protein or the encoding gene. Based on the characteristic that the cotton ARM protein or the encoding gene has the regulation of the resistance of plants to Verticillium wilt, in the embodiments of the present invention, after knocking out the GhARM gene, the resistance of cotton to Verticillium wilt is enhanced. It can be seen that the present invention provides a new path for plant breeding for resistance to Verticillium wilt. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly describe the technical solutions of the specific embodiments of the present invention, the following briefly introduces the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention and cannot be regarded as a limitation of the scope. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0023] Figure 1 It is a correlation result diagram of the expression level of GhARM and the disease index in Example 1;

[0024] Figure 2 It is a detection result diagram of the gene editing of the GhARM knockout line in Example 2;

[0025] Figure 3 It is a phenotypic identification result diagram of the GhARM knockout line for resistance to Verticillium wilt in Example 3; among them, (a) is the phenotypic result of indoor inoculation identification of the wild type Jin668 and the GhARM knockout line; (b) is the statistical result of the disease index of indoor inoculation identification of the wild type Jin668 and the GhARM knockout line; (c) is the phenotypic result of field Verticillium wilt identification of the wild type Jin668 and the GhARM knockout line; (d) is the statistical result of the agronomic traits of the wild type Jin668 and the GhARM knockout line. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] The present invention provides a cotton ARM protein, and the amino acid sequence is as shown in SEQ ID NO: 1 (MFTNDQRQQERTGQYGTSRQQYLQELVNQFQNTSDEETKEKIAANLANFAYDPYNYSFLRQLNVLELFLDCITEPNEKLMEFGIGGICNSCVDPANAAIITQCGGIPLVIKCLSSPVRNTVNYALGALYYLCNKSNREEILKPEVIDVIERYAAAQTVNASFSNLAKAFLDKHVS).

[0027] In the present invention, the cotton ARM protein has three tandemly arranged Armadillo (ARM) repeat domains (the three repeat domains are amino acid residues at positions 11-51, amino acid residues at positions 52-92, and amino acid residues at positions 93-133 respectively), and is a negative regulator of Verticillium wilt in plants. Its expression level is significantly negatively correlated with disease resistance, and the resistance of plants to Verticillium wilt is improved through negative regulation. In the examples of the present invention, 290 cotton samples were detected. The results showed that plants with a low expression level of the cotton ARM protein had strong disease resistance, while plants with a high expression level of the cotton ARM protein had weak disease resistance. In the examples of the present invention, the function of the cotton ARM protein was further verified by knocking out GhARM. The results showed that after knocking out GhARM, the resistance of cotton to Verticillium wilt was significantly improved compared with the wild-type line Jin668, and knocking out GhARM did not affect other agronomic traits of cotton. It can be seen that the cotton ARM protein is of great significance for cultivating disease-resistant plant varieties with excellent comprehensive traits.

[0028] The present invention provides a coding gene for the cotton ARM protein.

[0029] In the present invention, the nucleotide sequence of the coding gene is preferably as shown in SEQ ID NO: 2 (ATGTTTACCAATGATCAAAGGCAGCAAGAAAGAACTGGACAATATGGAA CTTCAAGGCAACAATATCTTCAGGAATTGGTGAATCAGTTTCAGAACACGTCTGATGAAGAAACAAAAGAAAAGATTGCTGCAAACTTGGCAAACTTTGCTTATGATCCATATAATTATTCTTTCTTGCGCCAGCTCAATGTTTTGGAACTCTTCCTAGATTGCATAACAGAACCAAACGAGAAGCTTATGGAATTTGGAATTGGAGGCATCTGCAATTCTTGTGTGGATCCAGCTAATGCTGCTATTATCACTCAG TGCGGTGGAATCCCTCTTGTCATTAAGTGTTTATCAAGCCCAGTCAGAAATACAGTGAATTATGCTCTGGGAGCCCTTTATTATCTTTGTAACAAGTCTAACAGGGAGGAGATTCTGAAGCCTGAAGTAATTGATGTCATCGAGAGGTATGCTGCAGCTCAAACTGTAAATGCAAGCTTCAGTAACCTGGCTAAAGCATTTCTTGACAAGCATGTATCTTGA). It should be understood that considering the degeneracy of codons, modification of the nucleotide sequence of the coding gene without changing the amino acid sequence also falls within the protection scope of the present invention. The coding gene is a Verticillium wilt-responsive gene, which improves the resistance of plants to Verticillium wilt through negative regulation, and knockout of the coding gene does not affect agronomic traits. The coding gene enriches the gene resource library for Verticillium wilt-resistant breeding.

