A Gossypium hirsutum GhbHLH147 gene and its application in regulating gossypol synthesis and / or growth and development of cotton

Abstract

The present invention relates to the field of genetic engineering technology, and particularly relates to a Gossypium hirsutum GhbHLH147 gene and its application in regulating gossypol synthesis and / or growth and development of cotton. The nucleotide sequence of the Gossypium hirsutum GhbHLH147 gene is shown in SEQ ID No.1. GhbHLH147 silencing results in a significant decrease in the expression levels of gossypol synthesis genes CDNC , CYP706B1 , CYP82D113 , DH‑1 , CYP71BE79 , and the content of free gossypol in cotton leaves also decreases significantly, indicating that GhbHLH147 may positively regulate gossypol synthesis.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly to a gene of upland cotton GhbHLH147 and its application in regulating gossypol synthesis and / or growth and development of cotton. Background Art

[0002] Cotton is one of the important cash crops in China. Its main by-product, cottonseed, is rich in resources such as oil and protein. However, gossypol in cottonseed is toxic to humans and non-ruminant animals, which greatly limits the development and utilization of cottonseed. With the intensification of global climate change, the issue of food security has become increasingly severe. Studying genes that regulate gossypol biosynthesis in cotton and cultivating cotton varieties with low gossypol in cottonseed to fully develop the edible value of cottonseed not only has important scientific significance but also broad application prospects.

[0003] Due to their sessile nature, plants have evolved a complete defense system during their interaction with the external environment, and the phytohormone jasmonic acid plays an important role in this process. One of the important reactions in which jasmonic acid signaling participates in regulating plant defense is the induction of the synthesis of secondary metabolites. The molecular basis for JA-induced accumulation of plant secondary metabolites is to enhance the expression of genes involved in secondary metabolite synthesis. Most bHLH transcription factors are induced by jasmonic acid signaling, and the bHLH family transcription factors GoPGF and GhMYC2 in cotton are involved in regulating gossypol biosynthesis. The role of bHLH transcription factors in the synthesis of plant secondary 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, Betula platyphylla, and Oryza sativa.

[0004] In recent years, exploring transcription factors that regulate gossypol biosynthesis has become a research hotspot among scholars at home and abroad. Pigment glands are storage tissues for terpenoid compounds such as gossypol. Therefore, transcription factors that regulate gland development in cotton often also affect the content of gossypol. GoPGF The silencing of GoSGF results in a decrease in the number of glands and a reduction in gossypol content. Further RNA-seq shows that genes related to secondary metabolism and terpenoid biosynthesis, jasmonic acid (JA) signal transduction, and genes of the WRKY and MYB transcription factor families are all significantly decreased. GhERF105 and GauGRAS1 The silencing of CYP706B1 results in a significant decrease in gossypol content in transgenic seeds after specific silencing, but the number of glands does not decrease, further indicating that gland formation and gossypol synthesis are uncoupled. At the same time, the MYB family transcription factor CGP1After silencing, cotton also exhibits a glandless phenotype, with the expression levels of gossypol biosynthesis genes downregulated and the gossypol level significantly reduced. However, further analysis shows that the number of glands does not decrease. It is speculated that CGP1 forms a heterodimer with GoPGF to regulate the synthesis of gossypol.

[0005] Some studies have shown that the roots of cotton are the main organs for gossypol synthesis. Therefore, some scholars believe that gossypol is mainly produced in the roots and transported to the above-ground parts of the plant. A recent study has shown that genes related to gossypol synthesis are highly expressed in the pigment glands of cotton, and the pigment glands are the sites for the synthesis and storage of gossypol and related terpenoid compounds; gland morphogenesis and terpenoid biosynthesis are two independent regulatory pathways; GoPGF has dual functions and regulates gland formation and the biosynthesis of terpenoids such as gossypol through downstream targets GhJUB1 simultaneously. Mining the GoPGF downstream genes is of great significance for clarifying the complex relationship between pigment glands and gossypol synthesis and promoting the comprehensive utilization of cotton.

