Application of GhBAM1 gene in reduction of cotton squaring and boll shedding rate

Abstract

The invention relates to application of a GhBAM1 gene in reduction of cotton squaring and boll shedding rate, the cotton squaring and boll shedding gene GhBAM1 is identified for the first time, and the gene has important value in theoretical research such as analysis of molecular mechanism of cotton squaring and boll shedding and the like. By knocking out the upland cotton endogenous gene GhBAM1, the cotton squaring and boll shedding rate can be reduced, the high-temperature stress resistance of cotton is improved, a high-yield and stable-yield cotton breeding material is created, a cotton gene resource library is enriched, and the important practical application significance is achieved for cotton breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene breeding, and particularly relates to application of a GhBAM1 gene in reducing a cotton bud and boll shedding rate. Background Art

[0002] Cotton is an important cash 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 production and life of the people. 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 continuous high temperature weather. This is the time when cotton flowers and bolls form, which is a key stage affecting the formation of cotton yield (Ma et al. Disrupted genome methylation in response to hightemperature has distinct affects on microspore abortion and antherindehiscence. Plant Cell. 2018, 30 (7): 1387-1403). Continuous high temperature weather above 35 ° C will have an adverse effect on the reproductive development of cotton, mainly manifested in anthers not opening, pollen vitality decreases, and bud boll shedding rate increases, which ultimately affects 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 temperature is an important cause of cotton bud and boll shedding. Discovering new heat-resistant genes and breeding excellent 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 huge development space for the realization of precision cotton breeding. Breeding new heat-resistant cotton varieties through genetic engineering technology can not only effectively increase cotton production and increase farmers' income, but also has important significance for promoting the development of my country's cotton textile industry and improving the competitiveness of my country's cotton in the international market.

[0004] The bud and boll abscission rate is an important yield trait of cotton. High temperature is the main reason affecting bud and boll abscission. High temperature above 35℃ usually inhibits photosynthesis, and photosynthesis stops when it exceeds 40℃. Insufficient photosynthesis can cause bud and boll abscission. On the other hand, high temperature can also lead to decreased pollen vitality, and more seriously, it can cause anthers to fail to crack, causing the ovary to fall off due to poor fertilization or inability to complete fertilization. Therefore, identifying the regulatory genes related to cotton bud and boll abscission is crucial for the cultivation of high temperature resistant cotton germplasm. Summary of the invention

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

[0006] The present invention identifies the gene GhBAM1 that regulates the bud and boll abscission rate of cotton based on CRISPR / Cas9 gene editing technology. Knocking out the GhBAM1 gene in cotton can significantly reduce the bud and boll abscission rate of cotton (the percentage of the number of buds and bolls abscission per plant to the sum of the number of effective bolls per plant and the number of buds and bolls abscission), thereby significantly increasing the cotton yield, indicating that the gene or the protein encoded by it plays an important role in controlling the bud and boll abscission rate of cotton.

[0007] The invention provides a gene GhBAM1 for regulating cotton bud and boll abscission rate, wherein the genomic nucleotide sequence is shown in SEQ ID NO:1, the CDS sequence is shown in SEQ ID NO:2, and the amino acid sequence is shown in SEQ ID NO:3.

[0008] It should be understood that, taking into account the degeneracy of gene expression regulatory regions and codons, modifications to the GhBAM1 gene sequence without changing the amino acid sequence also fall within the scope of protection of the present invention.

[0009] The present invention provides the use of the GhBAM1 gene and an agent for regulating the expression of GhBAM1 in any of the following:

[0010] A1) Regulate the cotton bud and boll shedding rate;

[0011] A2) Create products to control cotton bud and boll shedding rate;

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

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

[0014] A5) Regulating cotton production;

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

[0016] A7) Cultivate high-yield and heat-resistant cotton varieties;

[0017] A8) Identify or assist in identifying heat 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 reagent for regulating GhBAM1 expression includes sgRNA and / or CRISPR vector, and 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 the expression of GhBAM1, wherein the reagent for regulating the expression of GhBAM1 comprises sgRNA, the nucleotide sequence of the sgRNA is shown in SEQ ID NO:4 and / or SEQ ID NO:5, and the amino acid sequence encoded by the GhBAM1 gene is shown in SEQ ID NO:3.

