Application of GhHSTF2d gene of upland cotton in regulation and control of gossypol synthesis of cotton

Abstract

The invention relates to the technical field of gene engineering, in particular to application of a GhHSTF2d gene of upland cotton to regulation and control of gossypol synthesis of cotton. Along with silencing of the upland cotton GhHSTF2d, the expression quantity of gossypol synthesis genes CDNC, CYP706B1, CYP71BE79, CYP82D113 and DH-1 is remarkably reduced, the content of gossypol in cotton leaves is remarkably reduced, and it is indicated that the biosynthesis of gossypol is affected by the upland cotton GhHSTF2d by regulating and controlling the expression of the gossypol synthesis genes.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, in particular to upland cotton GhHSTF2d Application of genes in regulating cotton gossypol synthesis. Background Art

[0002] Cotton is one of the world's major cash crops, with fiber as its main product and cottonseed as its byproduct. Cottonseed is rich in protein and oil. If used as a food resource, the annual global cottonseed production can meet the protein needs of 500 million people. However, the presence of a large amount of anti-nutritional substance, gossypol, in cottonseed greatly limits its utilization. On the other hand, the presence of gossypol helps protect cotton from pests and diseases, and it has been used in clinical treatment as an anti-tumor drug and contraceptive.

[0003] Cottonpol belongs to the sesquiterpenoids. Its biosynthesis starts from the mevalonate pathway (MVA pathway), starting with farnesyl pyrophosphate (FPP) and ending with the polymerization of two molecules of semi-gossypol to form gossypol. The genes reported in the gossypol biosynthesis pathway mainly include CDNC, CYP706B1, CYP82D113, CYP71BE79, DH1, 2-OD-1, CYP736A196 and SPG. A recent study successfully identified and characterized the key protein GhDIR5 that controls the biosynthesis of left-handed and right-handed gossypol in cotton. By eliminating left-handed gossypol through gene editing technology, low-toxic or non-toxic cottonseed was obtained without significant effect on cotton's insect resistance, opening up a new path for the asymmetric synthesis of complex natural products. Although the research on the synthesis pathway of gossypol is relatively clear, the low-cottonpol plants obtained by knocking out the cottonpol synthesis gene cannot be promoted on a large scale due to instability in the field. Cotton contains other terpene compounds such as hemi-gossypol ketone and noctuidin that share the same synthesis pathway with gossypol. The structures of these substances are similar to those of gossypol and they are also insect-resistant.

[0004] Plants cannot move, so they have evolved complex and efficient molecular regulatory networks to resist or adapt to external stresses, in which the regulation of transcription factors plays a key role. Heat stress transcription factors (HSFs) play an important role in the response to abiotic stress, especially high temperature stress. At present, multiple members of the HSFs transcription factor family have been identified in some plants. For example, SIHsfA2 plays an important role in the response and tolerance of different abiotic stresses in tomatoes; Arabidopsis AtHsfB1 and AtHsfB2 act as inhibitors of heat-induced HSFs expression, regulating the heat tolerance of Arabidopsis; OsHsfA7 may participate in the response of transgenic rice to high salt or drought stress by regulating the target gene OsHsp24.1. Research on heat shock transcription factors in cotton also focuses on drought or heat stress. GhHRP affects cotton's heat stress response by finely regulating ethylene and auxin signal transduction. GhHSFA4a may regulate cotton's resistance to Verticillium wilt by participating in the synthesis of secondary metabolites and signal transduction, but there is a lack of direct evidence that it regulates the synthesis of secondary metabolites. Moreover, heat shock transcription factors have rarely been reported in the regulation of secondary metabolite synthesis in other plants.

[0005] The present invention not only provides a new perspective on the function of heat shock transcription factors in plant growth and development by verifying the function of GhHSTF2d in regulating cotton gossypol biosynthesis, but also draws a new pathway for the cotton gossypol biosynthesis regulatory network, providing a solid theoretical basis for the breeding of low-phenol cotton varieties. Summary of the invention

[0006] In order to solve the above problems, the present invention provides upland cotton GhHSTF2d The application of genes in regulating cotton gossypol synthesis has been GhHSTF2d Silencing of cottonpol biosynthesis genes CDNC , CYP706B1 , CYP71BE79 , CYP82D113 , DH- 1 The expression of α-cotton decreased significantly, and the gossypol content in cotton leaves decreased significantly, indicating that upland cotton GhHSTF2d The biosynthesis of cottonpol is affected by regulating the expression of cottonpol biosynthesis genes.