[0006] In the present invention, through differential analysis of the transcriptomes of glanded and glandless cotton, candidate genes GhbHLH147 were screened out. Preliminary studies have shown that virus-induced GhbHLH147 silencing leads to a decrease in the content of free gossypol in cotton leaves. By gene editing GhbHLH147 to create low-gossypol cotton varieties, new insights are provided into the molecular regulation mechanism of gossypol biosynthesis in cotton, the restriction of gossypol on the development and utilization of cotton seeds is lifted, and the cotton industry is upgraded. SUMMARY OF THE INVENTION

[0007] To solve the above problems, the present invention provides a Gossypium hirsutum GhbHLH147 gene and its application in regulating gossypol synthesis and / or growth and development of cotton, GhbHLH147 silencing of which results in a significant decrease in the expression levels of gossypol synthesis genes CDNC , CYP706B1 , CYP82D113 , DH-1 , CYP71BE79 , and a significant decrease in the content of free gossypol in cotton leaves, indicating that silencing of GhbHLH147 may inhibit gossypol synthesis.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides a Gossypium hirsutum GhbHLH147 gene, and the nucleotide sequence of the Gossypium hirsutum GhbHLH147 gene is as shown in SEQ ID No.1.

[0010] The present invention also provides a Gossypium hirsutum GhbHLH147A protein encoded by a gene, the amino acid sequence of the protein being as shown in SEQ ID No. 2.

[0011] The present invention also provides the upland cotton according to the above technical solution GhbHLH147 The application of the gene in regulating gossypol synthesis in cotton.

[0012] Preferably, silencing the upland cotton GhbHLH147 gene negatively regulates gossypol synthesis in cotton.

[0013] Preferably, silencing the upland cotton GhbHLH147 gene decreases the expression of gossypol synthesis genes in cotton.

[0014] Preferably, the cotton gossypol synthesis genes include CDNC gene, CYP706B1 gene, CYP82D113 gene, DH-1 gene, and CYP71BE79 gene, one or more of them.

[0015] The present invention also provides the upland cotton according to the above technical solution GhbHLH147 The application of the gene in regulating cotton leaf development.

[0016] Preferably, overexpressing the upland cotton GhbHLH147 gene causes the cotton leaves to curl during growth.

[0017] Advantages of the present invention:

[0018] GhbHLH147 The silencing of CDNC , CYP706B1 , CYP82D113 , DH-1 , CYP71BE79 results in a significant decrease in the expression levels of gossypol synthesis genes

[0019] Overexpressing the GhbHLH147 gene causes the leaves of cotton plants to curl. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments.

[0021] Figure 1 For GhbHLH147 phylogenetic analysis and amino acid sequence alignment of GhbHLH147 phylogenetic relationship analysis and sequence alignment of GhbHLH147 homologous evolution analysis of GhbHLH147 protein domain analysis ofGhbHLH147 Alignment of amino acid sequences with GhMYC2 , GoPGF and AtMYC2 ;

[0022] Figure 2 For GhbHLH147 expression pattern analysis; A, GhbHLH147 Analysis of expression levels 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 result of GhbHLH147 protein rabbit polyclonal antibody;

[0023] Figure 3 For GhbHLH147 subcellular localization results;

[0024] Figure 4 For GhbHLH147 changes in gossypol content and expression levels of key genes in gossypol synthesis after GhbHLH147 silencing; A, GhbHLH147 phenotypes of plants after silencing and control and positive plants; CLCRV: 00 negative control empty vector; CLCRV: GhbHLH147 experimental group; CLCRV: SU positive control SU yellow leaf phenotype; Bar = 10cm; B, plants after infiltration in the experimental group GhbHLH147 expression levels; C, GhbHLH147 changes in expression levels of key gossypol synthesis genes after *P<0.05,**P<0.01 silencing; D,

