Application of GhMYC3 gene and its encoded protein in cotton trait improvement

Abstract

The present invention discloses <h2 style=";text-align:left;direction:ltr">GhMYC3 The invention relates to the application of genes and their encoded proteins in improving cotton traits, which belongs to the technical field of crop science. The invention identifies a key gene from upland cotton that has both early maturity and disease resistance. <h2 style=";text-align:left;direction:ltr"> GhMYC3 The present invention creates <h2 style=";text-align:left;direction:ltr"> GhMYC3‑KO Edited strains, phenotypes and statistical analysis findings <h2 style=";text-align:left;direction:ltr"> GhMYC3‑KO The mutant plants showed obvious late flowering and disease susceptibility phenotypes. <h2 style=";text-align:left;direction:ltr"> GhMYC3 Phenotypic and statistical analysis of overexpressed transgenic cotton plants showed that the overexpressed plants showed obvious early flowering and disease resistance phenotypes. The research results of this project not only provide genetic resources for the improvement of excellent traits of cotton, but also provide new ideas for the effective aggregation of early maturity and disease resistance traits.

Description

Technical Field

[0001] The present invention belongs to the technical field of crop science and specifically relates to GhMYC3 Application of genes and their encoded proteins in cotton trait improvement. Background Art

[0002] Cotton early maturity is an important agronomic trait for improving the multiple cropping index and ensuring stable yield. The early maturity of cotton is affected by factors such as flowering time, boll opening rate, and cotton plant growth and development rate. Among many factors, achieving early maturity by regulating flowering time is a key and hot issue in the field of cotton molecular breeding. Cotton Verticillium wilt is mainly caused by Verticillium dahliae ( Verticillium dahliae ) soil-borne fungal diseases caused by infection harm cotton growth, and it is crucial to breed new cotton varieties that are resistant to Verticillium wilt.

[0003] Among the existing cotton germplasm resources, most early-maturing varieties have poor disease resistance, while most disease-resistant varieties are greedy for green and mature late, making it difficult to aggregate early-maturing and disease-resistant traits in the main varieties. Therefore, breaking the negative correlation between early maturity and disease resistance and obtaining cotton varieties that are both disease-resistant and early-maturing is an important goal of cotton genetic breeding. In recent years, research on cotton disease resistance and achieving early maturity through the regulation of flowering has been carried out, but these studies have mostly focused on the analysis of the mechanism of a single trait, and there are few reports on key gene-related research and regulatory pathways that regulate early maturity and disease resistance, resulting in a lack of excellent sites and germplasm resources for the aggregation of early maturity and disease resistance, making it difficult to achieve breeding goals. Summary of the invention

[0004] In view of the problems existing in the above-mentioned prior art, the present invention aims to provide GhMYC3 Application of genes and their encoded proteins in cotton trait improvement, GhMYC3 It is a member of the GhMYCs family and is involved in the jasmonic acid signaling pathway. This study first discovered GhMYC3 It also has the dual functions of early maturity and resistance to Verticillium wilt, providing genetic and germplasm resources for the synergistic improvement of early maturity and disease resistance in cotton.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides GhMYC3 Application of genes in cotton trait improvement, the GhMYC3 A gene is a DNA molecule as shown in i) or ii) or iii) below:

[0007] i) The CDS nucleotide sequence is the DNA molecule shown in SEQ ID NO.1;

[0008] ii) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 other than i);

[0009] iii) a DNA molecule that has 80% or more identity with the DNA fragment defined in i) or ii), and encodes a protein that is functionally equivalent to the protein shown in SEQ ID NO.2.

[0010] The cotton GhMYC3 The gene comes from upland cotton ( Gossypium hirsutum L .) Zhongmian 24 (ZM24) variety.

[0011] The cotton trait improvement includes: improving the early maturity trait and / or disease resistance trait of cotton.

[0012] The disease resistance trait is cotton's resistance to Verticillium wilt.

[0013] The cotton trait is improved by overexpression GhMYC3 The gene advances cotton's flowering time, promotes early maturity and improves cotton's resistance to Verticillium wilt.

[0014] In a second aspect, the present invention provides GhMYC3 Application of gene-encoded protein in cotton trait improvement, the GhMYC3 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0015] The cotton trait improvement includes: improving the early maturity trait and / or disease resistance trait of cotton.

[0016] The disease resistance trait is cotton's resistance to Verticillium wilt.

