GhbHLH147 gene of upland cotton and application of GhbHLH147 gene in regulation and control of synthesis and / or growth and development of gossypol of cotton

Abstract

The invention relates to the technical field of gene engineering, in particular to a GhbHLH147 gene of upland cotton and application of the GhbHLH147 gene to regulation and control of gossypol synthesis and / or growth and development of cotton. The nucleotide sequence of the GhbHLH147 gene of the upland cotton is as shown in SEQ ID No. 1. The silence of the GhbHLH147 leads to the significant reduction of the expression quantities of gossypol synthesis genes CDNC, CYP706B1, CYP82D113, DH-1 and CYP71BE79, and the significant reduction of the content of free gossypol in cotton leaves, which indicates that the GhbHLH147 may positively regulate the synthesis of gossypol.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, in particular to a kind of upland cotton GhbHLH147 Genes and their applications in regulating cotton gossypol synthesis and / or growth and development. Background Art

[0002] Cotton is one of the important economic crops in my country. Its main byproduct, cottonseed, is rich in oil, protein and other resources. However, the gossypol in cottonseed is toxic to humans and non-ruminant animals, which greatly limits the development and utilization of cottonseed. As global climate change intensifies, food security issues are becoming more and more serious. Researching genes that regulate cottonseed biosynthesis, cultivating cotton varieties with low gossypol, and fully developing the edible value of cottonseed not only has important scientific significance, but also has broad application prospects.

[0003] Due to the existence of fixation, plants have evolved a complete defense system in the process of interacting with the external environment, in which the plant hormone jasmonic acid plays an important role. One of the important reactions in which jasmonic acid signals participate in regulating plant defense is to induce the synthesis of secondary metabolites. The molecular basis for JA-induced accumulation of plant secondary metabolites is to enhance the expression of genes that synthesize secondary metabolites. Most bHLH transcription factors are induced to express by jasmonic acid signals. In cotton, bHLH family transcription factors GoPGF and GhMYC2 participate in regulating the biosynthesis of cottonpol. The role of bHLH transcription factors in the biosynthesis of plant metabolites has been widely reported. For example, bHLH transcription factors that regulate the synthesis of secondary metabolites exist in plants such as Catharanthus roseus, Arabidopsis thaliana, Aquilaria sinensis, B. leucophylla, and rice.

[0004] In recent years, the discovery of transcription factors that regulate the biosynthesis of cottonpol has become a hot topic for scholars at home and abroad. Pigment glands are storage tissues for terpenoids such as cottonpol, so transcription factors that regulate the development of cotton glands often also affect the content of cottonpol. GoPGF Silencing of caused a decrease in gland number and gossypol content, and further RNA-seq showed that genes related to secondary metabolism and terpenoid biosynthesis, jasmonic acid (JA) signal transduction, and genes of WRKY and MYB transcription factor families were significantly decreased. GoSGF The silencing of induced a decrease in the number of glands on the cotton stems, and the cottonpol content in the stems also decreased significantly. Genes involved in gland formation and cottonpol biosynthesis were significantly downregulated. GhERF105 as well as GauGRAS1 After silencing, the cottonpol content decreased. On the other hand, the key enzyme genes in the cottonpol biosynthesis pathway CYP706B1 After specific silencing, the cottonpol content in transgenic seeds was significantly reduced, but the number of glands did not decrease, further indicating that gland formation and cottonpol synthesis are uncoupled. CGP1The silenced cotton also showed a glandless phenotype, the expression of cottonphenol biosynthesis genes was downregulated, and the cottonphenol level was significantly reduced, but further analysis showed that the number of glands did not decrease. CGP1 It forms a heterodimer with GoPGF to regulate the synthesis of gossypol.