[0025] Figure 5 For GhbHLH147 overexpressing transgenic cotton; A, GhbHLH147 phenotype of overexpressing transgenic cotton; B, GhbHLH147 phenotype of abnormally developed leaves of overexpressing cotton plants; C, GhbHLH147 DNA positive identification of overexpressing transgenic cotton; D, GhbHLH147 overexpressing transgenic cotton GhbHLH147 expression level identification. Detailed implementation methods

[0026] The present invention provides a Gossypium hirsutum GhbHLH147 gene, and the nucleotide sequence of the Gossypium hirsutum GhbHLH147 gene is shown in SEQ ID No.1, specifically as follows:

[0027] ATGGCGTCCACAGTAATGAATCCATCGACAAACATGAATTCTGATCGAAGGAAGATGATGAAGAAGAAGAAGAAACCGATGATCAAAGAGAATCAGATCAACCAGAGCCACACCAGATGGAAATCAGAGACACAGCAGCAGATCTTTTCCTCGAAACTCGTTGAAGCTTTGAGTCAGCTCAGTCTGGAAAACGGTTGCACTCCTTCTCCTTCGGCTCCACGCGGTGGTCGAGCTGTACGTGAAGCTACTGATAAAGCTTTGGCCATCACGGCTAAAGGAAAAACAAGGTGGAGTCGAGCCATTTTGACCGGCCGGCTTAAGCTGAAGTTCCGAAAGCGGAAGAGACAGAGAGGATCCGCCGCTGCCGTGGCGGTCGTCACCAAGAGTAGCCGGTCGAAGAAACCGAGAGTTAGCGTCTCGAAATTAAAAGCGAGAAGTATACCGAACGTTCTAAGAAAAGTGAAAGTTCTTGGACGGTTAGTTCCCGGTTGCCGAAAGGAACCGTTACCGGTTATTCTTGAAGAAGCTACTGATTACATAGCGGCACTTGAGATGCAAGTTCGAGCCATGGCCACTCTCGCCGAGTTGCTATCTGGTTCCGCCGCCAGCTCCAGCTCAATTCCTCCGCCTCATTCGCCGCCGTCGATTAGGCAGTGA。

[0028] The present invention also provides a Gossypium hirsutum as described in the above technical solution GhbHLH147 The protein encoded by the gene, the amino acid sequence of the protein is shown in SEQ ID No. 2, specifically as follows:

[0029] MASTVMNPSTNMNSDRRKMMKKKKKPMIKENQINQSHTRWKSETQQQIFSSKLVEALSQLSLENGCTPSPSAPRGGRAVREATDKALAITAKGKTRWSRAILTGRLKLKFRKRKRQRGSAAAVAVVTKSSRSKKPRVSVSKLKARSIPNVLRKVKVLGRLVPGCRKEPLPVILEEATDYIAALEMQVRAMATLAELLSGSAASSSSIPPPHSPPSIRQ。

[0030] The present invention also provides the application of the upland cotton described in the above technical solution GhbHLH147 gene in regulating the gossypol synthesis of cotton. In the present invention, silencing the upland cotton GhbHLH147 gene preferably negatively regulates the gossypol synthesis of cotton. In the present invention, silencing the upland cotton GhbHLH147 gene preferably decreases the expression of the gossypol synthesis gene of cotton. In the present invention, the gossypol synthesis gene of cotton preferably includes CDNC gene, CYP706B1 gene, CYP82D113 gene, DH-1 gene and CYP71BE79 gene, one or more of them.

[0031] The present invention also provides the application of the upland cotton described in the above technical solution GhbHLH147 gene in regulating the leaf development of cotton. In the present invention, overexpressing the upland cotton GhbHLH147 gene preferably causes the cotton leaves to curl.