[0017] The cotton traits are improved by increasing the GhMYC3 The activity of the gene-encoded protein advances the flowering time of cotton, promotes early maturity and improves cotton's resistance to Verticillium wilt

[0018] The third aspect of the present invention provides a GhMYC3 The use of a recombinant gene expression vector or a genetically engineered bacterium in any of the following (1) to (3):

[0019] (1) Advance the flowering time of cotton and promote early maturity;

[0020] (2) Improve cotton's resistance to Verticillium wilt;

[0021] (3) Improvement of cotton germplasm resources.

[0022] In a fourth aspect, the present invention provides the use of the substance described in (1) or (2) below in the preparation of a drug for preventing and treating cotton Verticillium wilt:

[0023] (1) Improve cotton GhMYC3 Gene-expressed substances;

[0024] (2) Improve cotton GhMYC3 Genes encode substances that produce active proteins.

[0025] Beneficial effects of the present invention:

[0026] The present invention clones a cotton from upland cotton GhMYC3 Gene, and first study found: overexpression in cotton GhMYC3 The gene can not only advance the flowering time of cotton, shorten the cotton growth period, promote the early maturity of cotton, but also improve the resistance of cotton to Verticillium wilt. The research results of the present invention provide a new idea for the effective aggregation of early maturity and disease resistance traits in the improvement of cotton germplasm resources, and provide good germplasm materials for the synergistic improvement of early maturity and excellent disease resistance traits of cotton, which is helpful to achieve high and stable yield of cotton, and increase the total output and economic benefits of cotton. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 :Identification of flowering phenotypes of cotton lines; Figure 1 A in the middle is: GhMYC3-KO Analysis of mutation sites in transgenic plants; Figure 1 B in the middle is: WT, 35S:GhMYC3-3HA , GhMYC3 -KO transgenic plants GhMYC3 Expression level; Figure 1 C in the middle is: WT, 35S:GhMYC3-3HA ( OE1 and OE2 Two strains), GhMYC3-KO Flowering phenotype analysis of transgenic plants; Figure 1 D in the middle is: WT, 35S:GhMYC3-3HA , GhMYC3-KO Statistics of flowering time of transgenic plants.

[0028] Figure 2 :Analysis of disease resistance phenotype of cotton plants; Figure 2 A in the middle is: WT, GhMYC3-KO , 35S:GhMYC3-3HA Disease phenotype of transgenic plants after inoculation with Verticillium dahliae (Vd991); Figure 2 B in the middle is: WT, 35S:GhMYC3-3HA , GhMYC3- KO Disease index of transgenic plants after inoculation with Vd991; Figure 2 C in the middle is: WT, 35S:GhMYC3-3HA , GhMYC3-KO Expression level of bacteria in transgenic plants. DETAILED DESCRIPTION

[0029] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0030] As mentioned above, cotton is an important economic crop. The precocity of cotton is affected by factors such as flowering time and is one of the important factors restricting cotton yield. Cotton Verticillium wilt is a soil-borne fungal disease mainly caused by Verticillium dahliae infection, which is serious. Identifying genes that simultaneously regulate cotton precocity and disease resistance, aggregating precocity and disease resistance traits, shortening the cotton growth period, and cultivating cotton varieties that are both disease-resistant and precocious are important directions in cotton genetic research.

[0031] Based on this, the present invention provides GhMYC3 The application of the gene and its encoded protein in cotton trait improvement, the present invention clones a cotton GhMYC3 Gene, GhMYC3 The gene CDS nucleotide sequence is shown in SEQ ID NO.1, and is as follows:

[0032] SEQ ID NO.1:

[0033]

[0034] GhMYC3 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2, and is as follows:

[0035] SEQ ID NO.2:

[0036] MSSSSSSLITLGQDASPTLQQRLHFIVQSRPEWWVYSIFWQASRDVDGRVVLSWGDGYFRGTRDGTGKSINRLSPSKLGSSFERKRSGKDQVQAYFNEVMDVDRMVDGDVTDYEWYYT VSMTRSFAVGDGILGKAFGSGSHIWLGGDHELQLYQCERVREARMRGIQTLVCLPTSFGVVELGSSDIIMEDWGTLQLTKSIFSSGINNSLGSNQPAHDSQPQISTPSIPFVDFGMVSG DQKERILEDKQQVEPKKETTGLGRSSSESDGDFASADTEFNASGRSKKRGRKPGNGKESPINHVEAERQRRERLNHRFYALRSVVPNVSKMDKASLLSDAVAYIKELRSKIDKLEAQLL VQSEKSKLNPINVFENQTTKSAFDNTMKQSSTYWPKTVEVDVKIVGSEAMIRVRSPDIDHPAARLMDALRDLELPVHHASVSNVNDLMLQDVVVRVPTGIFITDEMLSTAILQRCTLN.

