GhSBP1 gene for regulating cotton fiber development, regulating method and breeding application

Abstract

The invention provides a GhSBP1 gene for regulating cotton fiber development, a regulation method and breeding application, and belongs to the technical field of gene engineering. The invention provides a GhSBP1 gene for regulating and controlling the development of cotton fibers. The CDS sequence of the GhSBP1 gene comprises a sequence as shown in SEQ ID No. 1. According to verification in the embodiment of the invention, the GhSBP1 gene is over-expressed in cotton, the fiber length of the over-expressed GhSBP1 cotton in different periods after flowering is longer than that of a wild type cotton, and the over-expressed GhSBP1 can significantly increase the fiber length and the number of initial fiber cells, which indicates that the GhSBP1 gene regulates and controls the fiber length of the fiber cells. The GhSBP1 gene is transferred into cotton, so that the elongation of cotton fiber cells can be promoted, the cell wall thickness of the cotton fiber cells can be increased, and the GhSBP1 gene has great significance in research and improvement of cotton fiber quality.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a GhSBP1 gene that regulates cotton fiber development, its regulation method, and its breeding applications. Background Technology

[0002] Cotton fibers develop from polar, elongated single cells of the ovule epidermis. Fiber development involves distinct and overlapping stages: initiation, elongation, secondary cell wall thickening, and maturation. The initiation and elongation stages of cotton fibers play a crucial role in the number, length, and thickness of fibers, ultimately determining cotton yield and length. The biosynthesis of cotton fiber cell length and number directly affects cotton fiber quality. Each stage of cotton fiber development is essential for the overall fiber phenotype, and its development is inseparable from gene regulation. Multiple specific genes participate in specific periods of cotton fiber development, forming a complex regulatory network. However, known regulations are limited, and all involved genes and their regulatory mechanisms are not yet fully understood. Therefore, a new way to regulate cotton fiber development is needed. Summary of the Invention

[0003] This invention provides a GhSBP1 gene that regulates cotton fiber development, a regulation method, and its breeding applications. The GhSBP1 gene can regulate cotton fiber development and can be applied to the breeding of transgenic cotton.

[0004] This invention provides a GhSBP1 gene that regulates cotton fiber development, wherein the GhSBP1 gene comprises any one of the following:

[0005] (1) The CDS includes the nucleotide sequence shown in SEQ ID No. 1;

[0006] (2) It has at least 75% homology with the CDS sequence shown in SEQ ID No.1 and has the function of the GhSBP1 gene;

[0007] (3) A sequence obtained by deleting, adding or replacing one or more nucleotides based on the CDS sequence shown in SEQ ID No.1, wherein the sequence has the function of the GhSBP1 gene.

[0008] The present invention also provides the protein encoded by the GhSBP1 gene.

[0009] The present invention also provides a biological material comprising the above-mentioned GhSBP1 gene and expressing the above-mentioned protein.

[0010] In a preferred embodiment of the present invention, the type of biomaterial includes a carrier, a bacterial strain, or a cell.

[0011] This invention also provides the application of the above-mentioned biomaterials in regulating cotton fiber development.

[0012] In a preferred embodiment of the present invention, the regulation includes overexpressing the GhSBP1 gene to promote cotton fiber development.

[0013] In a preferred embodiment of the present invention, promoting cotton fiber development includes at least one of the following: (a) increasing fiber length;

[0014] (b) Increase the cell length of mature cotton fiber cells;

[0015] (c) Increase the number of cells that initiate the fibers.

[0016] This invention also provides the application of the above-mentioned biological materials in the breeding of new cotton varieties.

[0017] The present invention also provides a method for breeding cotton varieties, comprising overexpressing the above-mentioned GhSBP1 gene in the genome of the target cotton variety.

[0018] In a preferred embodiment of the present invention, the overexpression method includes transforming a recombinant overexpression vector containing the GhSBP1 gene into a target cotton variety; the base vector of the recombinant overexpression vector includes pCAMBIA2300.