[0032] In order to further illustrate the present invention, the present invention will be described in detail below with reference to the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0033] Example 1

[0034] Cotton GhbHLH147 Gene cloning

[0035] Extract the total RNA of the leaves, roots, stems of the seedlings of CCRI 24 at 14 days of growth and the petals at 5 - 10 days of the flowering period in the field (TIANGEN Biotech Co., Ltd. Total RNA Extraction Kit for Polysaccharide and Polyphenol-rich Plants (DP441)). The experimental method refers to the instructions in the kit, and then use TransScript II One-srep gDNA Removal and cDNA sythesis SuperMIX (TransGen Biotech) to synthesize the first-strand cDNA. The specific steps are as follows:

[0036] Prepare the following reaction system on ice

[0037] Table 1 One-step reverse transcription system

[0038]

[0039] 1. Incubate at 50 °C for 15 min in a PCR instrument;

[0040] 2. After the reaction is completed, incubate at 85 °C for 5 min to terminate the reaction;

[0041] 3. Collect the cDNA solution obtained after the reaction is completed and store it in a -20 °C refrigerator.

[0042] Using the above cDNA as a template, clone the GhbHLH147 gene:

[0043] (I) Primer design

[0044] Bioinformatics analysis of the gene number Ghicr24_D01G026000.1 (in the 24 database) and the nucleotide sequence of the coding region of GhbHLH147, GhbHLH147 The nucleotide sequence of the gene is shown in SEQ ID No.1, its coding sequence length is 657 bp, and its amino acid sequence is shown in SEQ ID No.2, including 218 amino acids. Primers were designed using Primer Premier 5.0, and the primer information is shown in the following table.

[0045] Primer name Primer sequence (5'-3')

[0046] bHLH147-F (SEQ ID No.3): ATGGCGTCCACAGTAATGA;

[0047] bHLH147-R (SEQ ID No.4): TCACTGCCTAATCGACGG.

[0048] (II) Ordinary PCR amplification and PAGE electrophoresis

[0049] The high-fidelity amplification enzyme used for the gene was KOD-Plus-Neo purchased from Toyobo (Japan, KOD-401), and the amplification system is shown in the following table:

[0050] Table 2 Amplification system

[0051]

[0052] Centrifuge briefly and mix well, then perform PCR amplification on an eppendorf nexus SX1, and the program is as follows:

[0053] 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.

[0054] 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 electrophoresis at 220 V for 15 min, the gel was observed and photographed on a gel imager from Bio-RAD company ( Figure 2 as shown in A), where the DNA Maker was 2K (TransGen Biotech).

[0055] (III) Gel extraction of the target gene

[0056] The kit used was the FastPure® GelDNA Extraction Mini Kit purchased from Novoprotein (Nanjing, #DC301). The specific operation steps are as follows:

[0057] 1. After the PCR amplification products are electrophoresed, quickly cut the gel with the target gene band under ultraviolet light while wearing eye protection. Try to cut off the excess part as much as possible and keep the band intact, and then add it to a clean 2 mL centrifuge tube.

[0058] 2. First, add 500 μL of Buffer GDP to the centrifuge tube, then place it in a water bath at 55°C for 10 min, and invert the centrifuge tube up and down during this period until the gel is completely melted.

[0059] 3. Centrifuge briefly in a centrifuge to collect the droplets on the tube wall. Place the FastPure DNA Mini Columns-G adsorption column into a 2 mL Collection Tubes collection tube. If the solution in the centrifuge tube is ≤700 μL, it can be added to the adsorption column in full, and centrifuge at 12000 rpm for 1 min. If >700 μL, it can be added to the adsorption column in two portions. After the first centrifugation, put the adsorption column back into the collection tube and repeat centrifugation at 12000 rpm for 1 min.

[0060] 4. Discard the waste liquid. First, put the adsorption column back into the collection tube, then add 300 μL of Buffer GDP, let it stand at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min.

[0061] 5. Discard the waste liquid. First, put the adsorption column back into the collection tube, add 700 μL of Buffer GW (with an appropriate amount of absolute ethanol added) to the adsorption column, and centrifuge at 12000 rpm for 1 min.

[0062] 6. Repeat step 5.

[0063] 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.

[0064] 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 completely evaporate the residual washing solution.