[0005] Studies have shown that cotton roots are the main organ for the synthesis of gossypol, so some scholars believe that gossypol is mainly produced in the roots and transported to the aboveground part of the plant. A recent study showed that cotton pigment glands are highly expressed in genes related to gossypol synthesis, and pigment glands are the site of synthesis and storage of gossypol and related terpenoid compounds; gland morphogenesis and terpene biosynthesis are two independent regulatory pathways; GoPGF Has dual functions, through downstream targets GhJUB1 It also regulates gland formation and the biosynthesis of terpenoids such as cottonpol. GoPGF The downstream genes of cotton are of great significance for clarifying the complex relationship between pigment glands and cottonpol synthesis and promoting the comprehensive utilization of cotton.

[0006] The present invention screens candidate genes by differentially analyzing the transcriptomes of glandular and glandless cotton. GhbHLH147 Preliminary studies have shown that virus-induced GhbHLH147 Silencing leads to a decrease in the free gossypol content in cotton leaves. GhbHLH147 Create low-phenol cotton varieties, provide new insights into the molecular regulatory mechanism of cotton gossypol biosynthesis, remove the restrictions of gossypol on the development and utilization of cotton seeds, and promote the upgrading of the cotton industry. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a kind of upland cotton GhbHLH147 Genes and their use in regulating cotton gossypol synthesis and / or growth and development, GhbHLH147 Silencing of the cottonpol biosynthesis gene CDNC , CYP706B1 , CYP82D113 , DH-1 , CYP71BE79 The expression level of GhbHLH147 was significantly decreased, and the content of free gossypol in cotton leaves was also significantly decreased, indicating that silencing of GhbHLH147 may inhibit the synthesis of gossypol.

[0008] In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides a kind of upland cotton GhbHLH147 Gene, the upland cotton GhbHLH147 The nucleotide sequence of the gene is shown in SEQ ID No.1.

[0009] The present invention also provides a kind of upland cotton described in the above technical scheme GhbHLH147The protein encoded by the gene, the amino acid sequence of the protein is shown as SEQ ID No.2.

[0010] The present invention also provides the upland cotton described in the above technical solution GhbHLH147 Application of genes in regulating gossypol synthesis in cotton.

[0011] Preferably, silent upland cotton GhbHLH147 Genes negatively regulate gossypol synthesis in cotton.

[0012] Preferably, silent upland cotton GhbHLH147 Genetically down-regulates expression of cotton gossypol biosynthesis genes.

[0013] Preferably, the cotton gossypol synthesis gene comprises CDNC Gene, CYP706B1 Gene, CYP82D113 Gene, DH-1 Genes and CYP71BE79 One or more genes.

[0014] The present invention also provides the upland cotton described in the above technical solution GhbHLH147 Application of genes in regulating cotton leaf development.

[0015] Preferably, overexpression of upland cotton GhbHLH147 The gene causes cotton leaves to grow and curl.

[0016] Beneficial effects of the present invention: GhbHLH147 Silencing of the cottonpol biosynthesis gene CDNC , CYP706B1 , CYP82D113 , DH-1 , CYP71BE79 The expression level of GhbHLH147 was significantly decreased, and the content of free gossypol in cotton leaves was also significantly decreased, indicating that GhbHLH147 may positively regulate the synthesis of gossypol.