[0037] The present invention constructs a transgenic GhMYC3-KO Editing strains and GhMYC3 Phenotypic and statistical analysis of overexpressing transgenic cotton lines GhMYC3-KO The mutant plants showed obvious late flowering and disease-susceptible phenotypes, while the overexpressing plants showed obvious early flowering and disease-resistant phenotypes. The results of the study fully demonstrated that GhMYC3 It is a key gene that combines early maturity and disease resistance, and has important application value in improving excellent traits of cotton.

[0038] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments. The following detailed descriptions are all illustrative and are intended to provide further explanation of the present application rather than to limit the scope of the present invention.

[0039] The experimental materials used in the examples of the present invention without specific description are all conventional experimental materials in the art and can be purchased through commercial channels. If no specific experimental conditions and methods are specified in the examples of the present invention, conventional conditions are generally used.

[0040] Embodiment 1: GhMYC3 Construction of overexpression vector

[0041] 1. Obtain medium cotton 24 (ZM24) from the CotonFGD website (https: / / cottonfgd.net / ) GhMYC3 The CDS sequence of the gene (Ghicr24_A12G261800) is shown in SEQ ID NO.1. GhMYC3 The CDS sequence of the gene was determined, and amplification primers were designed for PCR amplification. The nucleotide sequences of the primers are shown in SEQ ID NO.3-SEQ ID NO.4:

[0042] GhMYC3 -CDS-F:GAGAGAACACGGGGGACGTCGACATGTCTTCCTCTTCTTCGTC (SEQ IDNO.3);

[0043] GhMYC3 -CDS-R: GAACATCGTATGGGTACATGGATCCATTCAACGTGCATCTCTGA (SEQ ID NO. 4).

[0044] The PCR product was ligated to 3HA The labeled vector pCambia2300 was transformed into E. coli. The specific operation steps are as follows:

[0045] (1) Take out the competent E. coli (DH5α) from the -80°C freezer and immediately place it on ice for 10 min to thaw;

[0046] (2) Take 5 µL of the ligation product and the competent E. coli, mix them evenly on ice, and let them stand on ice for 30 min;

[0047] (3) Heat shock in a 42°C water bath for 90 seconds and place on ice for 2 minutes;

[0048] (4) Add 1 mL of LB culture medium (without antibiotics) into the clean bench;

[0049] (5) Incubate at 37°C, 250 rpm for 1 h;

[0050] (6) Centrifuge at 4000 rpm for 1 min, discard the supernatant, and spread the remaining approximately 100 μL of solution onto resistant LB solid culture medium, invert it and place it in a 37°C incubator for overnight culture.

[0051] 2. Perform colony PCR. The specific steps are as follows

[0052] (1) Using a single colony on LB medium as a template, vector universal primers and fragment primers were used for colony PCR amplification. The reaction system is as follows;

[0053] Table 1: Reaction system

[0054]

[0055] (2) Perform agarose gel electrophoresis on the PCR product and find the positive clone based on the size of the target band;

[0056] (3) Add a single colony of the positive clone into LB liquid medium containing 50× kanamycin and culture overnight at 37°C, 230 rpm.

[0057] 3. Plasmid extraction and sequencing, the specific steps are as follows:

[0058] Pure Plasmid Mini Kit (Kangwei Reagent Company) was used to extract high-purity plasmid. The specific steps are as follows:

[0059] (1) Centrifuge the overnight culture at 12,000 rpm for 3 min, discard the supernatant, and retain the bacterial pellet;

[0060] (2) Add 250 μL of Buffer P1 (containing RNase A) to the precipitate and vortex to mix thoroughly.

[0061] (3) Add 250 μL of Buffer P2 to the centrifuge tube and gently invert it 4-6 times to make the solution become a viscous liquid;

[0062] Note: This step requires gentle mixing, not too intense mixing. In addition, the lysis time should not exceed 5 min.

[0063] (4) Add 350 μL of Buffer N3 to the centrifuge tube and invert it 5-10 times. A white flocculent precipitate will appear. Centrifuge at 13,000 rpm for 5 min.