[0022] The present invention also provides a method for reducing the bud and boll shedding rate of cotton, which achieves the purpose of increasing the number of effective bolls per cotton plant and reducing the bud and boll shedding rate of cotton by inhibiting and / or knocking out GhBAM1. Preferably, the GhBAM1 protein is knocked out by gene editing technology, or the expression of GhBAM1 is interfered with by RNAi technology to reduce the cotton boll shedding rate.

[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, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO:4 and / or SEQ ID NO:5.

[0026] Furthermore, the effect of inhibiting the expression of GhBAM1 in cotton is achieved by introducing a gene knockout vector into cotton. The preparation method of the gene knockout vector comprises the following steps:

[0027] 1) Using the pGTR plasmid as a template, the pGREB32-7s / cBAM1-1as primer pair was used to perform PCR amplification of the first target site to obtain a fragment containing sgRNA1;

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

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

[0030] 2) Using the pGTR plasmid as a template, the cBAM1-1s / cBAM1-2as primer pair was used to perform PCR amplification of the second target site to obtain a fragment containing sgRNA2;

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

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

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

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

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

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

[0037] Beneficial effects: The present invention identifies the cotton bud and boll abscission gene GhBAM1 for the first time. This gene has important value for theoretical research such as analyzing the molecular mechanism of cotton bud and boll abscission.

[0038] The invention can significantly reduce the bud and boll shedding rate of cotton to create high-yield and stable-yield cotton breeding materials, and has important practical application significance for cotton breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

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

[0041] Figure 2 Heat map of expression patterns of 13 annotated GhBAM1 genes in Upland cotton.

[0042] Figure 3 Effect of knocking out the GhBAM1 gene on cotton yield traits. (a) The phenotypes of wild-type Jin668 and three transgenic lines with GhBAM1 knockout; (b)-(d) The phenotypic statistics of wild-type Jin668 and three transgenic lines with GhBAM1 knockout in terms of the number of effective bolls per plant, the number of bolls shed per plant, and the rate of bolls shed per plant. DETAILED DESCRIPTION

[0043] The following examples are only used to more clearly illustrate the technical scheme of the present invention, and are therefore only used as examples, and cannot limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise stated, the reagents and materials used in the following examples are commercially available.

[0044] Example 1: Analysis of the function and expression pattern of GhBAM1 in upland cotton

[0045] (1) Analysis of GhBAM1 gene function

[0046] GhBAM1 belongs to the cotton leucine-rich repeat receptor-like kinase (LRR-RLK), which is the largest known transmembrane receptor kinase (RLK) in plants. LRR-RLK generally consists of three parts, namely the extracellular LRR domain, the transmembrane domain and the intracellular kinase domain, and is widely involved in various life activities of plants. RLK is a key protein kinase for plants to perceive environmental signals and is also the largest receptor protein family in plants, which is of great significance for plants to adapt to environmental changes.

[0047] (2) Expression pattern of GhBAM1

[0048] There are 15 annotated BAM1 genes in upland cotton. According to the gene expression pattern, the expression levels of Ghir_A02G016300, Ghir_A02G016290 and Ghir_D03G003280 genes are higher ( Figure 2 ). Among them, the Ghir_A02G016300 gene was significantly expressed in tissues related to reproductive development, such as anthers, ovules, and stigmas ( Figure 2 ), from which it can be inferred that the GhBAM1 gene is related to the reproductive development process of cotton. Its genomic nucleotide sequence is:

[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 carried out 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 PCR amplification procedure is preferably: 95°C pre-denaturation for 5 min; 95°C 30 sec, 55°C 30 sec, 72°C 150 sec, 3 cycles; 95°C 30 sec, 59°C 30 sec, 72°C 150 sec, 28 cycles; 72°C extension for 5 min. Subsequently, the first round of PCR products were used as templates and the full-length gene primers BAM1-F / BAM1-R were used for the second round of PCR amplification. The PCR amplification procedure is preferably: 95°C pre-denaturation for 3 min; 95°C 30 sec, 58°C 30 sec, 72°C 150 sec, 32 cycles; 72°C extension for 5 min. The second round of PCR products can be used for the construction of the full-length vector of the GhBAM1 gene after purification and recovery.

