Application of GhBAM1 gene in reducing the boll shedding rate of cotton flower

Abstract

The application relates to application of a GhBAM1 gene in reducing cotton bud boll shedding rate, and a cotton bud boll shedding gene GhBAM1 is identified for the first time, and the gene has important value for theoretical research on molecular mechanisms of cotton bud boll shedding. By knocking out an endogenous gene GhBAM1 of upland cotton, the cotton bud boll shedding rate can be reduced, the high-temperature stress resistance of the cotton can be improved, high-yield and stable-yield cotton breeding materials can be created, and a cotton gene resource library is enriched, and the application has important practical application significance for cotton breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant gene breeding technology, specifically involving the application of the GhBAM1 gene in reducing cotton bud and boll shedding rate. Background Technology

[0002] Cotton is an important economic crop in my country. Stabilizing cotton production is of great strategic significance for promoting the development of the textile industry and ensuring the safety of people's production and life. In recent years, the trend of global warming has become increasingly significant, and my country's main cotton-producing areas have also encountered varying degrees of sustained high temperatures. At this time, cotton is in the flowering and boll-forming stage, which is a critical stage affecting cotton yield (Ma et al. Disrupted genome methylation in response to high temperature has distinct effects on microspore abortion and antherindehiscence. Plant Cell. 2018, 30(7): 1387-1403). Sustained high temperatures above 35℃ will have adverse effects on the reproductive development of cotton, mainly manifested in anther dehiscence, decreased pollen viability, and increased bud and boll shedding rate, ultimately affecting cotton yield (Li et al. High temperature induces male sterility via MYB66-MYB4-Casein kinase I signaling in cotton. Plant Physiol. 2022, 189(4): 2091-2109).

[0003] High temperatures are a major cause of boll shedding in cotton. Discovering new heat-resistant genes and selecting superior heat-resistant germplasm resources are important goals for breeders. With the continuous improvement of the cotton reference genome and the development of cotton biotechnology, gene editing technology has become an important method for rapid genetic improvement of cotton, and has brought enormous development potential for precision cotton breeding. Breeding new heat-resistant cotton varieties through genetic engineering technology can not only effectively increase cotton yield and farmers' income, but also play a significant role in promoting the development of my country's cotton textile industry and enhancing the competitiveness of Chinese cotton in the international market.

[0004] Boll shedding rate is an important yield trait in cotton. High temperature is the main factor affecting boll shedding; temperatures above 35°C usually inhibit photosynthesis, and above 40°C, photosynthesis stops altogether. Insufficient photosynthesis can lead to boll shedding. On the other hand, high temperatures can also reduce pollen viability, and in more severe cases, prevent anther dehiscence, causing the ovary to detach due to poor fertilization or inability to complete fertilization. Therefore, identifying the regulatory genes related to cotton boll shedding is crucial for breeding heat-resistant cotton germplasm. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the cotton gene GhBAM1 in regulating cotton boll shedding. By knocking out the endogenous gene GhBAM1 in upland cotton, the boll shedding rate of cotton can be reduced, the cotton's ability to resist high temperature stress can be improved, the cotton gene resource library can be enriched, and technical support can be provided for the breeding of high temperature resistant cotton germplasm resources.

[0006] This invention identifies the gene GhBAM1, which regulates cotton boll shedding rate, based on CRISPR / Cas9 gene editing technology. Knocking out the GhBAM1 gene in cotton significantly reduces the boll shedding rate (the percentage of bolls shed per plant relative to the sum of the effective bolls and the shed bolls), thereby significantly increasing cotton yield. This indicates that the gene or its encoded protein plays an important role in controlling cotton boll shedding rate.

[0007] This invention provides a gene GhBAM1 that regulates the shedding rate of cotton buds and bolls. Its genomic nucleotide sequence is shown in SEQ ID NO:1, its CDS sequence is shown in SEQ ID NO:2, and its amino acid sequence is shown in SEQ ID NO:3.

[0008] It should be understood that, considering the gene expression regulatory region and the degeneracy of codons, modifying the GhBAM1 gene sequence without changing the amino acid sequence also falls within the scope of protection of this invention.

[0009] This invention provides the application of the GhBAM1 gene and reagents regulating GhBAM1 expression in any of the following:

[0010] A1) Regulate the shedding rate of cotton buds and bolls;

[0011] A2) Create products to regulate the shedding rate of cotton buds and bolls;

[0012] A3) Increase the number of effective bolls per cotton plant and reduce the boll shedding rate;

[0013] A4) Create products that increase the number of effective bolls per cotton plant and reduce the boll shedding rate of cotton buds;

[0014] A5) Regulating cotton production;

[0015] A6) Create products to regulate cotton production;

[0016] A7) Develop high-yield, heat-resistant cotton varieties;

[0017] A8) Identify or assist in the identification of high-temperature resistant cotton varieties.