[0019] Beneficial Effects: This invention provides a GhSBP1 gene that regulates cotton fiber development. The CDS sequence of the GhSBP1 gene includes the sequence shown in SEQ ID No. 1. In the embodiments, this invention verifies that overexpressing the GhSBP1 gene in cotton and collecting fibers at 5d, 10d, 15d, 20d, and mature stages reveals that the fiber length of cotton overexpressing GhSBP1 at different post-flowering stages is longer than that of wild-type cotton. By detecting fiber length, plant height, and the number of initial fiber cells in wild-type cotton and GhSBP1-overexpressing cotton, it was found that overexpression of GhSBP1 significantly increases fiber length and the number of initial fiber cells, indicating that the GhSBP1 gene regulates fiber length. Transforming the GhSBP1 gene into cotton can promote cotton fiber cell elongation and increase cotton fiber cell wall thickness, which is of great significance for the research and improvement of cotton fiber quality.

[0020] The GhSBP1 gene described in this invention can regulate downstream regulatory genes GhNRT1.5, GhEzrA, and GhSH3P2. In plants overexpressing the GhSBP1 gene, the expression levels of these three genes are significantly increased. Furthermore, this invention experimentally confirms that GhSBP1 specifically binds to the P4 fragment of the GhNRT1.5 promoter and directly binds to the L4 fragment of the promoter in vitro, forming a protein-DNA complex; GhSBP1 specifically binds to the L3 fragment of the GhEzrA promoter, and this binding disappears after mutation at the binding site; GhSBP1 specifically binds to the P2 fragment of the GhSH3P2 promoter and directly binds to the L2 fragment of the promoter of this gene in vitro. Attached Figure Description

[0021] Figure 1 Genotypes of GhSBP1 gene knockout plants;

[0022] Figure 2 Figure 1 shows the molecular identification results of GhSBP1 overexpressing transgenic cotton plants. A: Protein level identification of GhSBP1 overexpressing cotton lines; B: RNA level identification of GhSBP1 transgenic cotton; C: Fiber length of GhSBP1 transgenic cotton 15 days after flowering; D: Phenotypic image of mature fibers of GhSBP1 transgenic cotton; E: Statistical graph of mature fiber length of GhSBP1 transgenic cotton; F: Electron micrograph of fiber initiation on the day of flowering of GhSBP1 transgenic cotton; G: Statistical graph of the number of fibers initiation on the day of flowering of GhSBP1 transgenic cotton.

[0023] Figure 3 The diagram shows the interaction between GhSBP1 and GhNRT1.5. In the diagram, A: yeast one-hybrid assay, B: competitive EMSA assay, C: quantification of dual-luciferase assay of GhNRT1.5 full-length promoter LUC expression, D: quantification of dual-luciferase assay of GhNRT1.5 promoter fragment LUC expression, and E: direct binding EMSA assay.

[0024] Figure 4 The diagram shows the interaction between GhSBP1 and GhEzrA. In the diagram, A: yeast one-hybrid assay, B: competitive EMSA assay, C: quantification of dual-luciferase assay of GhEzrA full-length promoter LUC expression, D: quantification of dual-luciferase assay of GhEzrA promoter fragment LUC expression, and E: direct binding EMSA assay.

[0025] Figure 5This diagram illustrates the interaction between GhSBP1 and GhSH3P2. In the diagram, A represents the yeast one-hybrid assay, B represents the competitive EMSA assay, C represents the quantification of dual-luciferase assay for GhSH3P2 full-length promoter LUC expression, D represents the quantification of dual-luciferase assay for GhSH3P2 promoter fragment LUC expression, and E represents the direct binding EMSA assay. Detailed Implementation

[0026] This invention provides a GhSBP1 gene that regulates cotton fiber development, wherein the GhSBP1 gene comprises any one of the following:

[0027] (1) The CDS includes the nucleotide sequence shown in SEQ ID No. 1;

[0028] (2) It has at least 75% homology with the CDS sequence shown in SEQ ID No.1 and has the function of the GhSBP1 gene;

[0029] (3) A sequence obtained by deleting, adding or replacing one or more nucleotides based on the CDS sequence shown in SEQ ID No.1, wherein the sequence has the function of the GhSBP1 gene.