[0017] Overexpression GhbHLH147 Gene, cotton plant leaves grow and curl. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0019] Figure 1 for GhbHLH147 Evolutionary analysis and amino acid sequence comparison; GhbHLH147 Evolutionary relationship analysis and sequence comparison; A, GhbHLH147 Homology analysis of GhbHLH147 Protein domain analysis; C, GhbHLH147 andGhMYC2 , GoPGF and AtMYC2 Amino acid sequence alignment; Figure 2 for GhbHLH147 Analysis of expression patterns; A, GhbHLH147 Expression analysis in cotton roots, stems, leaves and flowers; B, WB verification results of GhbHLH147 rabbit polyclonal antibody; MW.peotein maker; 1. GhbHLH147 antigen protein (34.90KDa); 2. KSI tag protein (18.03KDa); C is the WB verification results of GhbHLH147 protein rabbit polyclonal antibody; Figure 3 for GhbHLH147 Subcellular localization results of Figure 4 for GhbHLH147 Silencing of cottonpol content and expression changes of key genes for cottonpol biosynthesis; A, GhbHLH147 Phenotypes of control and positive plants after silencing; CLCRV:00 negative control empty vector; CLCRV: GhbHLH147 Experimental group; CLCRV: SU positive control SU yellow leaf phenotype; Bar = 10cm; B, experimental group plants after infection GhbHLH147 The expression level of C, GhbHLH147 Expression changes of key cottonpol biosynthesis genes after silencing; D, GhbHLH147 Changes in cottonpol content in leaves of plants after silencing; values ​​are the mean ± standard deviation of three replicates, and * indicates significant differences from the values ​​of wild-type cotton seedlings: *P<0.05,**P<0.01 ; Figure 5 for GhbHLH147 Overexpression transgenic cotton; A, GhbHLH147 Phenotype of overexpressed transgenic cotton; B, GhbHLH147 Overexpressing cotton plants show abnormal leaf phenotype; C, GhbHLH147 Positive identification of overexpressed transgenic cotton DNA; D, GhbHLH147 Overexpression transgenic cotton GhbHLH147 Expression level identification. DETAILED DESCRIPTION

[0020] The present invention provides a kind of upland cotton GhbHLH147 Gene, the upland cotton GhbHLH147 The nucleotide sequence of the gene is shown in SEQ ID No. 1, and is as follows: .

[0021] The present invention also provides a kind of upland cotton described in the above technical scheme GhbHLH147 The protein encoded by the gene, the amino acid sequence of the protein is shown in SEQ ID No.2, and is as follows: MASTVMNPSTMNMNSDRRKMMKKKKKPMIKENQINQSHTRWKSETQQQIFSSKLVEALSQLSLENGCTPSPSAPRGGRAVREATDKALAITAKGKTRWSRAILTGRLKLK FRKRKRQRGSAAAVAVVTKSSRSKKPRVSVSKLKARSIPNVLRKVKVLGRLVPGCRKEEPLPVILEEATDYIAALEMQVRAMATLAELLSGSAASSSSIPPPPHSPPSIRQ.

[0022] The present invention also provides the upland cotton described in the above technical solution GhbHLH147 The application of genes in regulating cotton cotton phenol synthesis. In the present invention, the upland cotton is silenced GhbHLH147 The gene preferably negatively regulates cotton cottonpol synthesis. GhbHLH147 The gene preferably reduces the expression of cotton gossypol synthesis gene. In the present invention, the cotton gossypol synthesis gene preferably includes CDNC Gene, CYP706B1 Gene, CYP82D113 Gene, DH-1 Genes and CYP71BE79 One or more genes.

[0023] The present invention also provides the upland cotton described in the above technical solution GhbHLH147 The application of genes in regulating cotton leaf development. In the present invention, overexpression of upland cotton GhbHLH147 The gene is preferably used to cause cotton leaves to curl.

[0024] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1 cotton GhbHLH147 Gene cloning Total RNA was extracted from leaves, roots, stems of 14-day-old Zhongmian 24 seedlings and petals at 5-10 days of flowering in the field (Tiangen Biotechnology Co., Ltd. Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441)). The experimental method was referred to the instructions in the kit, and then the first-strand cDNA was synthesized using TransScript II One-srep gDNA Removal and cDNA sythesis SuperMIX (full gold). The specific steps are as follows: Prepare the following reaction system on ice: Table 1 One-step reverse transcription system 1. Incubate at 50°C for 15 min in a PCR instrument; 2. After the reaction is completed, incubate at 85°C for 5 min to terminate the reaction; 3. Collect the cDNA solution obtained after the reaction and store it in a -20℃ refrigerator.

[0026] Using the above cDNA as template, clone the GhbHLH147 gene: 1. Primer design Bioinformatics analysis of the gene number of GhbHLH147, Ghicr24_D01G026000.1 (Zhong24 database) and the nucleotide sequence of the coding region, GhbHLH147 The nucleotide sequence of the gene is shown in SEQ ID No. 1, and the coding sequence is 657 bp long. The amino acid sequence is shown in SEQ ID No. 2, and includes 218 amino acids. Primers were designed using Primer Premier 5.0, and the primer information is shown in the table below.