[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. The present invention constructs a CRISPR / Cas9 knockout vector specifically targeting the GhBAM1 gene, named pRGEB32-7-GhBAM1. According to the genomic sequence (SEQ ID NO: 1) and CDS sequence (SEQ ID NO: 2) of GhBAM1, two CRISPR / Cas9 target sites, sgRNA1 (SEQ ID NO: 4) and sgRNA2 (SEQ ID NO: 5), are designed at the first exon and the second exon thereof, respectively. pRGEB32-7-GhBAM1 contains two targets, sgRNA1 and sgRNA2, which can greatly increase the probability of knocking out the GhBAM1 gene.

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

[0063] According to the target sequence, cBAM1-1as (SEQ ID NO: 7), cBAM1-1s (SEQ ID NO: 8), cBAM1-2as (SEQ ID NO: 9) and inf cBAM1-2as (SEQ ID N NO: 11) primers were designed, pGREB32-7s (SEQ ID NO: 6) and inf pRGEB32-7s (SEQ ID NO: 10) were used as universal primers, and PCR amplification was performed twice with the above primers. The pRGEB32-7 empty vector was digested with BsaI, and gel electrophoresis was performed after digestion. The large fragment of the pRGEB32-7 empty vector was excised and recovered and purified, and the target fragment and the linearized expression vector were connected with Exnase enzyme to construct a GhBAM1 gene CRISPR / Cas9 vector.

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

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

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

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

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

[0069] The nucleotide sequence of the inf cBAM1-2as: 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). PCR1 was amplified using primers pGREB32-7s and cBAM1-1as. The amplification reaction system was ddH2O 16.1μL; Buffer 2μL; dNTP 0.3μL; primers 0.2μL each; high-fidelity DNA polymerase 0.2μL; pGTR template 1μL. The program of the amplification reaction was pre-denaturation at 95°C for 4 min; 3 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 20 sec, and 27 cycles of 95°C for 30 sec, 60°C for 30 sec, and 72°C for 20 sec; and extension at 72°C for 5 min.

[0071] PCR2 amplification was performed using cBAM1-1s and cBAM1-2as primers. The amplification reaction system was ddH2O16.1μL; Buffer 2μL; dNTP 0.3μL; primers 0.2μL each; high-fidelity DNA polymerase 0.2μL; pGTR template 1μL. The amplification reaction program was 95℃ pre-denaturation for 4min; 95℃30sec, 55℃30sec, 72℃20sec, 3 cycles, 95℃30sec, 60℃30sec, 72℃20sec, 27 cycles; 72℃ extension for 5min.

[0072] The PCR1 and PCR2 products were then amplified by overlapping extension PCR using inf pRGEB32-7s and inf cBAM1-2as primers. The amplification reaction system was 16.7 μL of ddH2O, 2 μL of Buffer, 0.3 μL of dNTP, 0.2 μL of each primer, 0.2 μL of high-fidelity DNA polymerase, and 0.2 μL of each fragment containing sgRNA1 and sgRNA2. The amplification reaction program was 95°C pre-denaturation for 4 min; 95°C for 30 sec, 59°C for 30 sec, 72°C for 20 sec, 28 cycles, and 72°C extension for 5 min. A fragment containing two sgRNAs was obtained.

[0073] The pRGEB32-7 empty vector was digested with BsaI at 37°C for 6 hours, and then subjected to gel electrophoresis. The large fragment of the pRGEB32-7 empty vector was recovered by gel digging. The target fragment amplified by the second PCR and the linearized expression vector were connected by In-fusion using Exnase enzyme. The constructed vector was named pRGEB32-7-GhBAM1 (see the vector map for details). Figure 1 ).