[0007] In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides upland cotton GhHSTF2d Application of genes in regulating cotton gossypol synthesis.

[0008] Preferably, silent upland cotton GhHSTF2d Genes negatively regulate gossypol synthesis in cotton.

[0009] Preferably, silent upland cotton GhHSTF2d Genetically down-regulates the expression of cottonpol biosynthesis genes.

[0010] Preferably, the cottonpol synthesis gene comprises CDNC Gene, CYP706B1 Gene, CYP71BE79 Gene, CYP82D113 Genes and DH-1 One or more genes.

[0011] Preferably, the upland cotton GhHSTF2d The nucleotide sequence of the gene is shown in SEQ ID No.1.

[0012] Preferably, the upland cotton GhHSTF2d The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.2.

[0013] Beneficial effects of the present invention: With the upland cotton GhHSTF2d Silencing of cottonpol biosynthesis genes CDNC , CYP706B1 , CYP71BE79 , CYP82D113 , DH-1 The expression of α-cotton decreased significantly, and the gossypol content in cotton leaves decreased significantly, indicating that upland cotton GhHSTF2d The biosynthesis of cottonpol is affected by regulating the expression of cottonpol biosynthesis genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] 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.

[0015] Figure 1 Gossypium hirsutum GhHSTF2d Analysis of gene expression patterns; A, analysis of the expression of GhHSTF2d in cotton roots, stems, leaves and flowers; B, WB verification results of GhHSTF2d rabbit polyclonal antibody, MW.peotein maker, GhHSTF2d antigen protein (24.54KDa); C, WB verification results of GhHSTF2d protein rabbit polyclonal antibody; Figure 2 Gossypium hirsutum GhHSTF2d Subcellular localization results of genes; Figure 3 Gossypium hirsutum GhHSTF2dGene silencing leads to a decrease in the content of gossypol in cotton leaves; A. ClcrV:SU is the positive control, ClcrV:00 is the negative control, and ClcrV:GhHSTF2d is the experimental group; B. Leaves of 15 cotton seedlings with the first true leaf just unfolded were injected and numbered 1-15, among which the expression of GhHSTF2d in 4, 5, 6, and 7 was extremely significantly decreased, and the expression of GhHSTF2d in 8 and 9 was significantly decreased; C. Expression of gossypol biosynthesis genes; D. Free gossypol content in cotton leaves. Asterisks indicate significant differences, and the detection method used Student's t-test (* P<0.05, ** P<0.01). DETAILED DESCRIPTION

[0016] The present invention provides upland cotton GhHSTF2d The application of genes in regulating cotton cotton phenol synthesis. In the present invention, the upland cotton GhHSTF2d The nucleotide sequence of the gene is shown in SEQ ID No. 1, and is as follows:

[0017] In the present invention, the upland cotton GhHSTF2d The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No. 2, and is as follows: MNPYFPVKEEYPSSSYSQSDDDAPRMMELPQPVEGLHDSGPPPFLTKTFDMVDDPSTNYIVSWSSGGSSFIVWDPHSFSTNLLPRYFKHNNFSSFVRQLNTYGFRKIDSDKWEFANEGFVRGQRDLLKSIRRRKTTTSQLPTSQQALGPCVEVGRFGLDGEVDRLRRDNQVLT MELVKLRQQQLSTRAYIQAIEERLQCTEKKQQQMMSFLARAIQNPSFLQQLMQQKERTKDLEEAMSRKRMRPIVQLPLGVDVGESSRGSDGTNPVKTKPLEFGDYGYQVTELEALALEMQGYGRTRRGQEESQNGHEHRESHDKELDEGFWEELLNEKFLELDIPGTELARD.

[0018] In the present invention, the silenced upland cotton GhHSTF2d The gene preferably negatively regulates cotton cottonpol synthesis. GhHSTF2d The gene preferably reduces the expression of the cottonpol synthesis gene. In the present invention, the cottonpol synthesis gene preferably includes CDNC Gene, CYP706B1 Gene, CYP71BE79 Gene, CYP82D113 Genes and DH-1 One or more genes.

[0019] 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.