[0018] Furthermore, the amino acid sequence encoded by the GhBAM1 gene is shown in SEQ ID NO:3.

[0019] Furthermore, the nucleotide sequence of the GhBAM1 gene is shown in SEQ ID NO:1, and the CDS sequence is shown in SEQ ID NO:2.

[0020] Furthermore, the reagents for regulating GhBAM1 expression include sgRNA and / or a CRISPR vector, wherein the nucleotide sequence of the sgRNA is shown in SEQ ID NO:4 and / or SEQ ID NO:5.

[0021] The present invention also provides a reagent for regulating GhBAM1 expression, the reagent comprising sgRNA, the nucleotide sequence of the sgRNA being shown in SEQ ID NO:4 and / or SEQ ID NO:5, and the amino acid sequence encoded by the GhBAM1 gene being shown in SEQ ID NO:3.

[0022] This invention also provides a method for reducing cotton boll shedding rate by inhibiting and / or knocking out GhBAM1, thereby increasing the number of effective bolls per cotton plant and reducing the cotton boll shedding rate. Preferably, the cotton boll shedding rate is reduced by knocking out the GhBAM1 protein through gene editing technology or by interfering with the expression of GhBAM1 through RNAi technology.

[0023] Furthermore, the amino acid sequence encoded by the GhBAM1 gene is shown in SEQ ID NO:3.

[0024] Furthermore, the nucleotide sequence of the GhBAM1 gene is shown in SEQ ID NO:1, and the CDS sequence is shown in SEQ ID NO:2.

[0025] Furthermore, the reagent for regulating GhBAM1 expression includes sgRNA, the nucleotide sequence of which is shown in SEQ ID NO:4 and / or SEQ ID NO:5.

[0026] Furthermore, by introducing a gene knockout vector into cotton to inhibit the expression of GhBAM1 in cotton, the method for preparing the gene knockout vector includes the following steps:

[0027] 1) Using pGTR plasmid as a template, the first target site was amplified by PCR using the pGREB32-7s / cBAM1-1as primer pair to obtain the fragment containing sgRNA1.

[0028] The nucleotide sequence of pGREB32-7s is 5'AAGCATCAGATGGGCAAACAAAGC ACCAGTGGTCTAG 3' (SEQ ID NO:6);

[0029] The nucleotide sequence of cBAM1-1as is 5'ACCGGTTAAATCGCACGTGAtgcaccagc cgggaat 3' (SEQ ID NO:7);

[0030] 2) Using pGTR plasmid as a template, the second target site was amplified by PCR using the cBAM1-1s / cBAM1-2as primer pair to obtain the fragment containing sgRNA2.

[0031] The nucleotide sequence of cBAM1-1s is: 5'TCACGTGCGATTTAACCGGTgttttagagc tagaaata 3' (SEQ ID NO:8);

[0032] The nucleotide sequence of cBAM1-2as is: 5'GTTTTCTGCCACAGACTAGTtgcaccag ccgggaat 3' (SEQ ID NO:9);

[0033] 3) Using the fragments containing sgRNA1 and sgRNA2 as templates, overlap extension PCR amplification was performed using the inf pRGEB32-7s / inf cBAM1-2as primer pair to obtain DNA fragments containing sgRNA1 and sgRNA2.

[0034] The nucleotide sequence of inf pRGEB32-7s is: 5'AAGCATCAGATGGGCAAACAA A 3' (SEQ ID NO: 10);

[0035] The nucleotide sequence of inf cBAM1-2as is: 5'ttctagctctaaaacGTTTTCTGCCACAG ACTAGT3' (SEQ ID NO:11);

[0036] 4) Insert the DNA fragment containing sgRNA1 and sgRNA2 into the backbone vector pRGEB32-7 to obtain the CRISPR / Cas9 vector.

[0037] Beneficial effects: This invention is the first to identify the cotton boll shedding gene GhBAM1. This gene is of great value for theoretical research on the molecular mechanism of cotton boll shedding.

[0038] This invention can significantly reduce the cotton boll shedding rate, thereby creating high-yield and stable-yield cotton breeding materials, which has important practical application significance for cotton breeding. Attached Figure Description

[0039] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 Map of the GhBAM1 gene knockout vector pRGEB32-7-GhBAM1.

[0041] Figure 2 Heatmap of expression patterns of 13 GhBAM1 genes annotated in upland cotton.