[0027] Primer Name Primer Sequence (5'-3') bHLH147-F (SEQ ID No. 3):ATGGCGTCCACAGTAATGA; bHLH147-R (SEQ ID No. 4): TCACTGCCTAATCGACGG.

[0028] 2. Conventional PCR amplification and PAGE electrophoresis The high-fidelity gene amplification enzyme used was KOD-Plus-Neo purchased from Toyobo (Japan, KOD-401), and the amplification system is shown in the following table: Table 2 Amplification system Mix by centrifugation, and then perform PCR amplification on eppendorfnexusSX1. The procedure is as follows: Pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 s, annealing at 55°C for 30 s, extension at 68°C for 2 min, 35 cycles; extension at 68°C for 5 min.

[0029] The PCR amplification products were electrophoresed on a 10% agarose gel. An appropriate amount of Gel Red staining solution was added during gel preparation. After 15 min at 220V, the gel was observed and photographed on a gel imager from Bio-RAD. Figure 2 In A), the DNA Maker is 2K (all gold).

[0030] 3. Recovery of target gene gel The kit used was FastPure® GelDNA Extraction Mini Kit purchased from Novozymes (Nanjing, #DC301). The specific steps are as follows: 1. After the electrophoresis of the PCR amplification product is completed, wear eye protection and quickly cut out the gel with the target gene band under ultraviolet light, try to remove the excess part and keep the band intact, and then add it to a clean 2 mL centrifuge tube.

[0031] 2. First add 500 μL of Buffer GDP to the centrifuge tube, then place it in a 55°C water bath for 10 min, turning the centrifuge tube upside down until the gel is completely dissolved.

[0032] 3. Quickly centrifuge the collection tube wall droplets, place the FastPure DNA Mini Columns-G adsorption column into the Collection Tubes 2 mL collection tube. If the solution in the centrifuge tube is ≤700 μL, add all of it to the adsorption column and centrifuge at 12000 rpm for 1 min. If it is >700 μL, add the adsorption column twice. After the first centrifugation, put the adsorption column back into the collection tube and repeat the centrifugation at 12000 rpm for 1 min.

[0033] 4. Discard the waste liquid, put the adsorption column back into the collection tube, then add 300 μL of Buffer GDP, let it stand at room temperature for 2 minutes, and centrifuge at 12000 rpm for 1 minute.

[0034] 5. Discard the waste liquid, put the adsorption column back into the collection tube, add 700 μL of Buffer GW (with appropriate amount of anhydrous ethanol added) into the adsorption column, and centrifuge at 12000 rpm for 1 min.

[0035] 6. Repeat step 5.

[0036] 7. Discard the waste liquid, put the adsorption column back into the collection tube, and centrifuge the empty tube at 12000 rpm for 2 min.

[0037] 8. Place the adsorption column into a new 1.5 mL centrifuge tube, open the lid and let it stand at room temperature for a few minutes to allow the residual rinse solution to evaporate completely.

[0038] 9. Add 30-50 μL of Elution Buffer preheated in a 55°C water bath to the center of the adsorption column, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 1 min, then put the adsorption column back into the centrifuge tube, add the solution at the bottom of the tube back to the adsorption column, let it stand and centrifuge again, and store the collected DNA in a -20°C refrigerator.

[0039] Will GhbHLH147 Gene and AtMYC2 Gene, GhMYC2 Gene, GoPGF Gene, GhMYC2 The homology evolution analysis was performed on the 7 homologous genes of the gene and the amino acid sequences of bHLH transcription factors reported in other plants to be involved in regulating the synthesis of secondary metabolites (derived from literature and NCBI search). The results are as follows Figure 1 As shown, GhbHLH147 Gene and AtMYC2 The genes that are most closely related in evolution,GhMYC2 Gene and AtMYC2 The gene is stronger, so GhbHLH147 Gene and GhMYC2 The gene has strong homology with GoPGF Gene homology is weak, and protein structure analysis shows GhbHLH147 Genes have only double helical domains, GhMYC2 Gene, GoPGF Gene, AtMYC2 The protein sequence comparison results showed that GhbHLH147 The gene may be an atypical bHLH transcription factor.