[0030] The CDS sequence of the GhSBP1 gene of the present invention is as shown in SEQ ID No.1: ATGCAAGACATCCACTTGATCGGAGGTGGCCGAGTATTCAGCGGCGGCGGTGGAGGTGGCGGAGGAGATAGAAGGTTAGGACTGCAACACCAACAACAAAACAACCAGGCACTCAAGTGCCCACGTTGCGACTCACTTAACACGAAGTTCTGTTACTACAACAACTACGATCTCTCTCAGCCACGTCATTTCTGCAAGAGCTGCCGTCGTTACTGGACCAAAGGTGGTGTCCTTCGTAACGTCCCCGTTGGTGGTGGTTGCCGCAAAGCCAAGCGTTCGAAAACCAAGCCTTCATCCGAATCCACGACAGTCGCCGCCGCGGCCCTTGCACAGCCACAATCACAGCAACACCTTGATCAACGTAAAGCGAATTCTCATTCTAGCAGCGACAGTTCGAGTCTTATTGCAGCTAATTCTAAGGTTGCAGTGACAAATAAAAACAACAACAACGATAGTTCTACAGGCGGCATGGCGGAGGCGGTGTCGGCAGTAACGTCTCATTCAAACAATATAAATGTAAGCGAGTCGAAGTTGTATGGAAACCCCAACAATTTAGTCTTCGAGGCAGGATTACTGGAACAAAGATCAGAATCGGGGATTTTTCCGGAGATTGGGAGTTTTACGAGCTTGGTTACTTCGTCAAACAATGAAACGTCGTCGTTTGGCTTCGGTACGGTATTAAATGGGCAAGGACAGTGGCGGCAGCAAAAGATGATGAGTGTTGGAGGGGAGGAAATCACCGGGGAATTGCTCGATCAGACGATGCAGGTTGAGCTTTCAAATTTGCATGGTAGATCGGAGAGTGGATTTGGACCGTTGATTGGCAAGGGTGTGGAGATCAACCGTTCTTTGATCTTCCTAATGATGTTGACCAATCGTATTGGAGTCAAAGTCAAGGTC。

[0031] The GhSBP1 gene described in this invention also includes genes that have at least 75% homology with the sequence shown in SEQ ID No. 1 and have the function of the GhSBP1 gene. For example, mutations, deletions, or additions of one or more nucleotides to the sequence shown in SEQ ID No. 1, as well as nucleotides obtained by artificial modification that have at least 75% identity with the nucleotide sequence shown in SEQ ID No. 1 of this invention, as long as they encode the protein GhSBP1 and have the function of the gene GhSBP1, are all nucleotide sequences of this invention and are equivalent to the sequences of this invention.

[0032] The present invention also provides the protein encoded by the GhSBP1 gene.

[0033] In one embodiment of the present invention, the protein has the amino acid sequence shown in SEQ ID No. 2: MQDIHLIGGGRVFSGGGGGGGGGDRRLGLQHQQQNNQALKCPRCDSLNTKFCYYNNYNLSQPRHFCKSCRRYWTKGGVLRNVPVGGGCRKAKRSKTKPSSESTTVAAAALAQPQSQQHLDQRKANSHSSSDSSSLIAANSNVAVTNKNNNNDSSTGGMAEAVSAVTSHSNNINVSESKLYGNPNNLVFEAGLLEQRSESGIFPEIGSFTSLVTSSNNETSSFGFGTVLNGQGQWRQQKMMSVGGEEITGELLDQTMQVELSNLHGRSESGFGPLDWQGCGDQPFFDLPNDVDQSYWSQSQWSDQEHTTLYLP. Of course, proteins that retain similar functions to the protein shown in SEQ ID No. 2 after the deletion, substitution, or addition of one or more amino acids based on the amino acid sequence shown in SEQ ID No. 2, as well as proteins obtained by modifying the above-mentioned proteins, are all within the scope of protection of this invention.

[0034] The present invention also provides a biological material comprising the above-mentioned GhSBP1 gene and expressing the above-mentioned protein.

[0035] The types of biological materials described in this invention include vectors, strains, or cells, wherein the vectors include recombinant overexpression vectors, and in one embodiment, the recombinant overexpression vector is based on pCAMBIA2300. The biological materials described in this invention can be recombinant strains; for example, in one embodiment, the coding sequence of GhSBP1 is first cloned into the pCAMBIA2300 vector and transformed into Agrobacterium to obtain a recombinant strain containing GhSBP1.

[0036] This invention also provides the application of the above-mentioned biomaterials in regulating cotton fiber development.