[0042] Figure 3 Effects of GhBAM1 gene knockout on cotton yield traits. (a) Phenotypes of wild-type Jin668 and three transgenic lines with GhBAM1 gene knockout; (b)-(d) Phenotypic statistics of wild-type Jin668 and three transgenic lines with GhBAM1 gene knockout in terms of effective boll number per plant, number of buds and bolls shed per plant, and bud and boll shedding rate per plant, respectively. Detailed Implementation

[0043] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art. Unless specifically stated, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless specifically stated, the reagents and materials used in the following embodiments are commercially available.

[0044] Example 1: Functional and expression pattern analysis of GhBAM1 in upland cotton

[0045] (1) GhBAM1 gene function analysis

[0046] GhBAM1 belongs to the leucine-rich repeat receptor-like kinase (LRR-RLK) family of cotton, and is the largest known transmembrane receptor-like kinase (RLK) in plants. LRR-RLKs generally consist of three parts: an extracellular LRR domain, a transmembrane domain, and an intracellular kinase domain, and are widely involved in various plant life activities. RLKs are key protein kinases for plants to sense environmental signals and are the largest receptor protein family in plants, playing a crucial role in plant adaptation to environmental changes.

[0047] (2) Expression pattern of GhBAM1

[0048] The BAM1 genes annotated in upland cotton mainly include 15 genes. Based on gene expression patterns, the expression levels of Ghir_A02G016300, Ghir_A02G016290, and Ghir_D03G003280 genes were found to be relatively high. Figure 2 The Ghir_A02G016300 gene is significantly expressed in reproductive development-related tissues such as anthers, ovules, and stigmas. Figure 2 Therefore, it can be inferred that the GhBAM1 gene is related to the reproductive and developmental processes of cotton. Its genomic nucleotide sequence is as follows:

[0049] ATGAGGTTACTCCTCCTCCTCCTCTTCCTTCTTCTTCACATTTCCCACT CCTATGCCGCCGGCACCG CCGCAAGAGCCGTTACGGAGCTTCGCGCACTTATCGCTGTTAAATCCTCCATTACCGACGACCCTCAATCTTACCT CTCAAACTGGAATGCAACAACTCCCCTTTGTTCATTCGCCGGAGTCACGTGCGATTTAACCGGTCGGCACGTGACA TCCATCGATTTAACTAACTTCACCCTGTCCGGAACCCTTTCCCCCTCGCTTGCCCACCTCCGTTTCCTCCAAAACA TCTCCGTCGCCGACAACAATCTTACCGGTCCTATCCCGACGGAGCTCGCCGTCCTATCCAATCTCCGTTATCTAAA CCTTTCCAACAATGTTTTCAATGGTTCCTTTCCTACCCAGCTTTCCCAGCTGAAAAATCTACAGGTACTCGATTTA TACAACAATAACATGACGGCGGAGTTGCCGGTTTCCGTCACGGAGCTTCCCAATTTGCGTCACTTGCATTTGGGAG GGAACTATTTCAGCGGTCAGATCCCGTCAAGTTACGGCCGTTGGGAGCATCTTGAATATTTAGCCGTTTCGGGTAA CGAACTCAGCGGTAAAATCCCACCCGAAATCGGCAACTTAACGAAGCTGAAGGAGTTGTACATTGGTTACTTCAAT AATTTTGAAGGTGGTTTGCCGCCGGAGATCGGGAACTTGTCGGAACTCGTTCGTTTTGACGCCGCTAACTGCATGT TATCCGGTGAAATACCGCCGGAGATCGGTAAGTTGCAGAAGCTCGACACGTTGTTCCTCCAGGTGAATGCACTGTC TGGCTCCTTAACTCCCGAGCTGGGAACCTTAAACAGCTTGAAGTCCATGGATTTATCGAACAATATGTTTACCGGT GAGATTCCAGCGAGTTTCGCTCAGCTCAAAAACTTGACTCTTCTCAATCTTTTCAGAAACAAGCTCCACGGACAGA TTCCTGACTTCATTGGTGAGTTGCCCGAGTTGGAGGTCTTACAGCTATGGGAAAATAACTTCACCGGAAGCATTCC TCAGAAGTTGGGCAGTAACAAAAAGCTTCAAGTTCTAGATCTTTCGTCGAACAAGTTAACGGGGACTTTGCCGCCG GACATGTGCTCCGGCAACACGTTGCAAACGTTGATTACTTTGGGTAACTTCTTGCTTGGTCCAATCCCAGAATCGT TGGGGAAATGTGAATCACTCAGTCGGATTCGTATGGGTGAAAATTATCTCAACGGGTCCATCCCTAAAGGCCTTTT GGGATTACCACAGCTTACACAAGTTGAGTTACAGGATAAC TATCTAACAGGGGAGTTCCCGGTCACTGATTCTTC CATTTCCGTGAATCTCGGCCAAATCAGCTTATCCAACAACCAACTTTCCGGGGCTTTACCGGCTAGCGTCGGTAAC TTTTCCGGTGTTCAAAAGCTTCTTCTCGATGGCAACAAGTTTTCGGGTCCAATCCCAGCTGAGATTGGGAAGTTGC AGCAACTTTCAAAGATTGATTTCAGTCATAACAAGTTTTCAGGATTGATCCCACCAGAAATTTGCAAATGCAAGCT GTTAACCTTTGTTGATCTTAGTCGAAACGAGCTTTCCGGTCGAATTCCGACTGAGATCACAAGTATGAGGATATTA AACTTCCTGAATCTGTCGAGAAATCATCTCCTGGGTAGTATCCCTTCTTCAATATCCACTATGCAAAGCTTAACTT CCGTTGATTTCTCGTATAATAATCTCTCCGGTTTGGTTCCTGGCAGTGGTCAGTTTAGTTACTTCAACTACACCTC ATTTTTGGGGAACCCTGAGCTGTGTGGTCCTTATTTGGGGCCTTGCAAAGATGGGGTTGCTAAAGGAACACATGAA ACTCATGTTAAAGGTGGACTCTCTGCATCTTTGAAGCTTTTGCTCGTAATAGGCCTGCTTGTCTTCTCCATCTTGT TCGCAGTCGCAGCTATAATCAAAGCACGGTCCTTGAAGAAAGCGAGCGATGCTCGGGCTTGGAAGTTAACCGCGTT CCAGCGCTTGGAATTCACTTGTGATGATGTTTTGGATTGTTTGAAGGAGGATAACATTATAGGCAAAGGAGGTGCT GGGATTGTGTACAAGGGATCCATGCCTAGTGGTGACCAGGTCGCCGTTAAAAGGTTACCGGCTATGAGCCGAGGGT CTTCCCATGATCATGGATTCAGTGCTGAGATACAAACTCTGGGGAGGATTAGGCACAGGCATATTGTGAGACTGTT GGGTTTTTGCTCAAATCATGAAACCAATCTCTTGGTTTATGAGTATATGCCTAATGGGAGTTTAGGAGAGGTTCTT CATGGGAAGAAAGGGGGTCATTTGCACTGGGATACCAGATACAAGATCGCGGTCGAGGCGGCTAAGGGACTATGCT ACCTTCATCATGATTGTTCCCCTTTGATAGTCCACCGGGATGTGAAATCGAACAATATCCTCCTCGACTCAGAGTT TGAAGCCCATGTTGCTGATTTTGGCCTTGCTAAATTCTTGCAAGATTCTGGCACTTCCGAATGCATGTCCGCCATA GCTGGTTCATACGGATACATAGCTCCAG GTATATACCATTCTTTTTAACTAAAGGGTTCAAAGAACTTGATGTTTCATTTGACTCTTAATGATTTGCGATTGGATCATGCATGAAGCATTAATACCTACCAATAATTTCCTCTGGACTTTGGTTTTGACTTTGTGTTACCATGATTTGGTGCTATTGAAGTTCATATTTTCATCATTTCAATCAATATATGCAGCTTTCGAAGTACATCAATTTAGTTATAGTCTGACCAGCCAAATTTTGGTGGTTCCAACTGTCCAGTAGTAATTTCCCTTAATATTTCATATAATTCCCCTGTATATTTTTGCAG AATACGCCTACACACTGAAGGTAGATGAGAAGAGTGACGTG TATAGCTTTGGTGTAGTCCTGTTAGAACTAGTCTGTGGCAGAAAACCGGTAGGGGAGTTCGGTGATGGAGTCGATA TTGTTCAATGGGTTCGAAAAATGACAGACTCAAACAAAGAAAGTGTCCTCAAAGTCCTTGATCCCAGACTCCCATC AGTTCCTCTCCAGGAAGTGATGCACGTATTCTACGTCGCCATGCTTTGCGTCGAAGAACAAGCTGTAGAGCGACCA ACGATGAGGGAAGTGGTTCAAATCCTAACCGAACTCCCAAAACCACCAAACTCAAAACAGGGAGACACCACAATCA ATGAGTCCCCAACATCACCATCACCAGACACAACTCTAGACTCACCTACAACAACAATCACAAAGGACCCAAAAGA CCAGCAACAACAGCCCCCAGCACCTAAATCAACACCACCTGATCTTCTTAGCATTTAA .(SEQ ID NO:1, underlined indicates exon);

[0050] Its CDS sequence is shown in SEQ ID NO:2.