[0040] Quantitative fluorescence analysis GhbHLH147 Gene expression levels in cotton roots, stems, leaves and flowers ( Figure 2 B), the specific method is as follows: The reagents used were Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (Shanghai, #11184ES08) from Yisheng Company, and the PCR system was as follows: Table 3 System

[0041] Table 4 PCR reaction program on the LightCycler 480Ⅱ (Roche) system The primer information used is as follows: qPCR-GhbHLH147-F (SEQ ID No. 5): AGCAGCAGATCTTTTCCTCGAA; qPCR-GhbHLH147-R (SEQ ID No. 6): GCTCGACTCCACCTTGTTTTTC; Quantitative results such as Figure 2 As shown in B, GhbHLH147 The gene is expressed at a relatively high level in cotton leaves. Polyclonal antibodies against the GhbHLH147 protein were prepared in living rabbits (the relevant experiments were conducted by Wuhan Pujian Biotechnology Co., Ltd.). The results are as follows: Figure 2 As shown in middle C, the size of GhbHLH147 protein is approximately 34.90 KDa.

[0042] Example 2 GhbHLH147 Subcellular localization of genes Will GhbHLH147 The coding region sequence of the gene was cloned into the plant expression vector pB121 to obtain p35S- GhbHLH147-eGFP vector (constructed and synthesized by Shanghai Bioengineering Co., Ltd.). The constructed vector was transformed into Agrobacterium tumefaciens GV3101 (Weidi, AC1001), and the specific steps are as follows: 1. Add 0.01-1 µg pCAMBIA2300 plasmid to 100 µL of Agrobacterium competent medium, gently stir the bottom of the tube to mix, let stand on ice for 5 min, treat with liquid nitrogen for 5 min, and bathe in 37°C water for 5 min. To improve the transformation efficiency, repeat the operation once, and finally bathe in ice for 5 min.

[0043] 2. Under sterile conditions, add 700 μL of LB liquid culture medium without added antibiotics, invert and culture at 220 rpm in a 28°C constant temperature incubator for 2-3 h.

[0044] 3. Centrifuge at 6000 rpm for 1 min, then aspirate part of the supernatant and leave about 100 μL LB liquid medium in the clean bench, resuspend the bacterial pellet, spread it on the LB solid medium plate containing 50 μg / mL kanamycin and 50 μg / mL rifampicin resistance, and culture it in a constant temperature incubator at 28℃ for 2-3 days.

[0045] Then the obtained GV3101 strain was cultured in LB medium supplemented with 50 μg / mL kanamycin (Solarbio, K8020) and 50 μg / mL rifampicin (Solarbio, R8011) at 200 r / min and 28°C in a shaker for 16 h. The cultured bacterial solution was centrifuged at 5000 rpm for 10 min in a centrifuge (eppendorf, 5424R), and the obtained precipitate was resuspended with a resuspension solution of 10 mmol / LMgCL2 (Shengang, B300598), 10 mmol / L 2-(4-morpholino) ethanesulfonic acid (Solarbio, M8010), and 150 μmol / L acetosyringone (Shengang, A601111). OD 600 The value was adjusted to 1.0, and tobacco leaves were injected and cultured in the dark for 16 h, then cultured in the light at 28°C for 48 h, and fluorescence was observed under a laser confocal microscope. The experiment had three independent biological replicates.

[0046] The results are as follows Figure 3 As shown, GhbHLH147 The gene may be located in the chloroplast, and GhbHLH147 The gene may be located on the cell membrane in the cell and appear as discontinuous dots on the cell membrane, showing a type of atypical bHLH transcription factor.