[0037] The regulation described in this invention includes promoting cotton fiber development after overexpression of the GhSBP1 gene, including at least one of the following: (a) increasing fiber length;

[0038] (b) Increase the cell length of mature cotton fiber cells;

[0039] (c) Increase the number of cells that initiate the fibers.

[0040] This invention uses upland cotton as the cotton type, and in one embodiment, upland cotton variety J668 is used as an example. In this invention, after the GhSBP1 gene is expressed, the expression levels of its downstream genes GhNRT1.5, GhEzrA, and GhSH3P2 are significantly increased. Therefore, the GhSBP1 gene regulates the expression of GhNRT1.5, GhEzrA, and GhSH3P2, and there is a protein-protein interaction between them.

[0041] This invention also provides the application of the above-mentioned biological materials in the breeding of new cotton varieties.

[0042] The GhSBP1 gene described in this invention can regulate the development of cotton fibers, especially the length and quality of cotton fibers. Therefore, by controlling the GhSBP1 gene, it is possible to cultivate new cotton varieties with target fiber length and quality.

[0043] The present invention also provides a method for breeding cotton varieties, comprising overexpressing the above-mentioned GhSBP1 gene in the genome of the target cotton variety.

[0044] The present invention does not specifically limit the overexpression method, which includes introducing the gene GhSBP1, as shown in SEQ ID No. 1, into recipient cotton to obtain transgenic cotton with growth and development different from the recipient cotton. In one embodiment of the present invention, the overexpression method includes transforming a recombinant overexpression vector containing the GhSBP1 gene into a target cotton variety; the base vector of the recombinant overexpression vector includes pCAMBIA2300.

[0045] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of the GhSBP1 gene for regulating cotton fiber development, its regulatory method, and its breeding applications, should not be construed as limiting the scope of protection of the present invention.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified; quantitative experiments are performed in triplicate, and the average value is taken; cotton fiber tissue 10 days after flowering is referred to as 10DPA in this text. Alternating light and dark culture means alternating between light and dark culture, and the specific culture cycle can be 16 hours of light culture and 8 hours of dark culture.

[0047] In the following examples, the cotton variety is Ji 668 (J668), and the public can obtain this biomaterial through ordinary means.

[0048] In the following examples, Agrobacterium tumefaciens GV3101 is a commercially available product and is described in the literature (Xiao Weimin, Zhao Mingchen, Zou Min, Su Chenggang, Du Xiaobing. Effects of Arabidopsis thaliana injury and inoculation with Agrobacterium tumefaciens GV3101 on transcription. Journal of Agricultural Biotechnology, 2013, 21(5):537-545.).

[0049] In the following examples, plasmid pCAMBIA2300 was a commercially available product and is described in the literature (Gong Yuanyong, Feng Yongkun, Ni Wanchao, et al. Construction and verification of plant expression vector pCAMBIA2300-35S-GUS-CaMVterm[J]. China Biotechnology Journal, 2013, 33(03):86-91.DOI:10.13523 / j.cb.20130314.). All other vector plasmids were purchased from Wuhan Tianwen Biotechnology Co., Ltd.

[0050] Example 1: Obtaining and phenotypic analyzing GhSBP1 transgenic cotton plants

[0051] I. Preparation of GhSBP1-overexpressing recombinant Agrobacterium

[0052] 1. Primers were designed based on the CDS sequence (SEQ ID No. 1) of GhSBP1. The primer sequences are shown in SEQ ID No. 3 and SEQ ID No. 4. cDNA from 10DPA fibers in cotton, where GhSBP1 expression was relatively high, was used as a PCR template. The CDS sequence of GhSBP1 was amplified using a PCR amplification system of 2×Phanta Flash Master Mix (Dye Plus) (Vazyme, China).

[0053] GhSBP1-pC2300S-F (SEQ ID No. 3): GATGATAAATGAGGATCCATGCAAGACATCCACTTGATCG;

[0054] GhSBP1-pC2300S-R (SEQ ID No. 4): CTGCAGGTCGACTCTAGAGACCTTGACTTTGACTCCAATACG.

[0055] 2. The GhSBP1 target gene fragment obtained in step 1 was ligated into the vector pCAMBIA2300 using homologous recombination to obtain the recombinant plasmid pCAMBIA2300-GhSBP1.