[0030] Based on the characteristics that the cotton ARM protein or the coding gene has the property of regulating the resistance of plants to Verticillium wilt, the present invention provides an application of the cotton ARM protein or the coding gene as a target in at least one of the following works: creating plant varieties with Verticillium wilt resistance and / or yield advantages, identifying or assisting in identifying Verticillium wilt-resistant cotton varieties, regulating the Verticillium wilt resistance of plants, and increasing the yield of plants.

[0031] In the present invention, the cotton ARM protein or the coding gene preferably improves the resistance of plants to Verticillium wilt through negative regulation.

[0032] In the embodiments of the present invention, 290 cotton samples were detected. The results showed that plants with low expression levels of cotton ARM protein had strong disease resistance, while plants with high expression levels of cotton ARM protein had weak disease resistance. In the embodiments of the present invention, the function of cotton ARM protein was verified by knocking out GhARM. The results showed that after knocking out GhARM, the resistance of cotton to Verticillium wilt was significantly improved compared with the wild-type line Jin668, and knocking out GhARM did not affect other agronomic traits of cotton, which was beneficial to increasing cotton yield. Therefore, the cotton ARM protein or the cotton coding gene is used to create plant varieties with resistance to Verticillium wilt and / or yield advantages, identify or assist in identifying cotton varieties resistant to Verticillium wilt, regulate the resistance of plants to Verticillium wilt, and increase plant yield.

[0033] The present invention provides a reagent for interfering with the expression of the cotton ARM protein or the coding gene, including an sgRNA targeting the cotton ARM protein or a gene derivative product containing the sgRNA; the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 3 (AUGAUCAAAGGCAGCAAGAA).

[0034] In the present invention, the sgRNA has high cleavage efficiency, low off-target rate, and high mutation efficiency when combined with the CRISPR system, which is beneficial to obtaining more positive clones. In the embodiments of the present invention, multiple sgRNAs were designed for the cotton ARM protein. The results showed that the sgRNAs of the present invention could obtain more positive clones of knocked-out cotton ARM protein. In the embodiments of the present invention, the sgRNA was used to perform frameshift mutations on the ARM protein, and GhARM knockout lines (GhARM#KO-1 and GhARM#KO-2) were obtained. The GhARM knockout lines had significantly enhanced resistance to Verticillium wilt compared with the wild-type line, and there were no significant differences in other agronomic traits from the wild-type line.

[0035] In the present invention, the gene derivative product containing the sgRNA preferably includes at least one of the following: expression cassette, recombinant vector, and recombinant bacterium. The backbone vector of the recombinant vector preferably includes a CRISPR-Cas vector. The present invention does not make any special limitation on the type of CRISPR-Cas vector, and any commonly used CRISPR-Cas vector in the art can be adopted. In the examples of the present invention, pRGEB32-HtKt is used as the backbone vector, and the sgRNA targeting the cotton ARM protein is inserted into the backbone vector to obtain a recombinant vector. The construction of pRGEB32-HtKt refers to the prior art (Ramadan M, Alariqi M, Ma Y, Li Y, Liu Z, Zhang R, Jin S, Min L, Zhang X. Efficient CRISPR / Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton. Plant Methods. 2021 Feb 8;17(1):16.). The recombinant vector obtained by using pRGEB32-HtKt as the backbone vector can efficiently knockout the cotton ARM protein. The recipient bacterium of the recombinant bacterium preferably includes Agrobacterium, and more preferably includes the Agrobacterium strain GV3101. The recombinant bacterium efficiently transfers the sgRNA targeting the cotton ARM protein into the recipient plant by infecting the cotton recipient plant, thereby knocking out the cotton ARM protein and improving the Verticillium wilt resistance of the plant.

[0036] The present invention provides an application of the reagent in at least one of the following operations:

[0037] Creating plant varieties with Verticillium wilt resistance and / or yield advantages, improving the Verticillium wilt resistance of plants, and increasing the yield of plants.

[0038] In the examples of the present invention, the function of the cotton ARM protein was verified by knocking out GhARM. The results showed that after knocking out GhARM, the Verticillium wilt resistance of cotton was significantly improved compared with the wild-type line Jin668, and knocking out GhARM did not affect other agronomic traits of cotton, which was beneficial to increasing the cotton yield. Therefore, the cotton ARM protein, the cotton coding gene, or the reagent can be used to create plant varieties with Verticillium wilt resistance and / or yield advantages, improve the Verticillium wilt resistance of plants, and increase the yield of plants.