[0020] Example 1 Cloning of cotton GhHSTF2d gene: 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 MonScript™RTIII Super Mix with dsDNase (Two-Step) purchased from Mona (Suzhou, #MR05201). The specific steps are as follows: 1. Prepare the following reaction system on ice: Table 1 System 2. Mix gently and centrifuge for 30 seconds. 3. Incubate at 37°C for 2 min in a PCR instrument to eliminate contamination from genomic DNA; 4. Incubate at 55°C for 5 min to inactivate DNase I and temporarily place on ice.

[0021] 2. Synthesis of the first-strand cDNA 1. Prepare the following reaction system on ice: Table 2 System 2. Mix gently and centrifuge for 30 seconds in a small centrifuge; 3. Incubate at 50°C for 15 min in a PCR instrument; 4. After the reaction is completed, incubate at 85°C for 5 min to terminate the reaction; 5. Collect the cDNA solution obtained after the reaction and store it in a -20℃ refrigerator.

[0022] The cotton cDNA was used as a template to clone the cotton GhHSTF2d gene.

[0023] 1. Primer design Bioinformatics analysis of the gene number of GhHSTF2d, Ghicr24_A11G135000.1 (Zhong24 database) and the nucleotide sequence of the coding region, GhHSTF2d The nucleotide sequence of the gene is shown in SEQ ID No. 1, and the length of its coding sequence is 1038 bp. Its amino acid sequence is shown in SEQ ID No. 2, and includes 345 amino acids. Primers were designed using Primer Premier 5.0, and the primer information is shown in the following table: Table 3 Primers 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 4 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 58°C for 30 s, extension at 68°C for 2 min, 35 cycles; extension at 68°C for 5 min.

[0024] 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 1 In A), the DNA Maker is 2K (all gold).

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 6. Repeat step 5.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] Fluorescence quantitative experiments were used to analyze the expression levels of the GhHSTF2d gene in cotton roots, stems, leaves and flowers ( Figure 1 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 5 System The PCR reaction was performed on the LightCycler 480Ⅱ (Roche) system with the following procedure: Table 6 Program The primer information used is as follows: qPCR-GhHSTF2d-F (SEQ ID No. 3):TCTCAGCTCCCAACATCTCAAC; qPCR-GhHSTF2d-R (SEQ ID No. 4): CTGCTGCTTCTTTCTGTGCAT; Quantitative results such as Figure 1 As shown in B, GhHSTF2d The gene is expressed at a relatively high level in cotton leaves. Polyclonal antibodies against GhHSTF2d protein were prepared in living rabbits (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 GhHSTF2d protein is approximately 24.54 KDa.

[0035] Example 2 Subcellular localization of GhHSTF2d gene: Will GhHSTF2d The coding region sequence of p35S- GhHSTF2d-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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] The results are as follows Figure 2 As shown, GhHSTF2d may be localized in the nucleus and cell membrane.

[0040] Example 3 Virus-induced gene silencing of the GhHSTF2d gene The silencing sequence of GhHSTF2d was determined using an online website (http: / / vigs.solgenomics.net / ), and primers were designed for PCR amplification. The primer sequences are as follows: CLCrV-F (SEQ ID No.5): TGCCTGCAGACTAGTCAGAATCCGTCATTTCTGC CLCrV-R (SEQ ID No.6): ACCTAGGGGCGCGCCATGGCTCTCTCGATGCTCA 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 7 Primer information 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.

[0041] Table 8 Standard solution 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.

[0042] The chromatographic conditions are shown in the following table: Table 9 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.

[0043] The results are as follows Figure 3As shown in the data, with the silencing of GhHSTF2d, the expression levels of cottonpol biosynthesis genes CDNC, CYP706B1, CYP71BE79, CYP82D113, and DH-1 decreased extremely significantly, and the cottonpol content in cotton leaves decreased significantly, indicating that GhHSTF2d may affect the biosynthesis of cottonpol by regulating the expression of cottonpol biosynthesis genes.

[0044] 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. Upland cotton GhHSTF2d Application of genes in regulating cotton cottonpol synthesis, the upland cotton GhHSTF2d 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 GhHSTF2d Genes negatively regulate gossypol synthesis in cotton.

3. The use according to claim 2, characterized in that: Silent Upland Cotton GhHSTF2d Genetically down-regulates the expression of cottonpol biosynthesis genes.

4. The use according to claim 3, characterized in that: The cottonpol synthesis gene includes CDNC Gene, CYP706B1 Gene, CYP71BE79 Gene, CYP82D113 Genes and DH-1 One or more genes.

5. The use according to claim 1, characterized in that: Upland cotton GhHSTF2d The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.2.

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