[0056] 3. The recombinant plasmid PC2300S-GhSBP1 obtained in step 2 was introduced into Agrobacterium tumefaciens LBA4404 to obtain GhSBP1 overexpressing recombinant Agrobacterium.

[0057] II. Obtaining Transgenic Plants

[0058] The GhSBP1-overexpressing recombinant Agrobacterium obtained in step one was used to infect the hypocotyl segment of cotton seedlings of Ji 668 (J668) to obtain embryogenic callus tissue. After differentiation, sterile seedlings were obtained and transplanted into sterile substrate soil. Positive seedlings were identified and positive plants were obtained. Through multigeneration propagation, T3 homozygous transgenic plants, namely GhSBP1-overexpressing cotton lines, were obtained.

[0059] III. Identification of Transgenic Plants

[0060] 1. Identification of GhSBP1 overexpression in cotton

[0061] Using GhSBP1 overexpressing cotton (GhSBP1-OE) as the experimental group and wild-type cotton Ji668 (WT) as the control group, DNA was extracted from cotton leaves of each group and identified by PCR using primers.

[0062] GhSBP1-F (SEQ ID No. 5): GATGATAAATGAGGATCCATGCAAGACAT CCACTTGATCG;

[0063] GhSBP1-R (SEQ ID No. 6): GACCTTGACTTTGACTCCAATACG.

[0064] See results Figure 2 As shown in Figure A, the GhSBP1 transcription level in the GhSBP1 overexpressing plants was overexpressed compared to the wild-type Ji668, indicating that the GhSBP1 overexpression vector has been transferred into cotton.

[0065] Wild-type cotton 'Ji 668' (WT) was used as the control group, and GhSBP1-overexpressing cotton (GhSBP1-OE) was used as the experimental group. Gene expression levels of GhSBP1-OE and wild-type cotton 'Ji 668' were analyzed. Fiber tissue (10DPA) collected 10 days after flowering was ground with liquid nitrogen, and RNA was extracted from each group and its concentration was determined. Reverse transcription was performed using a reverse transcription kit to obtain cDNA from 10DPA, and the relative expression level of GhSBP1 was detected by qRT-PCR.

[0066] GhSBP1-F (SEQ ID No. 5): GATGATAAATGAGGATCCATGCAAGACAT CCACTTGATCG;

[0067] GhSBP1-R (SEQ ID No. 6): GACCTTGACTTTGACTCCAATACG.

[0068] In addition, wild-type cotton Ji668 (WT) was used as the control group and GhSBP1-OE was used as the experimental group. Western blotting experiments were performed on GhSBP1 overexpressing cotton and wild-type cotton Ji668, respectively.

[0069] The results are as follows Figure 2 In the wild-type cotton variety Ji 668, the relative expression level of the GhSBP1 gene was 0.0006, while the average relative expression level of GhSBP1 overexpressing cotton was 1.13. The relative expression level of GhSBP1 overexpressing cotton was significantly higher than that of wild-type cotton 10 days after flowering, further indicating that the GhSBP1 overexpression vector has been transferred into cotton.

[0070] Western Blot results are as follows: Figure 2 As shown in Figure C, both wild-type cotton (Ji 668) and GhSBP1-overexpressing cotton normally express the housekeeping gene Actin protein. Wild-type cotton (Ji 668) cannot express the flag-tagged GhSBP1 protein, while the GhSBP1 overexpression vector normally expresses the flag-tagged GhSBP1 protein. This result further indicates that the GhSBP1 overexpression vector has been transferred into cotton and is functioning normally.

[0071] 2. Identification of GhSBP1 gene knockout cotton

[0072] Design sgRNA based on the GhSBP1 sequence:

[0073] sgRNA1 (SEQ ID No. 7): GAACTTCCCAAGATGGTCTA;

[0074] sgRNA2 (SEQ ID No. 8):ATGACACTGTAGCTTTCACC.

[0075] Custom-designed single-guide RNA (sgRNA) was cloned into the Cas9-encoded plant expression vector pU2301F (Wuhan Tianwen Biotechnology Co., Ltd.), and then delivered into Jin668 cells to obtain GhSBP1 gene-edited cotton plants using CRISPR-Cas9 technology.