[0047] Example 3 Virus-induced gene silencingGhbHLH147 Gene The online website (http: / / vigs.solgenomics.net / ) was used to determine GhbHLH147 The silenced sequence was amplified by PCR using primers designed as follows: CLCrV-bHLH147-F (SEQ ID No. 7): TGCCTGCAGACTAGTATGGAAATCAGAGACACAG; CLCrV-bHLH147-R (SEQ ID No. 8): ACCTAGGGGCGCGCCACGCTAACTCTCGGTTTC; Obtain the target fragment according to the above gene cloning related methods and recover it, and connect it to the pCLCRVA vector. After transformation of Agrobacterium, shake the bacteria and mix it with the pCLCRVB resuspension. After 3 hours of rest, inject it into the cotyledons of 14-day-old cotton seedlings, among which the control (ck) is injected with CLCRV empty vector. After the positive plants show yellow leaf phenotype, extract the total RNA of each seedling leaf, and use the above fluorescence quantitative PCR detection method to identify the expression of key genes for cottonpol synthesis. The quantitative primers are as follows: Table 5 Primers At the same time, the content of terpenoid compounds such as gossypol in the dried cotton leaves was measured (measured by Hainan Baisui Biotechnology Co., Ltd.), and the determination method was as follows: The reagents used in the experiment, methanol (Shanghai, #M116118), phosphoric acid (Shanghai, #P112025), acetonitrile (Shanghai, #A104440), formic acid (Shanghai, #F112034), and ethanol (Shanghai, #E111993), were all chromatographic grade and purchased from Aladdin Company.

[0048] Table 6 Solution configuration Take 10 g of sample into a centrifuge tube, add 10 mL of anhydrous ethanol to each tube, shake for 2 min, let stand at room temperature for 5 min, aspirate 5 mL of supernatant, blow dry with nitrogen, finally make up to volume with 1 mL of anhydrous ethanol and filter under sterile conditions using a 0.45 µm inorganic filter membrane.

[0049] Table 7 Chromatographic conditions Add different concentrations of cotton pol standard working solution in sequence, calculate the peak height and peak area, and then draw a standard curve. Inject the sample solution into the liquid chromatograph, calculate the concentration of cotton pol in the sample after drawing the standard curve.

[0050] The results are as follows Figure 4 As shown, GhbHLH147 Gene silencing leads to the production of cottonpol CDNC Gene, CYP706B1 Gene, CYP82D113 Gene, DH-1 Gene, CYP71BE79 The expression of the gene was significantly decreased, and the content of free gossypol in cotton leaves was also significantly decreased, indicating that silencing GhbHLH147 The gene may negatively regulate the synthesis of cottonpol.

[0051] Example 4 Build GhbHLH147 Gene overexpression transgenic cotton The GhbHLH147 overexpression vector was constructed and transferred into Agrobacterium to infect Baimian No. 1 cotton. Weimi Biotechnology (Jiangsu) Co., Ltd. constructed the vector and created related transgenic cotton materials. The method is as follows: Mature dry cotton seeds without fiber were immersed in 75% ethanol, 6% H2O2, and sterile distilled water for surface sterilization, and then immersed in sterile MSB medium to allow the seeds to swell and germinate. The cotyledons on the hypocotyl were removed, the meristems were separated under a dissecting microscope and immediately placed in the infection medium for pretreatment, and then transferred to Agrobacterium for ultrasonic treatment and oscillated at room temperature for 50 min to improve the efficiency of gene delivery. The infected explants were dried on sterile filter paper for 10 minutes, and then cultured in a dark environment on the culture medium for 2-3 days, and then the explants were transplanted to the recovery medium for 7 days. The elongated roots of the explants were cut off and transferred to the bud induction medium for continued culture, subcultured every 2 weeks, and subcultured 3 to 4 times. When the resistant buds grew to 5-6 cm on the selection medium, the elongated buds were cut off and transferred to the rooting medium for continued culture, and then the rooted T0 plants were planted and cultured in the greenhouse.