[0051] Its amino acid sequence is: MRLLLLLLFLLLHISHSYAAGTAARAVTELRALIAVKSS ITDDPQSYLSNWNATTPLCSFAGVTCDLTGRHVTSIDLTNFTLSGTLSPSLAHLRFLQNISVADNNLTGPIPTELAVLSNLRYLNLSNNVFNGSFPTQLSQLKNLQVLDLYNNNMTAELPVSVTELPNLRHLHLGGNYFSGQIPSSYGRWEHLEYLAVSGNELSGKIPPEIGNLTKLKELYIGYFNNFEGGLPPEIGNLSELVRFDAANCMLSGEIPPEIGKLQKLDTLFLQVNALSGSLTPELGTLNSLKSMDLSNNMFTGEIPASFAQLKNLTLLNLFRNKLHGQIPDFIGELPELEVLQLWENNFTGSIPQKLGSNKKLQVLDLSSNKLTGTLPPDMCSGNTLQTLITLGNFLLGPIPESLGKCESLSRIRMGENYLNGSIPKGLLGLPQLTQVELQDNYLTGEFPVTDSSISVNLGQISLSNNQLSGALPASVGNFSGVQKLLLDGNKFSGPIPAEIGKLQQLSKIDFSHNKFSGLIPPEICKCKLLTFVDLSRNELSGRIPTEITSMRILNFLNLSRNHLLGSIPSSISTMQSLTSVDFSYNNLSGLVPGSGQFSYFNYTSFLGNPELCGPYLGPCKDGVAKGTHETHVKGGLSASLKLLLVIGLLVFSILFAVAAIIKARSLKKASDARAWKLTAFQRLEFTCDDVLDCLKEDNIIGKGGAGIVYKGSMPSGDQVAVKRLPAMSRGSSHDHGFSAEIQTLGRIRHRHIVRLLGFCSNHETNLLVYEYMPNGSLGEVLHGKKGGHLHWDTRYKIAVEAAKGLCYLHHDCSPLIVHRDVKSNNILLDSEFEAHVADFGLAKFLQDSGTSECMSAIAGSYGYIAPEYAYTLKVDEKSDVYSFGVVLLELVCGRKPVGEFGDGVDIVQWVRKMTDSNKESVLKVLDPRLPSVPLQEVMHVFYVAMLCVEEQAVERPTMREVVQILTELPKPPNSKQGDTTINESPTSPSPDTTLDSPTTTITKDPKDQQQQPPAPKSTPPDLLSI (as shown in SEQ ID NO:3).

[0052] (3) Cloning of GhBAM1 gene

[0053] PCR amplification was performed using primers 5UTR-BAM1-F / 3UTR-BAM1-R (SEQ ID NO.12 - 15) containing the 5'UTR and 3'UTR of the GhBAM1 gene. The specific primer sequences are as follows:

[0054] 5UTR-BAM1-F: 5'TCCATCGCAGGGAAAATGAGGTTAC 3' (SEQ ID NO:12)

[0055] 3UTR-BAM1-R: 5'CCCCCTTTGAAACATTGACAACCTT 3' (SEQ ID NO:13)

[0056] BAM1-F:5'ATGAGGTTACTCCTCCTCCTCCTCT 3'(SEQ ID NO:14)

[0057] BAM1-R:5'TTAAATGCTAAGAAGATCAGGTGGT 3'(SEQ ID NO:15)

[0058] The preferred PCR amplification program is: 95℃ pre-denaturation for 5 min; 3 cycles of 95℃ for 30 sec, 55℃ for 30 sec, and 72℃ for 150 sec; 28 cycles of 95℃ for 30 sec, 59℃ for 30 sec, and 72℃ for 150 sec; and a final extension at 72℃ for 5 min. Subsequently, using the first-round PCR product as a template, a second-round PCR amplification is performed using the full-length gene primers BAM1-F / BAM1-R. The preferred PCR amplification program is: 95℃ pre-denaturation for 3 min; 32 cycles of 95℃ for 30 sec, 58℃ for 30 sec, and 72℃ for 150 sec; and a final extension at 72℃ for 5 min. The purified and recovered second-round PCR product can be used for the construction of the full-length GhBAM1 gene vector.