[0064] (5) Transfer the supernatant after centrifugation to the adsorption column, centrifuge at 13000 rpm for 30 s, pour out the liquid in the collection tube, and put the adsorption column back into the collection tube;

[0065] (6) Add 150 μL PB Buffer to the adsorption column and centrifuge at 13,000 rpm for 30 s;

[0066] (7) Add 400 μL PW Buffer (containing ethanol) to the adsorption column, centrifuge at 13,000 rpm for 1 min, and discard the waste liquid in the collection tube;

[0067] (8) Repeat step (7);

[0068] (9) Centrifuge at 13,000 rpm for 2 min, pour out the liquid in the collection tube, and place the adsorption column at room temperature for about 5 min to completely dry;

[0069] (10) Place the adsorption column in a new 1.5 mL centrifuge tube, add 50 μL EBBuffer to the middle of the adsorption membrane, leave it at room temperature for 2 min, centrifuge it at 13,000 rpm for 1 min, collect the plasmid solution into a centrifuge tube, and send it to a sequencing company for sequencing.

[0070] The vector with the correct sequence was identified by sequencing. GhMYC3 Gene overexpression vector, denoted as 35Sp: GhMYC3-3HA .

[0071] Example 2: Agrobacterium transformation and acquisition of overexpression positive plants

[0072] 1. The freeze-thaw method was used to obtain 35Sp:GhMYC3-3HA The specific steps for introducing GV3101 Agrobacterium are as follows:

[0073] (1) Preparation of competent Agrobacterium

[0074] S1: Streak the GV3101 Agrobacterium strain stored in the laboratory on solid YEP medium containing 10 μg / mL rifampicin for two days, pick a single colony from the medium, inoculate it into 3 mL YEP liquid medium containing 10 μg / mL rifampicin, and culture it at 28°C, 230 rpm shaking overnight;

[0075] S2: Pipette 1 mL of the above bacterial solution into 200 mL YEP liquid medium containing 10 μg / mL rifampicin, and culture at 28°C, 230 rpm on a shaker for about 16 h until OD 600 is 0.6;

[0076] S3: Centrifuge at 4000 rpm for 10 min to collect the cells and discard the supernatant;

[0077] S4: Add 100 mL TE buffer to resuspend the cells, centrifuge at 4000 rpm for 10 min, and discard the supernatant;

[0078] S5: Resuspend the cells in 10 mL YEP liquid medium (containing 25% glycerol), divide into 1.5 mL centrifuge tubes, quickly freeze in liquid nitrogen, and store at -80°C for later use.

[0079] (2) Freeze-thaw Agrobacterium transformation

[0080] S1: Take out the stored Agrobacterium GV3101 competent cells from the -80°C freezer and immediately place them on ice for 10 min to thaw;

[0081] S2: Add 10 μL to the competent cells 35Sp:GhMYC3-3HA Plasmid, placed on ice for 10 min;

[0082] S3: Freeze the competent cells in liquid nitrogen for 1 min;

[0083] S4: Take out the competent cells from liquid nitrogen and immediately place them in a 37°C water bath for 3 min, then cool the competent cells on ice for 2 min;

[0084] S5: Add 1 mL of YEP medium in a clean bench and culture at 28°C with a shaker at 180 rpm for 3 h;

[0085] S6: Centrifuge at 4000 rpm for 1 min, collect the cells, discard about 900 μL of the supernatant, and mix the remaining 100 ul of culture medium by pipetting and applying it on a plate containing 100× kanamycin (K + ) on LB plates containing antibiotics and incubate at 28°C for 2 days;

[0086] S7: Pick a single colony and inoculate it into 5 mL of rifampicin and 100× kanamycin (K + ) in YEP medium at 28°C in the dark for 48 h;

[0087] S8: Pick a single colony to prepare a template, perform colony PCR identification, shake the positive clone with the correct identification sequence, and save the strain for later use.

[0088] 2. Contain 35Sp:GhMYC3-3HA The positive cloned Agrobacterium of the vector was used to infect cotton (ZM24) explants. After inoculation, callus was induced to differentiate into seedlings. Positive plants were screened and obtained. 35Sp:GhMYC3-3HA Overexpression lines, from 35Sp:GhMYC3- 3HA Two high-expressing plants (OE1 and OE2) were identified from the overexpression lines for subsequent experiments. The specific steps for infecting cotton explants are as follows:

[0089] S1: Seed delinting: First, cotton seeds collected from the field are ginned to remove cotton fibers to obtain cotton seeds with lint. The cotton seeds are placed in a 40°C oven for drying, and then the cotton fibers are removed using a cotton seed delinting machine.