[0039] The present invention provides a method for improving the Verticillium wilt resistance of plants, which is to knockout or inhibit the cotton ARM protein or the coding gene.

[0040] In the present invention, the method of knocking out or suppressing preferably includes gene editing techniques, more preferably includes any one of the following: RNA interference, virus-induced gene silencing, and CRISPR / Cas technology. The knocking out or suppressing is preferably to introduce the reagent into the cotton receptor plant. The method of introduction preferably includes Agrobacterium-mediated transformation. After introducing into the cotton receptor plant, positive plants are preferably subjected to molecular identification. The primers for the molecular identification preferably include the forward primer shown in SEQ ID NO: 6 and the reverse primer shown in SEQ ID NO: 7. In the examples of the present invention, the gene derivative product containing the sgRNA is introduced into the cotton receptor plant, and after molecular identification, the GhARM knockout lines (GhARM#KO-1 and GhARM#KO-2) are obtained. The detection of the disease index shows that the Verticillium wilt resistance of the GhARM knockout lines is significantly higher than that of the wild-type lines, indicating that knocking out or suppressing the cotton ARM protein or the coding gene can improve the plant's resistance to Verticillium wilt.

[0041] The present invention provides a method for identifying the resistance of a plant to Verticillium wilt, detecting the expression level of the cotton ARM protein or the coding gene in the plant, and judging the resistance of the plant to Verticillium wilt according to the level of the expression: the expression level of the cotton ARM protein or the coding gene is positively correlated with the resistance of the plant to Verticillium wilt.

[0042] In the present invention, the reagent for detecting the expression level of the coding gene includes the forward primer shown in SEQ ID NO: 4 and the reverse primer shown in SEQ ID NO: 5.

[0043] In the present invention, the cotton is preferably upland cotton. In the examples of the present invention, jin668 is taken as an example to illustrate the cotton ARM protein, the coding gene thereof, and their application in improving the resistance of plants to Verticillium wilt. In the examples of the present invention, the reagents, methods, and equipment for preparing the recombinant vector, recombinant bacteria, and sgRNA are conventional reagents, methods, and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available as usual.

[0044] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below in conjunction with the drawings and examples, but they cannot be understood as limiting the protection scope of the present invention.

[0045] Example 1

[0046] Statistics of the disease index of cotton plants and detection of the expression level of GhARM

[0047] 290 natural population materials of upland cotton were planted in the natural Verticillium wilt nursery in Korla, Xinjiang. Two biological replicates were set for each material. According to the cotton disease grading standard in the national standard (GB / T 22101.5—2009), the single plants were statistically analyzed respectively. After counting the disease grades of single plants, the disease index was calculated by formula I.

[0048] The cotton disease grading standard is divided into 5 levels: level 0, the cotton plant is healthy without diseased leaves; level 1, the cotton plant has symptoms on less than 1 / 4 of the leaves; level 2, the cotton plant has symptoms on more than 1 / 4 and less than 1 / 2 of the leaves; level 3, the cotton plant has symptoms on more than 1 / 2 and less than 3 / 4 of the leaves; level 4, the cotton plant has symptoms on more than 3 / 4 of the leaves.

[0049] Disease index (DI) = [Σ (number of diseased plants × corresponding disease level) / (total number of plants × highest disease level)] × 100. Formula I. Using the single-plant cDNA of 290 upland cotton populations as a template, qRT-PCR amplification was carried out with the forward primer shown in SEQ ID NO: 4 (AAGTGTTTATCAAGCCCAGTCAGA) and the reverse primer shown in SEQ ID NO: 5 (GCTTCAGAATCTCCTCCCTGTTAG) to detect the expression level of GhARM. The reagents used for qRT-PCR were purchased from Yesheng, and the product name was One Step Multiplex Quantitative PCR Mix Enzyme, and the product number was 13197ES|.

[0050] The reaction system was:

[0051] V MPBuffer 12 μl

[0052] One Step qRT-PCR Enzyme Mix 1 μl

[0053] Primer / Probe Mix (10 μM) 2 μl

[0054] Template cDNA 5 μl

[0055] RNase-free water 5 μl

[0056] The PCR reaction program was:

[0057] 50 °C 10 min × 1

[0058] 95 °C 5 min × 1

[0059] 95 °C 15 s; 60 °C 30 s × 45.