[0076] The results are as follows Figure 1 As shown, the genotyping of GhSBP1 knockout cell lines obtained through CRISPR-Cas9 gene editing is based on Sanger sequencing. Nucleotide deletions are indicated by red dashed lines. Nucleotide insertions are indicated by red letters. The phenotypes of knockout and overexpression plants differ significantly; the fiber length of cotton from GhSBP1 knockout plants is significantly longer. Figure 2 (B and C) and fibroblast length ( Figure 2 The levels of D and E in the expression were significantly lower than in the overexpressing plants, and the initial fibroblast density of the overexpressing plants was also much higher than that of the knockout plants. Figure 2 (G and F).

[0077] IV. Fiber Phenotypic Analysis of GhSBP1 Transgenic Cotton

[0078] Ten mature fiber plants from the GhSBP1 overexpressing cotton line and the wild-type cotton line Ji 668 obtained in step three were randomly selected. Fifteen cotton bolls were randomly taken from each plant. The fibers were pulled several times to make them parallel, and attached fibers were removed. The fiber bundles were placed on a black felt board, and their lengths were measured with a ruler. The phenotype of mature cotton fibers in each group was recorded, fiber lengths were counted, mature fiber fineness was identified, and twist was counted. Then, cross-sectional sections of mature fibers were prepared, and the number of fiber cells in each group was counted. The phenotype of mature plant height in each group was determined, and the height of mature plants was counted.

[0079] Cotton fibers were selected from different stages of development in wild-type plants and GhSBP1-overexpressing plants, and their lengths were statistically analyzed. The results are as follows: Figure 2 C and Figure 2 As shown in Figure D, wild-type fibers were significantly shorter than those of GhSBP1-overexpressing plants at different stages of fiber development.

[0080] In addition, the fiber length statistics are as follows Figure 2 China D and Figure 2 As shown in Figure E, the average mature fiber length of wild-type plants was 25.75 mm, while the fiber length of GhSBP1-overexpressing plants was 28.45 mm. The fiber length of overexpressing plants increased by 10% compared to wild-type plants. GhSBP1-OE increased fiber length, demonstrating that the GhSBP1 gene regulates fiber cell development.

[0081] V. GhSBP1 Interaction Detection

[0082] The GhSBP1 coding sequence was inserted into the JG45 vector, and the GhNRT1.5, GhEzrA, and GhSH3P2 promoters were cloned into the LacZi vector. The recombinant JG45 and LacZi vectors were co-transformed into yeast strain EGY48, while co-transformation of EGY48 with empty vector JG45 and LacZi served as a negative control. After culturing in SD / -Trp / -Ura medium for 3-4 days, the colonies were transferred to chromogenic medium for color development (cultured at 30°C in the dark).

[0083] The GhSBP1 coding sequence was inserted into the pGreenII 62-SK vector, and the promoters and fragments of GhNRT1.5, GhEzrA, and GhSH3P2 were cloned into the pGreenII 0800-LUC vector. After the recombinant vectors were transformed into Agrobacterium, they were co-infiltrated with Nicotiana benthamiana, cultured in the dark for 1 day and then exposed to light for 1 day. The luminescence intensity of luciferase was detected using an in vivo imaging system (Xenogen IVIS 100), and the enzyme activity was quantified using a dual luciferase reporter system.

[0084] Primers were designed based on the sequences of GhNRT1.5, GhEzrA, and GhSH3P2:

[0085] GhNRT1.5-1R (SEQ ID No.9): ATACAATGATACGTGGTCGTGATG;

[0086] GhNRT1.5-2F (SEQ ID No.10): CATCACGACCACGTATCATTGTAT;

[0087] GhEzrA-1R (SEQ ID No. 11): ATATATATATATTCACGTATAAGCAAAATT GTAACA;

[0088] GhEzrA-2F (SEQ ID No. 12): TGTTACAATTTTGCTTATACGTGAATATAT ATATAT;

[0089] GhSH3P2-1R (SEQ ID No. 13): CCAAGAAAGATACGTGGTCTAATGAG; GhSH3P2-2F (SEQ ID No. 14): CTCATTAGACCACGTATCTTTCTTGG;

[0090] Primers for the pGreenII 62-SK vector were designed based on the GhSBP1 sequence:

[0091] pGreenII 62-SK-F (SEQ ID No. 15): GACGCACAATCCCACTATCC;

[0092] pGreenII62-SK-GhSBP1-F (SEQ ID No. 16):TCCCCCGGGCTGCAGGAATTCATGGCAACAAGCAAAGCTGAA;

[0093] pGreenII62-SK-GhSBP1-R (SEQ ID No. 17): GGTACCGGGCCCCCCCTC GAGTTAGCTAGTTTTAATAATTTGAGCCTTCT;

[0094] Yeast one-hybrid primers were designed based on the sequences of GhNRT1.5, GhEzrA, and GhSH3P2 and the LacZi vector:

[0095] Laczi-GhSH3P2-P1-F (SEQ ID No. 18): TATTGGATCGGAATTCTGAGTTAATTTTTTTTTCAATTTTATGAA;

[0096] Laczi-GhSH3P2-P1-R (SEQ ID No. 19): GAGCACATGCCTCGAGCTCTTAAATTGGTAATTATTATTTTTTCT;

[0097] Laczi-GhNRT1.5-P1-F (SEQ ID No. 20): TATTGGATCGGAATTCCTTCGTGGTGTCAGAATCTAGATCA;

[0098] Laczi-GhNRT1.5-P1-R (SEQ ID No. 21): GAGCACATGCCTCGAGGATGCATTCTCAACATACGAGAAGC;

[0099] Laczi-GhEzrA-P1-F (SEQ ID No. 22): TATTGGATCGGAATTCGCTCCATCATGGCCGGCCACTAT;

[0100] Laczi-GhEzrA-P1-R (SEQ ID No. 23): GAGCACATGCCTCGAGTTTATTCATACTGGAAGGAAGTC;

[0101] EMSA experimental probes for GhNRT1.5, GhEzrA, and GhSH3P2:

[0102] Labeled probe, EMSA-GhNRT1.5-bio (SEQ ID No. 24): AGGTCATCACGACCGTACATCATTGTATCAACA;

[0103] The labeled wild-type probe, EMSA-GhNRT1.5 (SEQ ID No. 25): AGGTCATCACGACCGTACATCATTGTATCAACA;

[0104] Unlabeled mutant competitive probe, EMSA-GhNRT1.5-m (SEQ ID No. 26): AGGTCATCACGACCACGTATCATTGTATCAACA;

[0105] Labeled probe, EMSA-GhEzrA-F-bio (SEQ ID No. 27): ATTGGGTCTGAAGT GTTACAATTTTGCTTATGTACGAATATATATATATTTA;

[0106] Unlabeled wild-type probe, EMSA-GhEzrA (SEQ ID No. 28): ATTGGGTCTGA AGTGTTACAATTTTGCTTATGTACGAATATATATATATTTA;

[0107] Unlabeled mutant competitive probe, EMSA-GhEzrA-m (SEQ ID No. 29): ATTGGG TCTGAAGTGTTACAATTTTGCTTATACGTGAATATATATATATTTA;

[0108] Labeled probe, EMSA-GhSH3P2-bio (SEQ ID No. 30): TTCGTAGTAGCCTCA TTAGACCGTACATCTTTCTTGGTATATCCAGATAAT;

[0109] Unlabeled wild-type probe, EMSA-GhSH3P2 (SEQ ID No. 31): TTCGTAGTAG CCTCATTAGACCGTACATCTTTCTTGGTATATCCAGATAAT;

[0110] Unlabeled mutant competitive probe, EMSA-GhSH3P2-m (SEQ ID No. 32): TTCGT AGTAGCCTCATTAGACCACGTATCTTTCTTGGTATATCCAGATAAT;

[0111] The results are as follows Figure 3 As shown, GhSBP1 specifically binds to the P4 fragment of the GhNRT1.5 promoter ( Figure 3 (A) and this binding depends on a specific site marked with a black ellipse (wild-type colonies appear blue, and the interaction disappears after mutation of the binding site). EMSA experiment ( Figure 3 (E) Further evidence shows that GhSBP1 directly binds to the promoter L4 fragment in vitro, forming a protein-DNA complex (band translocation).