[0059] Example 2: Validation of GhBAM1 knockout in cotton

[0060] (1) Construction of pRGEB32-7-GhBAM1 vector

[0061] There are 13 GhBAM1 genes in cotton. This invention constructs a CRISPR / Cas9 knockout vector specifically targeting the GhBAM1 gene, named pRGEB32-7-GhBAM1. Based on the GhBAM1 genome sequence (SEQ ID NO:1) and CDS sequence (SEQ ID NO:2), two CRISPR / Cas9 targets, sgRNA1 (SEQ ID NO:4) and sgRNA2 (SEQ ID NO:5), were designed at exon 1 and exon 2, respectively. pRGEB32-7-GhBAM1 contains both sgRNA1 and sgRNA2 targets, which can significantly increase the probability of knocking out the GhBAM1 gene.

[0062] A CRISPR / Cas9 vector for knocking out the GhBAM1 gene was constructed, with the backbone vector being pRGEB32-7.

[0063] Primers cBAM1-1as (SEQ ID NO:7), cBAM1-1s (SEQ ID NO:8), cBAM1-2as (SEQ ID NO:9), and inf cBAM1-2as (SEQ ID NO:11) were designed based on the target sequence. Universal primers pGREB32-7s (SEQ ID NO:6) and inf pRGEB32-7s (SEQ ID NO:10) were used. Two PCR amplifications were performed using the above primers. The pRGEB32-7 empty vector was digested with BsaI. After digestion, gel electrophoresis was performed to recover and purify the large fragment of the pRGEB32-7 empty vector. The target fragment and the linearized expression vector were ligated using Exnase enzyme to construct the GhBAM1 gene CRISPR / Cas9 vector.

[0064] The nucleotide sequence of pGREB32-7s is 5'AAGCATCAGATGGGCAAACAAAGC ACCAGTGGTCTAG 3' (SEQ ID NO:6);

[0065] The nucleotide sequence of cBAM1-1as is 5'ACCGGTTAAATCGCACGTGAtgcaccagc cgggaat 3' (SEQ ID NO:7);

[0066] The nucleotide sequence of cBAM1-1s is: 5'TCACGTGCGATTTAACCGGTgttttagagc tagaaata 3' (SEQ ID NO:8);

[0067] The nucleotide sequence of cBAM1-2as is: 5'GTTTTCTGCCACAGACTAGTtgcaccag ccgggaat 3' (SEQ ID NO:9);

[0068] The nucleotide sequence of inf pRGEB32-7s is: 5'AAGCATCAGATGGGCAAACAA A 3' (SEQ ID NO: 10);

[0069] The nucleotide sequence of inf cBAM1-2as is: 5'ttctagctctaaaacGTTTTCTGCCACAG ACTAGT3' (SEQ ID NO:11);

[0070] The GhBAM1-gRNA sequence driven by the GhU6-7 promoter was obtained by overlapping PCR (Wang et al. High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR / Cas9 system. Plant Biotechnol J. 2018, 16(1):137-150). PCR amplification was performed using primers pGREB32-7s and cBAM1-1as. The amplification reaction system consisted of 16.1 μL ddH2O; 2 μL buffer; 0.3 μL dNTPs; 0.2 μL each primer; 0.2 μL high-fidelity DNA polymerase; and 1 μL pGTR template. The amplification reaction program was as follows: 95℃ pre-denaturation for 4 min; 95℃ for 30 sec, 55℃ for 30 sec, 72℃ for 20 sec, 3 cycles; 95℃ for 30 sec, 60℃ for 30 sec, 72℃ for 20 sec, 27 cycles; extension at 72℃ for 5 min.

[0071] PCR2 amplification was performed using cBAM1-1s and cBAM1-2as primers. The amplification reaction system consisted of 16.1 μL ddH2O; 2 μL buffer; 0.3 μL dNTPs; 0.2 μL each primer; 0.2 μL high-fidelity DNA polymerase; and 1 μL pGTR template. The amplification reaction program was as follows: 95℃ pre-denaturation for 4 min; 3 cycles of 95℃ for 30 sec, 55℃ for 30 sec, and 72℃ for 20 sec; 27 cycles of 95℃ for 30 sec, 60℃ for 30 sec, and 72℃ for 20 sec; and extension at 72℃ for 5 min.

[0072] The products of PCR1 and PCR2 were then amplified by overlap extension PCR using inf pRGEB32-7s and inf cBAM1-2as primers. The amplification reaction system consisted of 16.7 μL ddH2O, 2 μL buffer, 0.3 μL dNTPs, 0.2 μL each primer, 0.2 μL high-fidelity DNA polymerase, and 0.2 μL each of the fragments containing sgRNA1 and sgRNA2. The amplification program was as follows: 95℃ pre-denaturation for 4 min; 95℃ for 30 sec, 59℃ for 30 sec, 72℃ for 20 sec, 28 cycles, followed by an extension at 72℃ for 5 min. Fragments containing two sgRNAs were obtained.