[0090] S2: Disinfection of cotton seeds: Select a number of full cotton seeds, place them in a sterile triangular bottle, add 3% hydrogen peroxide (H2O2), shake at 28℃ for 16 hours, wait until the seeds break and turn white; then plant the white seeds on the seed culture medium. The formula of the seed culture medium is: 37.5mol / L of macroelements (mother solution), 15g / L sucrose, 6g / L agar powder. Adjust the pH to about 7.0. After one week of cultivation, when the cotton seedlings grow to 10cm, prepare to cut the hypocotyl.

[0091] S3: Explant preparation: Take cotton seedlings with good growth, and cut the hypocotyls of the cotton not close to the cotyledons and roots into small segments of 0.5 cm in length in a clean bench.

[0092] S4: Soak the cut hypocotyls in Agrobacterium for 1 hour, then blot the non-wound surface of the hypocotyls dry on sterile absorbent paper and culture them in an environment of 22℃~25℃.

[0093] Embodiment 3: GhMYC3 Construction of gene knockout vector and GhMYC3-KO Obtaining transgenic plants

[0094] 1. Use the primers shown in SEQ ID NO.5-SEQ ID NO.6 to clone the specific fragment shown in SEQ ID NO.1 from the ZM24 cDNA library, connect the PCR product to the pKSE401 vector, and transform Escherichia coli.

[0095] GhMYC3 -KO-F (SEQ ID NO.5):

[0096] AGAGTCGAAGTAGTGATTGTTGGGGTCCAGTTTCGAGAGGGTTTTAGAGCTAGAAATA;

[0097] GhMYC3 -KO-R (SEQ ID NO.6):

[0098] TATTTCTAGCTCTAAAACCCCCGAAGGATGTAGGAACAATCACTACTTC.

[0099] 2. After colony PCR, plasmid extraction and sequencing, the vector with the correct sequence is identified by sequencing. GhMYC3The gene knockout vector is denoted as WMC016-GhMYC3 carrier.

[0100] 3. The obtained WMC016-GhMYC3 The vector was introduced into Agrobacterium strain LBA4404, containing WMC016-GhMYC3 Agrobacterium tumefaciens, which is a positive clone of the vector, was used to infect cotton (ZM24) explants to obtain homozygous knockout mutants. GhMYC3-KO Transgenic plants.

[0101] Embodiment 4: GhMYC3 Identification of flowering phenotypes of transgenic plants

[0102] WT (ZM24), 35S:GhMYC3-3HA Overexpression plants (OE1 and OE2) and GhMYC3-KO The transgenic plants were planted in the experimental field of Anyang Cotton Research Institute in Henan Province in the spring of 2021 and 2022 (late April), with 15 plants per row and 80 cm between rows. The flowering phenotype of cotton was observed, and the flowering time was calculated based on the opening of the first flower.

[0103] The experimental results show that GhMYC3-KO The flowering time of transgenic plants was significantly delayed compared with that of wild type WT. 35S: GhMYC3-3HA The flowering time of overexpression plants (OE1 and OE2) was significantly advanced compared with the wild type WT ( Figure 1 D). The experimental results show that GhMYC3 Genes that positively regulate cotton flowering time, overexpression GhMYC3 Genes can advance the flowering time of cotton, promote early maturity of cotton, and shorten the growing period of cotton.

[0104] Embodiment 5: GhMYC3 Transgenic cotton plants enhance resistance to Verticillium dahliae

[0105] 1. GhMYC3 Identification of disease resistance phenotypes of transgenic cotton lines

[0106] The wild-type WT (ZM24), 35S:GhMYC3-3HA Overexpression plants (OE1 and OE2), GhMYC3-KOTransgenic plants were planted in nutrient soil (containing vermiculite), covered with a protective cover, and placed in a long day (LD) (16h light / 8h dark) culture room. The temperature of the culture room was set at 24°C and the humidity was set at 60%. They were planted and cultivated for 20 days. When the second true leaf of the cotton plant was flat, the cotton plant was treated with a suspension of Verticillium dahliae spores. During the treatment, all the capillary roots of the cotton plant were immersed in the suspension of Verticillium dahliae spores. The growth of the cotton plant was observed three weeks after the treatment. The disease index (%) was calculated according to the formula: DI (disease index) = (Σ (number of each disease level) * (number of plants) / (highest disease level (4) * total number of plants)) * 100.