[0112] Through competitive EMSA ( Figure 3 (B) Verification of its binding sequence specificity: The unlabeled wild-type cold probe competitively inhibited the binding of the biotin-labeled L4 fragment to GhSBP1, while the binding site mutant probe (L4m) lost its competitive ability. Dual-luciferase reporter system showed that GhSBP1 significantly activated the transcription of the full-length GhNRT1.5 promoter. Figure 3 In C, LUC expression level p<0.001, but after mutating the GTAC binding site in the promoter, transcriptional activation ability was completely lost. Figure 3 In the D mutant, LUC expression was p<0.001. Yeast one-hybrid assay ( Figure 3 Image A shows that GhSBP1 specifically binds to a key segment of the GhEzrA promoter (the binding site is marked by a black oval), and this binding disappears after mutation at the binding site. A dual-luciferase reporter system confirmed that GhSBP1 has a highly significant transcriptional activation effect on the full-length GhEzrA promoter. Figure 3 In the middle B, LUC expression level p<0.001; when the GTAC binding site in the promoter was mutated, the transcriptional activation ability was completely lost. Figure 3 In the C-cell mutant, LUC expression was p<0.001.

[0113] EMSA experiment ( Figure 4 (D) further proves that GhSBP1 directly binds to the L3 segment of the GhEzrA promoter (strip shift), while the competing EMSA ( Figure 4 (E) Verification of its sequence specificity: Unlabeled wild-type cold probes can competitively inhibit the binding of biotin-labeled L4 fragments to GhSBP1, but the binding site mutant probe (L3m) has a significantly reduced competitive efficiency.

[0114] Yeast one-hybrid experiment ( Figure 5(A) Confirms that GhSBP1 specifically binds to the P2 fragment of the GhSH3P2 promoter (the binding site is marked with a black oval), and the interaction disappears after mutation of the binding site. EMSA experiment ( Figure 5 (B) Further evidence shows that GhSBP1 directly binds to the L2 segment of the gene promoter in vitro (band translocation). Dual-luciferase reporter assays showed that when the GTAC binding site in the GhSH3P2 promoter was mutated, the transcriptional activation capacity of GhSBP1 was significantly reduced. Figure 5 In the C-cell mutant, LUC expression was *p<0.001 / p<0.01 / *p<0.05); while GhSBP1 significantly activated the full-length promoter of GhSH3P2. Figure 5 In the middle D, LUC expression *p<0.001 / p<0.01). Competitive EMSA ( Figure 5 (E) Verification of binding specificity: Unlabeled wild-type cold probes can competitively inhibit the binding of GhSBP1 to the biotin-labeled L2 fragment, but the competitive efficiency of binding site mutant probes (L2m) is significantly reduced.

[0115] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A GhSBP1 gene that regulates cotton fiber development, characterized in that, The GhSBP1 gene includes any of the following: (1) The CDS includes the nucleotide sequence shown in SEQ ID No. 1; (2) It has at least 75% homology with the CDS sequence shown in SEQ ID No.1 and has the function of the GhSBP1 gene; (3) A sequence obtained by deleting, adding or replacing one or more nucleotides based on the CDS sequence shown in SEQ ID No.1, wherein the sequence has the function of the GhSBP1 gene.

2. The protein encoded by the GhSBP1 gene as described in claim 1.

3. A biological material comprising the GhSBP1 gene of claim 1 and expressing the protein of claim 2.

4. The biomaterial according to claim 3, characterized in that, The types of biomaterials include carriers, strains, or cells.

5. The application of the biomaterial described in claim 3 or 4 in regulating cotton fiber development.

6. The application according to claim 5, characterized in that, The regulation includes promoting cotton fiber development by overexpressing the GhSBP1 gene.

7. The application according to claim 6, characterized in that, The promotion of cotton fiber development includes at least one of the following: (a) increasing fiber length; (b) Increase the cell length of mature cotton fiber cells; (c) Increase the number of cells that initiate the fibers.

8. The application of the biomaterial described in claim 3 or 4 in the breeding of new cotton varieties.

9. A method for breeding cotton varieties, characterized in that, This includes overexpressing the GhSBP1 gene of claim 1 in the genome of the target cotton variety.

10. The method according to claim 9, characterized in that, The overexpression method includes transforming a recombinant overexpression vector containing the GhSBP1 gene into a target cotton variety; the base vector of the recombinant overexpression vector includes pCAMBIA2300.

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