[0060] The correlation between the expression level of GhARM and the disease index was statistically analyzed, and the statistical results are shown in Figure 1 . Figure 1It is shown that among 290 materials, those with low GhARM expression levels have low disease indices, while those with high GhARM expression levels have high disease indices.

[0061] Example 2

[0062] Construction method of GhARM knockout lines

[0063] 1) Recombinant vector construction

[0064] To verify the regulatory role of GhARM in cotton resistance to Verticillium wilt, the present invention uses the knockout vector pRGEB32-HtKt (Ramadan M, Alariqi M, Ma Y, Li Y, Liu Z, Zhang R, Jin S, Min L, Zhang X. Efficient CRISPR / Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton. Plant Methods. 2021 Feb 8;17(1):16.) to knockout GhARM in cotton.

[0065] For the CDS sequence of GhARM, target sgRNA was designed through the website http: / / crispr.hzau.edu.cn / CRISPR2 / . The nucleotide sequence of the sgRNA is shown in SEQ ID NO: 3. The DNA sequence of the sgRNA was synthesized, and BsaI digestion adapters were added to both ends of the DNA sequence during synthesis. The 5'-end digestion adapter is ttcccggctggtgca (SEQ ID NO: 8), and the 3'-end digestion adapter is gttttagagctagaa (SEQ ID NO: 9). Using the DNA with digestion adapters as a template for PCR amplification, a PCR product was obtained.

[0066] The PCR reaction system is: 5 μl of 2×Rapid Taq MasterMix, 0.2 μl each of 10 μM forward primer and reverse primer, 1 μl of DNA template, and ddH 2 O 3.6 μl;

[0067] The nucleotide sequence of the forward primer is the same as that of the 5'-end digestion adapter, and the nucleotide sequence of the reverse primer is reverse complementary to that of the 3'-end digestion adapter.

[0068] The PCR reaction program is: 95°C for 5 min; 95°C for 30 s, 58°C for 30 s, 72°C for 10 s, 32 cycles; 72°C for 1 min.

[0069] After purifying the PCR product with a purification kit, the purified PCR product was ligated to the linearized pRGEB32-HtKt vector through an infusion ligation reaction, and then transformed into the competent Escherichia coli Top10 strain. Subsequently, the transformed Top10 strain was spread on an LB culture plate containing kanamycin and incubated overnight in the dark at 37°C. Then, single colonies were picked into LB liquid medium and cultured on a shaker at 37°C. The single colonies were detected, and the positive clones were sent to the company for sequencing. Plasmids were extracted from the correctly sequenced single colonies to obtain recombinant vectors. After transforming the recombinant vectors into the competent Agrobacterium tumefaciens strain GV3101 by electroporation, recombinant bacteria were obtained for subsequent Agrobacterium-mediated cotton genetic transformation.

[0070] 2) Agrobacterium-mediated cotton genetic transformation

[0071] The transgenic receptor cotton variety was "jin668". Seeds with plump grains were disinfected and sterilized, placed in a glass bottle containing sterile seedling medium, and cultured in the dark at 28°C for about 5 days. Vigorous sterile seedlings were selected, and the hypocotyls were cut into 0.8-cm segments, infected with the activated recombinant bacteria, dried, and then placed in a plate containing co-culture 2,4-D medium and cultured in the dark for 36-48 h, with this process carried out in the dark. Then, the infected cotton hypocotyls were placed in DK callus induction medium and cultured in a 28°C light incubator with a normal photoperiod, and subcultured every about 25 days. When embryogenic callus appeared, it was transferred to differentiation medium to further grow into embryoids, and subcultured until differentiated into small seedlings, which were then transferred to rooting medium until healthy roots grew out and then transplanted into nutrient soil for culture to obtain GhARM knockout line plants.

[0072] 3) Molecular detection of GhARM knockout lines

[0073] The transgenic positive detection was carried out on the GhARM knockout line plants. Using the leaf DNA of the T0 single plants and the T1 and T2 generation lines of the GhARM knockout line plants as templates, PCR amplification was performed with the forward primer shown in SEQ ID NO: 6 (ggagtgagtacggtgtgcTTACAAAACAGAAGTGCAAACT) and the reverse primer shown in SEQ ID NO: 7 (gagttggatgctggatggTATTGTTGCCTTGAAGTTCC), and the amplified products were sequenced. The GhARM knockout lines GhARM#KO-1 and GhARM#KO-2 were obtained, and the editing results of GhARM#KO-1 and GhARM#KO-2 were as Figure 2 shown.