[0074] The reaction product was used to transform the competent E. coli cell TOP10. After culturing for 10 to 12 hours, a single clone was picked for PCR positive detection. The primers used were U6-7s and inf cBAM1-2as. The PCR reaction conditions were: 94℃ pre-denaturation for 5min; 94℃30sec, 58℃30sec, 72℃1min, 30 cycles; 72℃ extension for 5min. The positive single clone was expanded and the plasmid was extracted to obtain the gene knockout plasmid pRGEB32-7-GhBAM1( Figure 1 ).

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

[0076] (2) Vector transformation of Agrobacterium

[0077] The pRGEB32-7-GhBAM1 plasmid vector was transformed into Agrobacterium strain GV3101 by electroporation, and cultured inverted in a bacterial incubator at 28°C for 2-3 days. A single clone colony was picked and inoculated into LB liquid culture medium containing 100 mg / L kanamycin and rifampicin and shaken at 150 rpm and 28°C for 24 hours. The bacterial solution was tested for positive results using U6-7s and inf cBAM1-2as primers. The positive bacterial solution can be used for genetic transformation of cotton after expansion.

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

[0079] A. Sterile seedling culture: Select seeds with full grains and normal development (recipient material is Jin668), peel off the cottonseed seed coat, soak in 2% sodium hypochlorite solution for 10 minutes, then rinse with sterile water for more than 3 times, inoculate on sterile seedling culture medium, and after the seeds turn white, insert the roots into the culture medium for seedling support, seal the medium and culture in a dark incubator at 28℃ for 5-6 days.

[0080] B. Select seedlings with good growth conditions and cut the hypocotyls 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 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 culture in the dark in a constant temperature incubator at 20°C for 36-48 hours;

[0081] C. Obtaining regenerated materials: The 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). Cultured in a light culture room at 28°C (16 h of light + 8 h of darkness), subcultured with fresh callus induction medium every 20-30 days.

[0082] D. When the callus tissue grows into rice-like particles, it is transferred to differentiation medium (MS medium + IBA 0.5 mg / L + KT0.1 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) and further differentiated into embryoids.

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

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

[0085] For the 22 independently transformed transgenic positive cotton lines obtained, DNA was extracted from the leaves of T0, T1 and T2 transgenic positive plants, and primers B1-BAM1-F: 5'-CATTACCGACGACCCTCAATCT-3' (SEQ ID NO: 17); B1-BAM1-R: 5'-GAGATGTTTTGGAGGAAACGGA-3' (SEQ ID NO: 18) were designed to PCR amplify the gene sequence at the first target site of GhBAM1, and primers B2-BAM1-F: 5'-AATACGCCTACACACTGAAGGTAGA-3' (SEQ ID NO: 19); B2-BAM1-R: 5'-CTTTGTTTGAGTCTGTCATTTTTCG-3' (SEQ ID NO: 20) were designed to amplify the gene sequence at the first target site of GhBAM1. NO:20), PCR amplified the gene sequence at the second target site of GhBAM1, and screened and isolated the gene knockout vector and the plants GhBAM1_KO#1 to KO#5 with homozygous mutation of GhBAM1 gene by Sanger sequencing, and carried out field phenotypic identification. After cotton harvest, the number of effective bolls per plant, the number of bolls per plant, and the rate of bolls per plant of the GhBAM1 gene knockout lines GhBAM1_KO#1 to KO#5 and the wild-type Jin668 plants were measured and counted. The results are shown in Figure 3 .Depend on Figure 3 It can be seen that the number of effective bolls per plant in the strain with GhBAM1 gene knocked out is significantly higher than that of the wild type Jin668, and the number of bolls per plant and the bolls per plant shedding rate are significantly lower than those of the wild type control plants. That is, the present invention identified the association between GhBAM1 gene and bolls shedding in cotton for the first time, which is of great value for theoretical research such as analyzing the molecular mechanism of bolls shedding in cotton; by knocking out this gene, the bolls shedding rate of cotton can be significantly reduced, and the ability of cotton to resist high temperature stress can be improved, so as to create high-yield and stable-yield cotton breeding materials, which has important practical application value for cotton breeding.