[0107] Among them, the severity of the disease is divided into five levels: Grade 0, Grade 1, Grade 2, Grade 3, and Grade 4. The specific standards for the classification of different severity levels are as follows:

[0108] Level 0: No yellowing of leaves, the whole plant is healthy. Level 1: Less than 25% of the leaf area is yellowing, and the plant looks healthy overall. Level 2: About 25%-65% of the leaf area is yellowing, and the plant is unhealthy. Level 3: About 65%-85% of the leaf area is yellow and wilting, and the whole plant has wilted. Level 4: The whole cotton plant is completely wilted and dead.

[0109] The preparation method of Verticillium dahliae spore suspension is as follows:

[0110] S1: First, in a sterilized clean bench, pick up 2 mm thick pieces of Verticillium wilt pathogen Vd991 from Czapek solid medium with a sterilized 1 mL pipette tip. 2 The size was inoculated into Czapek's liquid medium for expansion culture.

[0111] S2: Place the inoculated bacteria in a shaker at 25°C, set the speed to 150 r / min, and culture for 5 days.

[0112] S3: Clean the container required for filtration, filter the bacterial solution in the Czapek medium with four layers of gauze, remove the hyphae, and obtain the spore solution of Verticillium dahliae.

[0113] S4: Use a pipette to draw a small amount of spore liquid and drop it on the grid of the hemocytometer. Cover it with a coverslip and absorb the excess liquid with absorbent paper. Observe under an ordinary optical microscope to determine the spore concentration in the spore liquid. When the spore concentration is 1×10 7 When the spore suspension of Verticillium dahliae is 5.34 μg / mL, the preparation of the spore suspension of Verticillium dahliae is completed.

[0114] Table 2: Czapek's medium recipe

[0115]

[0116] The experimental results show that GhMYC3 The gene overexpression cotton lines (OE1 and OE2) showed obvious disease resistance phenotypes compared with WT, while the GhMC3-KO mutant plants showed obvious disease susceptible phenotypes compared with WT. GhMYC3 Genes enhance cotton plants' resistance to Verticillium dahliae.

[0117] 2. GhMYC3 Detection of the expression level of Verticillium dahliae in transgenic cotton lines

[0118] The leaves of plants inoculated with Verticillium dahliae for 22 days were taken to extract RNA and reverse transcribe to obtain cDNA. Three technical replicates and three biological replicates were performed for qRT-PCR for each experimental material. The primers used for the quantification of Verticillium dahliae are as follows:

[0119] VdELF-F (SEQ ID NO.7): CATTGATATCGCACTGTGG;

[0120] VdELF-R (SEQ ID NO. 8): TGGAGATACCAGCCTCGAAC.

[0121] qRT-PCR results showed: GhMYC3-KO The expression level of Verticillium dahliae in the plants was significantly higher than that in the WT plants. 35S:GhMYC3-3HA The expression level of Verticillium dahliae in transgenic plants (OE1 and OE2) was significantly lower than that in WT plants ( Figure 2 qRT-PCR results further showed that GhMYC3 The gene confers significant resistance to Verticillium wilt.

[0122] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification made within the spirit and principle of the present application shall be deemed to be appropriate.

[0123] Equivalent replacements, improvements, etc. should all be included in the protection scope of this application.

Claims

1. Application of GhMYC3 gene in cotton trait improvement, characterized in that: The CDS nucleotide sequence of the GhMYC3 gene is shown in SEQ ID NO.1; The cotton trait improvement includes: improving the early maturity trait and / or disease resistance trait of cotton; The disease resistance trait is: cotton's resistance to Verticillium wilt caused by Verticillium dahliae.

2. The use according to claim 1, characterized in that: Overexpression of the GhMYC3 gene advanced the flowering time of cotton, promoted early maturity, and improved cotton's resistance to Verticillium wilt caused by Verticillium dahliae.

3. Application of the protein encoded by the GhMYC3 gene in improving cotton traits, characterized in that: The amino acid sequence of the protein encoded by the GhMYC3 gene is shown in SEQ ID NO.2; The cotton trait improvement includes: improving the early maturity trait and / or disease resistance trait of cotton; The disease resistance trait is: cotton's resistance to Verticillium wilt caused by Verticillium dahliae.

4. The use according to claim 3, characterized in that: By increasing the activity of the protein encoded by the cotton GhMYC3 gene, the flowering time of cotton can be advanced, early maturity can be promoted, and the resistance of cotton to Verticillium wilt caused by Verticillium dahliae can be improved.

Images