[0073] The empty pRGEB32-7 vector was digested with BsaI at 37°C for 6 hours. Following digestion, gel electrophoresis was performed to recover the large fragment of the empty pRGEB32-7 vector. The target fragment from the second PCR amplification was then infused with Exnase and ligated into the linearized expression vector. The constructed vector was named pRGEB32-7-GhBAM1 (vector image shown). Figure 1 ).

[0074] The reaction product was transformed into *E. coli* competent cells TOP10. After culturing for 10–12 hours, single clones were picked for PCR positive detection. Primers used were U6-7s and inf cBAM1-2as. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min; 30 cycles of 94℃ for 30 sec, 58℃ for 30 sec, and 72℃ for 1 min; extension at 72℃ for 5 min. Positive single clones were amplified and plasmids were extracted to obtain the gene knockout plasmid pRGEB32-7-GhBAM1 (…) for transformation. Figure 1 ).

[0075] U6-7s: 5'TGTGCCACTCCAAAGACATCAG 3' (SEQ ID NO:16, sequence on vector)

[0076] (2) Transformation of Agrobacterium tumefaciens by vector

[0077] The pRGEB32-7-GhBAM1 plasmid vector was electroporated to transform Agrobacterium strain GV3101. The culture was incubated upside down in a bacterial incubator at 28°C for 2-3 days. Single colonies were picked and inoculated into LB liquid medium containing 100 mg / L kanamycin and rifampin. The medium was shaken at 150 rpm and 28°C for 24 h. The bacterial culture was positively detected using U6-7s and inf cBAM1-2as primers. The positive bacterial culture was then propagated and used for genetic transformation of cotton.

[0078] (3) Agrobacterium-mediated genetic transformation of cotton

[0079] A. Sterile seedling culture: Select plump and normally developed seeds (recipient material is Jin668), peel off the cottonseed seed coat, soak in 2% sodium hypochlorite solution for 10 minutes, rinse with sterile water more than 3 times, inoculate on sterile seedling culture medium, and after the seeds show white sprouts, insert the roots into the culture medium to support the seedlings, seal the opening and place in a 28℃ dark constant temperature incubator for 5-6 days.

[0080] B. Select seedlings with good growth and cut the hypocotyl into small segments of about 0.8 cm. Infect them with activated Agrobacterium containing the GhBAM1 knockout vector, discard the bacterial solution, and blow dry. Spread the hypocotyls flat on a co-culture medium (MS medium + 2,4-D 0.1 mg / L + KT 0.1 mg / L + 30 g / L glucose + 2.6 g / L Phytagel, pH: 5.85~5.95) with filter paper and incubate in the dark at 20℃ for 36~48 h.

[0081] C. Obtaining regenerated material: Infected hypocotyls were inoculated into DK callus induction medium (MS medium + 2,4-D 0.1 mg / L + KT 0.1 mg / L + 30 g / L glucose + 2.6 g / L Phytagel + kanamycin 50 mg / L + cephalosporin 400 mg / L, pH: 5.85–5.95). The culture was carried out at 28°C in a light-controlled culture chamber (16 h light + 8 h dark), and subcultured every 20–30 days using fresh callus induction medium.

[0082] D. When the callus tissue grows into rice-grain-sized granules, it is transferred to differentiation medium (MS medium + IBA 0.5 mg / L + KT 0.1 mg / L + glutamine 1.0 g / L + asparagine 0.5 g / L + 30 g / L glucose + 2.6 g / L Phytagel, pH: 6.1~6.2) to further differentiate into embryoids.

[0083] E. The differentiated seedlings are subcultured on rooting medium (1 / 2 MS medium + 15 g / L glucose + 2.6 g / L Phytagel, pH: 6.1-6.2) until they grow into well-rooted seedlings.