[0065] 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 12000 rpm for 1 min, then put the adsorption column back into the centrifuge tube, re - add the solution at the bottom of the tube to the adsorption column, let it stand and centrifuge again, and store the collected DNA in a - 20 °C refrigerator.

[0066] The GhbHLH147 gene and AtMYC2 gene, GhMYC2 gene, GoPGF gene, GhMYC2 gene, the amino acid sequences of 7 homologous genes of the Figure 1 gene and other bHLH transcription factors reported to be involved in regulating the synthesis of secondary metabolites in other plants (from literature and NCBI retrieval) were subjected to a homologous phylogenetic analysis. The results are as GhbHLH147 shown. The AtMYC2 gene has the closest phylogenetic relationship with the GhMYC2 gene. The AtMYC2 gene has a relatively strong GhbHLH147 gene and GhMYC2 gene, so the GoPGF gene has a relatively strong homology with the GhbHLH147 gene and a relatively weak homology with the GhMYC2 gene. Protein structure analysis shows that the GoPGF gene only has a helix - loop - helix domain. The protein sequence alignment results of the AtMYC2 gene, GhbHLH147 gene indicate that the

[0067] gene may be an atypical bHLH transcription factor. GhbHLH147 The relative expression levels of the Figure 2 gene in cotton roots, stems, leaves and flowers were analyzed by fluorescence quantitative experiments (

[0068] in B). The specific method is as follows:

[0069] Table 3 Reaction system

[0070]

[0071] Table 4 PCR reaction procedure on the LightCycler480Ⅱ (Roche) system

[0072]

[0073] The primer information used is as follows:

[0074] qPCR-GhbHLH147-F (SEQ ID No.5): AGCAGCAGATCTTTTCCTCGAA;

[0075] qPCR-GhbHLH147-R (SEQ ID No.6): GCTCGACTCCACCTTGTTTTTC;

[0076] The quantitative results are as shown in Figure 2 B below. The gene has a relatively high expression level in cotton leaves. A polyclonal antibody against the GhbHLH147 protein was prepared in live rabbits (relevant experiments were conducted by Wuhan Pujian Biotechnology Co., Ltd.). The results are as shown in GhbHLH147 C below. The size of the GhbHLH147 protein is approximately 34.90KDa. Figure 2

[0077] Example 2

[0078] GhbHLH147 Subcellular localization of the gene

[0079] GhbHLH147 The coding region sequence of the gene was cloned into the plant expression vector pB121 to obtain the p35S- GhbHLH147 -eGFP vector (constructed and synthesized by Shanghai Sangon Biotech Co., Ltd.). The constructed vector was transformed into Agrobacterium tumefaciens GV3101 (Vidi, AC1001). The specific steps are as follows:

[0080] 1. Add 0.01 - 1 μg of the pCAMBIA2300 plasmid to 100 μL of Agrobacterium competent cells, gently pipette the bottom of the tube to mix, incubate on ice for 5 min, treat with liquid nitrogen for 5 min, incubate in a water bath at 37°C for 5 min. To improve the transformation efficiency, this operation can be repeated once, and finally incubate on ice for 5 min.

[0081] 2. Under sterile conditions, add 700 μL of LB liquid medium without added antibiotics, invert and incubate in a 28°C constant temperature incubator with shaking at 220 rpm for 2 - 3 h.

[0082] ​​3. Centrifuge at 6000 rpm for 1 min. Then, in a laminar flow hood, aspirate some of the supernatant and leave about 100 μL of LB liquid medium. Resuspend the cell pellet and spread it on an LB solid medium plate containing 50 μg / mL kanamycin and 50 μg / mL rifampicin resistance. Incubate in a constant temperature incubator at 28°C for 2 - 3 d.