[0086] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. Use of the GhBAM1 gene or an agent for regulating GhBAM1 expression in any of the following: A1) Regulate the cotton bud and boll shedding rate; A2) Create products to control cotton bud and boll shedding rate; A3) Increase the number of effective bolls per cotton plant and reduce the rate of cotton bud and boll shedding; A4) Create products that increase the number of effective bolls per cotton plant and reduce the rate of cotton bud and boll shedding; A5) Regulating cotton production; A6) Create products to regulate cotton production; A7) Cultivate high-yield and heat-resistant cotton varieties; A8) Identify or assist in identifying heat resistant cotton varieties.

2. The use according to claim 1, characterized in that: The amino acid sequence encoded by the GhBAM1 gene is shown in SEQ ID NO:

3.

3. The use according to claim 2, 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.

4. The use according to any one of claims 1 to 3, characterized in that: The reagent for regulating GhBAM1 expression includes sgRNA and / or CRISPR vector, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO:4 and / or SEQ ID NO:

5.

5. An agent for regulating the expression of GhBAM1, characterized in that: The reagent for regulating GhBAM1 expression includes sgRNA, the nucleotide sequence of the sgRNA is shown in SEQ ID NO:4 and / or SEQ ID NO:5, and the amino acid sequence encoded by the GhBAM1 gene is shown in SEQ ID NO:

3.

6. A method for reducing the bud and boll shedding rate of cotton, characterized in that: By inhibiting and / or knocking out GhBAM1, the purpose of increasing the number of effective bolls per cotton plant and reducing the cotton bud and boll shedding rate can be achieved.

7. The method according to claim 6, characterized in that The amino acid sequence encoded by the GhBAM1 gene is shown in SEQ ID NO:

3.

8. The method according to claim 7, 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.

9. The method according to any one of claims 6 to 8, characterized in that: The reagent for regulating GhBAM1 expression includes sgRNA, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO:4 and / or SEQ ID NO:

5.

10. The method according to any one of claims 6 to 8, characterized in that: The effect of inhibiting the expression of GhBAM1 in cotton is achieved by introducing a gene knockout vector into cotton. The preparation method of the gene knockout vector comprises the following steps: 1) Using the pGTR plasmid as a template, the pGREB32-7s / cBAM1-1as primer pair was used to perform PCR amplification of the first target site to obtain a fragment containing sgRNA1; The nucleotide sequence of the pGREB32-7s is 5'AAGCATCAGATGGGCAAACAAAGC ACCAGTGGTCTAG 3' (SEQ ID NO: 6); The nucleotide sequence of the cBAM1-1as is 5'ACCGGTTAAATCGCACGTGAtgcaccagc cgggaat 3' (SEQ ID NO: 7); 2) Using the pGTR plasmid as a template, the cBAM1-1s / cBAM1-2as primer pair was used to perform PCR amplification of the second target site to obtain a fragment containing sgRNA2; The nucleotide sequence of cBAM1-1s: 5'TCACGTGCGATTTAACCGGTgttttagagc tagaaata 3' (SEQ ID NO: 8); The nucleotide sequence of the cBAM1-2as: 5'GTTTTCTGCCACAGACTAGTtgcaccag ccgggaat 3' (SEQ ID NO: 9); 3) Using the fragment containing sgRNA1 and the fragment containing sgRNA2 as templates, overlap extension PCR amplification was performed with the inf pRGEB32-7s / i nf cBAM1-2as primer pair to obtain DNA fragments containing sgRNA1 and sgRNA2; The nucleotide sequence of the inf pRGEB32-7s: 5'AAGCATCAGATGGGCAAACAA A3' (SEQ ID NO: 10); The nucleotide sequence of the inf cBAM1-2as: 5'ttctagctctaaaacGTTTTCTGCCACAG ACTAGT 3' (SEQ ID NO: 11); 4) Inserting the DNA fragment containing sgRNA1 and sgRNA2 into the backbone vector pRGEB32-7 to obtain a CRISPR / Cas9 vector.

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