[0052] The plants were then transplanted to a greenhouse for cultivation and grown to Figure 5 As shown in Figure A, DNA was extracted from young leaves using the following method: Cotton DNA Extraction and Identification Prepare relevant solutions before the experiment. The formula of cotton DNA lysis buffer is as follows: Table 8 Cotton DNA Lysis Buffer Formula Here are the steps: 1. Weigh 3-5 g of hairy roots into a sterilized mortar. Pre-cool the mortar with liquid nitrogen. Continue to add liquid nitrogen to quickly grind it into powder, then transfer it to a 2 mL centrifuge tube without RNase, add 800 μL of CTAB lysis buffer preheated at 65°C, and place it in a 65°C water bath for lysis for 30-40 min. Take it out and mix it by inversion every 10 min.

[0053] 2. Add 800 μL of a mixture of chloroform and isoamyl alcohol. Pre-cool the isoamyl alcohol on ice, then mix by inverting until no layers are formed. Centrifuge at 4°C, 12,000 rpm for 10 min.

[0054] 3. Pipette the supernatant into a new sterile 2 mL centrifuge tube. The tip of the pipette can be cut off.

[0055] 4. Add equal volumes of chloroform and isoamyl alcohol mixture and invert the tube to mix the solution.

[0056] 5. Repeat steps 4 and 5.

[0057] 6. Use a pipette tip to transfer the supernatant to a new RNase-free 1.5 mL centrifuge tube.

[0058] 7. Add 0.8 times the volume of pre-cooled isoamyl alcohol solution on ice, turn it upside down several times, and white flocculent precipitate should be produced. Let it stand for 30 minutes.

[0059] 8. Use a sterile pipette tip to pick up the DNA and transfer it to a new sterile 1.5 mL centrifuge tube.

[0060] 9. Pour out the anhydrous ethanol and dry overnight, add ddH2O to dissolve the DNA until it is completely dissolved, and store at 4°C for later use Using DNA as a template, primers were designed to amplify AADA for positive identification. The sequence was a sequence of AADA resistance in the vector, with a length of 435 bp. The primer information is as follows: Table 9 Primers The results are as follows Figure 5 As shown, DNA Maker was purchased from Quanshijin Company at 2K, and 8 strains were identified by DNA positive GhbHLH147 Gene overexpression transgenic positive cotton, including OE-GhbHLH147-1 (abbreviated as OB1), OE-GhbHLH147-2 (abbreviated as OB2), OE-GhbHLH147-1 (abbreviated as OB3) GhbHLH147 The expression level of the gene increased significantly and an obvious phenotype appeared, and the leaves of the plant curled up.

[0061] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A type of upland cotton GbHH147 A gene characterized by Upland cotton GbHH147 The nucleotide sequence of the gene is shown in SEQ ID No. 1, and the upland cotton GbHH147 The gene has the function of regulating the synthesis of cotton gossypol.

2. The upland cotton of claim 1 GbHH147 The protein encoded by the gene is characterized in that The amino acid sequence of the protein is shown in SEQ ID No. 2, and the protein has the function of regulating the synthesis of cotton gossypol.

3. The upland cotton of claim 1 GbHH147 Application of genes in regulating gossypol synthesis in cotton.

4. The use according to claim 3, characterized in that: Silent Upland Cotton GbHH147 Genes negatively regulate gossypol synthesis in cotton.

5. The use according to claim 4, characterized in that: Silent Upland Cotton GbHH147 Genetically down-regulates expression of cotton gossypol biosynthesis genes.

6. The use according to claim 5, characterized in that: The cotton gossypol synthesis gene comprises CDNC Gene, CYP706B1 Gene, CYP82D113 Gene, DH-1 Genes and CYP71BE79 One or more genes.

7. The upland cotton of claim 1 GbHH147 Application of genes in regulating cotton leaf development.

8. The use according to claim 7, characterized in that: Overexpression of upland cotton GbHH147 Gene causes cotton leaves to curl.

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