[0083] Then, culture the obtained GV3101 strain in LB medium supplemented with 50 μg / mL kanamycin (Solarbio, K8020) and 50 μg / mL rifampicin (Solarbio, R8011) at 200 r / min in a shaker at 28°C for 16 h. Centrifuge the cultured bacterial solution at 5000 rpm for 10 min in a centrifuge (eppendorf, 5424R). Resuspend the obtained pellet with a resuspension solution containing 10 mmol / L MgCl2 (Sangon Biotech, B300598), 10 mmol / L 2-(4-morpholino) ethanesulfonic acid (Solarbio, M8010), and 150 μmol / L acetosyringone (Sangon Biotech, A601111). Adjust the OD 600 value to 1.0, inject it into tobacco leaves, culture in the dark for 16 h, and then culture under light at 28°C for 48 h. Observe the fluorescence under a laser confocal microscope. The experiment has three independent biological replicates.

[0084] The results are as Figure 3 shown, GhbHLH147 the gene may be localized in the chloroplast. At the same time, GhbHLH147 the gene may be localized on the cell membrane in the cell, and it shows discontinuous dots on the cell membrane, showing a type of atypical bHLH transcription factor.

[0085] Example 3

[0086] Virus-induced gene silencing GhbHLH147 gene

[0087] Use the online website (http: / / vigs.solgenomics.net / ) to determine GhbHLH147 the silencing sequence, design primers for PCR amplification, and the primers are as follows:

[0088] CLCrV-bHLH147-F (SEQ ID No.7):

[0089] TGCCTGCAGACTAGTATGGAAATCAGAGACACAG;

[0090] CLCrV-bHLH147-R (SEQ ID No.8):

[0091] ACCTAGGGGCGCGCCACGCTAACTCTCGGTTTC;

[0092] 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:

[0093] Table 5 Primers

[0094]

[0095] 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:

[0096] 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.

[0097] Table 6 Solution configuration

[0098]

[0099] 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.

[0100] Table 7 Chromatographic conditions

[0101]

[0102] 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.

[0103] 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-1Gene CYP71BE79 The expression level of the gene decreased significantly, and the content of free gossypol in cotton leaves also decreased significantly, indicating that silencing GhbHLH147 the gene may negatively regulate the synthesis of gossypol.

[0104] Example 4

[0105] Construct GhbHLH147 transgenic cotton with overexpressed

[0106] Construct an overexpression vector of GhbHLH147 and transfer it into Agrobacterium tumefaciens to infect Coker 100 cotton. The vector was constructed and related transgenic cotton materials were created by Weimi Biotechnology (Jiangsu) Co., Ltd. The method is as follows:

[0107] Immerse mature dry cotton seeds without fibers successively in 75% ethanol, 6% H2O2, and sterile distilled water for surface sterilization, and then soak them in sterile MSB medium to allow the seeds to imbibe and germinate. Remove the cotyledons on the hypocotyl, isolate the meristem under a dissecting microscope and immediately place it in the infection medium for pretreatment, and then transfer it to Agrobacterium tumefaciens. After ultrasonic treatment, shake it at room temperature for 50 min to improve the gene delivery efficiency. Dry the infected explants on sterile filter paper for 10 minutes, and then culture them in the dark on the medium for 2 - 3 days. Subsequently, transfer the explants to the recovery medium for 7 days. Cut off the elongated roots of the explants and transfer them to the bud induction medium for continuous culture, subculturing once every 2 weeks for 3 - 4 times. When the resistant buds grow to 5 - 6 cm on the selection medium, cut off the elongated buds and transfer them to the rooting medium for continuous culture. Subsequently, plant the rooted T0 plants in the greenhouse for cultivation.

[0108] Subsequently, transplant the plants to the greenhouse for cultivation. When they grow to Figure 5 as shown in Figure A in

[0109] Cotton DNA Extraction and Identification

[0110] Prepare relevant solutions before the experiment. The formula of the cotton DNA lysis buffer is as follows:

[0111] Table 8 Formula of Cotton DNA Lysis Buffer

[0112]

[0113] The steps are as follows:

[0114] 1. Weigh 3 - 5 g of hairy roots into a sterilized mortar. The mortar has been pre-cooled with liquid nitrogen in advance. Continue to add liquid nitrogen and quickly grind it into a powder. Then transfer it to a 2 mL centrifuge tube without RNase, add 800 μL of CTAB lysis buffer pre-warmed at 65°C, place it in a water bath at 65°C for lysis for 30 - 40 min, and take it out and invert it every 10 min to mix evenly.