[0074] Example 3

[0075] Phenotypic identification of Verticillium wilt resistance of GhARM knockout lines

[0076] 1) Indoor disease resistance identification of GhARM knockout lines

[0077] Germinate the seeds of Jin668 (wild - type line), GhARM#KO - 1 and GhARM#KO - 2 and hydroponically culture them for 10 days. During this period, activate the Verticillium dahliae strain v991, pick colony blocks and inoculate them into Czapek's culture solution, and incubate them in the dark on a shaker at 25°C for 3 - 4 days. Filter the bacterial solution with double - layer gauze, microscopically examine the spore concentration with a hemocytometer and dilute it to 5×10 5 spore / mL. Dip the roots of the hydroponic cotton seedlings for 2 minutes and plant them in a substrate with a volume ratio of nutrient soil to vermiculite of 2:1, and culture them under normal photoperiod.

[0078] Symptoms began to appear on the 9th day after inoculation, as shown in Figure 3 (a). According to the cotton disease grading standard in the national standard (GB / T22101.5—2009), statistically analyze each single plant. After counting the disease grades of single plants, calculate the disease index of Jin668 and GhARM knockout lines through formula I, and the results are shown in Figure 3 (b).

[0079] 2) Field disease resistance identification of GhARM knockout lines

[0080] Plant Jin668 and GhARM#KO - 1 in the natural Verticillium dahliae disease nursery in Korla, Xinjiang. Investigate the Verticillium dahliae disease index in late July, and statistically analyze agronomic traits such as plant height, height of the first fruiting branch node, number of vegetative branches, lint percentage, etc. in mid - August. The results are shown in Figure 3 (c) and (d).

[0081] The results of indoor and field disease resistance identification of GhARM knockout lines show that the disease index of GhARM knockout lines is significantly lower than that of Jin668, and there is no significant difference in agronomic traits between GhARM knockout lines and Jin668. This indicates that after knocking out GhARM, the resistance of cotton to Verticillium dahliae is significantly improved, and knocking out GhARM does not affect other agronomic traits of cotton.

[0082] Although the above - mentioned embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. Other embodiments can be obtained based on this embodiment without creative work, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A cotton ARM protein, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. A gene encoding the cotton ARM protein according to claim 1.

3. Use of the cotton ARM protein according to claim 1 or the encoding gene according to claim 2 as a target in at least one of the following tasks: Create plant varieties with resistance to Verticillium wilt and / or yield advantages, identify or assist in identifying Verticillium wilt-resistant cotton varieties, regulate plant resistance to Verticillium wilt and increase plant yield.

4. The use according to claim 3, characterized in that: The cotton ARM protein or the encoding gene improves the plant's resistance to Verticillium wilt through negative regulation.

5. An agent for intervening the expression of the cotton ARM protein according to claim 1 or the encoding gene expression according to claim 2, characterized in that: It includes an sgRNA targeting cotton ARM protein or a gene-derived product comprising the sgRNA; The nucleotide sequence of the sgRNA is shown in SEQ ID NO:

3.

6. The reagent according to claim 5, characterized in that The gene-derived product containing the sgRNA includes at least one of the following: an expression cassette, a recombinant vector and a recombinant bacterium.

7. Use of the reagent according to claim 5 or 6 in at least one of the following tasks: Create plant varieties with resistance to Verticillium wilt and / or yield advantages, improve plant resistance to Verticillium wilt and increase plant yield.

8. A method for improving plant resistance to Verticillium wilt, characterized in that: Knock out or inhibit the expression of the cotton ARM protein according to claim 1 or the encoding gene according to claim 2.

9. A method for identifying plant resistance to Verticillium wilt, characterized in that: Detect the expression level of the cotton ARM protein according to claim 1 or the encoding gene according to claim 2 in the plant, and judge the plant's resistance to Verticillium wilt according to the expression level: the expression level of the cotton ARM protein or the encoding gene is positively correlated with the plant's resistance to Verticillium wilt.

10. The method according to claim 9, characterized in that: The reagent for detecting the expression amount of the coding gene includes a forward primer shown in SEQ ID NO: 4 and a reverse primer shown in SEQ ID NO: 5.

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