[0084] (4) Identification and phenotype of GhBAM1 knockout progeny

[0085] DNA was extracted from leaves of 22 independently transformed transgenic positive cotton lines (T0, T1, and T2 generations). Primers B1-BAM1-F: 5'-CATTACCGACGACCCTCAATCT-3' (SEQ ID NO: 17); B1-BAM1-R: 5'-GAGATGTTTTGGAGGAAACGGA-3' (SEQ ID NO: 18) were designed for PCR amplification of the gene sequence at the first target site of GhBAM1. Primers B2-BAM1-F: 5'-AATACGCCTACACACTGAAGGTAGA-3' (SEQ ID NO: 19); B2-BAM1-R: 5'-CTTTGTTTGAGTCTGTCATTTTTCG-3' (SEQ ID NO: 18) were designed for PCR amplification of the gene sequence at the first target site of GhBAM1. NO:20), the gene sequence at the second target site of GhBAM1 was amplified by PCR. Sanger sequencing was used to screen and isolate plants GhBAM1_KO#1 to KO#5 with gene knockout vectors and homozygous mutations in the GhBAM1 gene for field phenotypic identification. After cotton harvest, the number of effective bolls per plant, the number of buds and bolls dropped per plant, and the bud and boll drop rate of the above GhBAM1 gene knockout lines GhBAM1_KO#1 to KO#5 and wild-type Jin668 plants were measured and statistically analyzed. The results are shown in […]. Figure 3 .Depend on Figure 3 It was found that the number of effective bolls per plant in the GhBAM1 gene knockout line was significantly higher than that in the wild-type Jin668, while the number of boll sheddings and the boll shedding rate per plant were significantly lower than those in the wild-type control. This invention is the first to identify the association between the GhBAM1 gene and cotton boll shedding, which is of great value for theoretical research on the molecular mechanism of cotton boll shedding. By knocking out this gene, the boll shedding rate of cotton can be significantly reduced, and the cotton's resistance to high-temperature stress can be improved, thus creating high-yielding and stable-yielding cotton breeding materials, which has important practical application value for cotton breeding.

[0086] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. Application of the GhBAM1 gene in any of the following: A1) Increase the number of effective bolls per cotton plant and reduce the boll shedding rate; A2) Create products that increase the number of effective bolls per cotton plant and reduce the boll shedding rate of cotton buds; By knocking out the cotton GhBAM1 gene, the effective number of bolls per cotton plant is increased, and the boll shedding rate is reduced; wherein the amino acid sequence encoded by the GhBAM1 gene is shown in SEQ ID NO:

3.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the GhBAM1 gene is shown in SEQ ID NO:1, and the CDS sequence is shown in SEQ ID NO:

2.

3. The application according to any one of claims 1-2, characterized in that, The cotton GhBAM1 gene was knocked out using a CRISPR vector containing sgRNA, the nucleotide sequence of which is shown in SEQ ID NO:4 and / or SEQ ID NO:

5.

4. A method for reducing cotton boll shedding rate, characterized in that, The purpose of knocking out the GhBAM1 gene is to increase the number of effective bolls per cotton plant and reduce the boll shedding rate; the amino acid sequence encoded by the GhBAM1 gene is shown in SEQ ID NO:

3.

5. The method according to claim 4, characterized in that, The nucleotide sequence of the GhBAM1 gene is shown in SEQ ID NO:1, and the CDS sequence is shown in SEQ ID NO:

2.

6. The method according to any one of claims 4-5, characterized in that, The cotton GhBAM1 gene was knocked out using a CRISPR vector containing sgRNA, the nucleotide sequence of which is shown in SEQ ID NO:4 and / or SEQ ID NO:

5.

7. The method according to any one of claims 4-5, characterized in that, The GhBAM1 gene in cotton is knocked out by introducing a gene knockout vector into cotton. The preparation method of the gene knockout vector includes the following steps: 1) Using pGTR plasmid as a template, the first target site was amplified by PCR using the pGREB32-7s / cBAM1-1as primer pair to obtain the fragment containing sgRNA1. The nucleotide sequence of pGREB32-7s is 5' AAGCATCAGATGGGCAAACAAAGCACCAGTGGTCTAG 3' (SEQ ID NO:6); The nucleotide sequence of cBAM1-1as is 5' ACCGGTTAAATCGCACGTGAtgcaccagccgggaat 3' (SEQ ID NO:7). 2) Using pGTR plasmid as a template, the second target site was amplified by PCR using the cBAM1-1s / cBAM1-2as primer pair to obtain the fragment containing sgRNA2. The nucleotide sequence of cBAM1-1s is: 5' TCACGTGCGATTTAACCGGTgttttagagctagaaata 3' (SEQ ID NO:8); The nucleotide sequence of cBAM1-2as is: 5' GTTTTCTGCCACAGACTAGTtgcaccagccgggaat 3' (SEQ ID NO:9); 3) Using the fragments containing sgRNA1 and sgRNA2 as templates, overlap extension PCR amplification was performed using the inf pRGEB32-7s / inf cBAM1-2as primer pair to obtain DNA fragments containing sgRNA1 and sgRNA2. The nucleotide sequence of inf pRGEB32-7s is: 5' AAGCATCAGATGGGCAAACAAA 3' (SEQ ID NO:10); The nucleotide sequence of inf cBAM1-2as is: 5' ttctagctctaaaacGTTTTCTGCCACAGACTAGT 3' (SEQ ID NO:11); 4) Insert the DNA fragment containing sgRNA1 and sgRNA2 into the backbone vector pRGEB32-7 to obtain the CRISPR / Cas9 vector.

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