[0115] 2. Add 800 μL of a mixed solvent of chloroform and isoamyl alcohol. Pre-cool the isoamyl alcohol on ice, and then invert it up and down until it is in a non-layered state. Centrifuge at 12000 rpm at 4°C for 10 min.

[0116] 3. Aspirate the supernatant into a new sterile 2 mL centrifuge tube. The pipette tip can be cut off.

[0117] 4. Add an equal volume of chloroform and isoamyl alcohol mixture, and invert it up and down to mix the solution evenly.

[0118] 5. Repeat steps 4 and 5.

[0119] 6. Use a pipette tip without a tip to transfer the supernatant to a new 1.5 mL centrifuge tube without RNase.

[0120] 7. Add 0.8 times the volume of isoamyl alcohol solution pre-cooled on ice, and invert it several times. At this time, white flocculent precipitates should be produced, and let it stand for 30 min.

[0121] 8. Use a sterile pipette tip to pick out the DNA into a new sterile 1.5 mL centrifuge tube.

[0122] 9. Pour out the absolute ethanol and dry it overnight. Add ddH2O to dissolve the DNA until it is completely dissolved, and store it at 4°C for later use.

[0123] Using the DNA as a template, design primers to amplify AADA for positive identification. The sequence is a segment of the AADA resistance in the vector, with a length of 435 bp. The primer information is as follows:

[0124] Table 9 Primers

[0125]

[0126] The results are as Figure 5 shown. The DNA Maker is 2K purchased from TransGen Biotech Co., Ltd. 8 strains of DNA positive identification were obtained for the overexpression transgenic positive cotton of the gene. Among them, in OE-GhbHLH147-1 (abbreviated as OB1), OE-GhbHLH147-2 (abbreviated as OB2), and OE-GhbHLH147-1 (abbreviated as OB3) GhbHLH147 gene overexpression transgenic positive cotton, among which OE-GhbHLH147-1 (abbreviated as OB1), OE-GhbHLH147-2 (abbreviated as OB2), OE-GhbHLH147-1 (abbreviated as OB3) GhbHLH147The expression level of the gene increased significantly and obvious phenotypes appeared, and the leaves of the plants curled up.

[0127] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Upland cotton <h2 style=";text-align:left;direction:ltr">GhbHLH147 The application of a gene in regulating cotton gossypol synthesis is characterized in that: Silent Upland Cotton <h2 style=";text-align:left;direction:ltr"> GhbHLH147 The gene negatively regulates cotton gossypol synthesis; Upland cotton <h2 style=";text-align:left;direction:ltr"> GhbHLH147 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that: Silent Upland Cotton <h2 style=";text-align:left;direction:ltr"> GhbHLH147 Genetically down-regulates the expression of cotton gossypol biosynthesis genes; The cotton gossypol synthesis gene is <h2 style=";text-align:left;direction:ltr"> CDNC Gene, <h2 style=";text-align:left;direction:ltr"> CYP706B1 Gene, <h2 style=";text-align:left;direction:ltr"> CYP82D113 Gene, <h2 style=";text-align:left;direction:ltr"> DH-1 Genes and <h2 style=";text-align:left;direction:ltr"> CYP71BE79 One or more genes.

3. Upland cotton <h2 style=";text-align:left;direction:ltr"> GhbHLH147 The application of the gene in regulating the development of cotton leaves is characterized in that: Overexpression of upland cotton <h2 style=";text-align:left;direction:ltr"> GhbHLH147 Gene causes cotton leaves to curl; Upland cotton <h2 style=";text-align:left;direction:ltr"> GhbHLH147 The nucleotide sequence of the gene is shown in SEQ ID No.1.

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