Application of GH_D13G0533 gene in regulating plant gland development

Abstract

The present invention provides an application of the GH_D13G0533 gene in regulating the development of plant glands, and belongs to the field of genetic engineering technology. The nucleotide sequence of the GH_D13G0533 gene of the present invention is shown in SEQ ID NO.1. Based on the comparative analysis of the transcriptome data of leaves of two sets of nearly isogenic lines of cotton pigment glands (Zhong 12 and Zhong 12 are obviously absent, Liao 7 and Liao 7 are obviously absent), the present invention obtains a gene (GH_D13G0533) that is highly expressed in cotton glandular varieties and lowly expressed in glandless varieties. Functional analysis was performed using real-time fluorescence quantitative PCR (RT‑qPCR), virus-induced gene silencing (VIGS) technology and subcellular localization, and the results showed that the GH_D13G0533 gene is involved in the glandular development of stems of glandular materials of upland cotton. The present invention provides gene resources for breeding glandular varieties of glandless seeds and glandular plants.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to the application of GH_D13G0533 gene in regulating the development of plant glands. Background Art

[0002] Cotton is an important cash crop and natural fiber crop in my country, and cottonseed is an important source of protein and oil. However, the gossypol toxin in cottonseed limits the comprehensive development and utilization of cottonseed. At the same time, gossypol is a natural antitoxin unique to cotton, which has an antagonistic effect on certain fungi, diseases and insect pests. Breeding low-phenol cotton varieties has always been the goal and direction pursued by cotton breeders.

[0003] At present, many experts and scholars have conducted extensive research on the molecular mechanism of gland development, and have cloned genes related to cotton gland development, such as GoPGF (GhMYC2-like / CGF3), CGF1, CGF2, GauGRAS1, GoSPGF, GhERF105, etc. However, the cotton gland regulation network is still not very clear. Therefore, it is particularly important to explore the genes related to cotton gland development and cottonphenol synthesis and explore their molecular mechanisms. Summary of the invention

[0004] The purpose of the present invention is to provide an application of the GH_D13G0533 gene in regulating the development of plant glands, and to provide a theoretical basis for the mining of genes related to cotton gland development and the breeding of gland-free materials.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides application of GH_D13G0533 gene in regulating plant gland development. The nucleotide sequence of the GH_D13G0533 gene is shown in SEQ ID NO.1.

[0007] SEQ ID NO.1: ATGGCATCTTTCAATTGCTTTGCTTTAGCATTCTTGA TGGCTTTGTCATTTGCAAGCATTGATGTTGGGGTAGCAGCTCGTCACCTTCTGCAGCAGCCTCAAACGCCAAGTTTGTTAAAAAAAACATTCCCACCATTGCCTTCTTTCCCTAAACCATCATTCCCAAGGCCATCCATACCATCATTCCCAAAGCCATCCATACCATCATTCCCAAA GCCATCCATACCATCATTCCCAAGGCCTGGGGTGCTTCCTCCACTTCCTTCCAGACTTCCAAGTCGACCAAAGGCCACACTGGCCCCTCTGCCAAGCATTCCCTCGGTCCCCCAAATCCCAACTGCAATTCCCTCTATTCCTTTCTTTTCTCCACCACTTTCTCATTCTACTCCTTGA

[0008] Preferably, the amino acid sequence encoded by the GH_D13G0533 gene is shown in SEQ ID NO.2.

[0009] SEQ ID NO.2: MASFNCFALAFLMALSFASIDVGVAARHLLQQPQTP SLLKKTFPPLPSFPKPSFPRPSIPSFPKPSIPSFPKPSIPSFPRPGVLPPLPSRLP SRPKATLAPLPSIPSVPQIPTAIPSIPFFSPPLSHSTP

[0010] Preferably, the plant comprises cotton.

[0011] Preferably, the application is to silence or knock out the GH_D13G0533 gene in plants, thereby inhibiting the development of plant glands.

[0012] The present invention provides a method for inhibiting cotton gland development and reducing the cottonpol content in cotton, comprising the following steps:

[0013] (1) digesting the plasmid vector with a restriction endonuclease to obtain a plasmid vector skeleton; recovering and purifying the GH_D13G0533 gene amplification product containing the restriction endonuclease site to obtain a recovered product;

[0014] (2) connecting the plasmid vector backbone with the recovered product to obtain a silencing vector plasmid and transferring the silencing vector plasmid into Agrobacterium;

[0015] (3) Infecting cotton plants with the Agrobacterium obtained in step (2) to obtain gene-silenced plants that inhibit cotton gland development and reduce the cottonpol content in cotton.

[0016] Preferably, the restriction endonucleases in step (1) are EcoRI and BamHI, the plasmid vector is TRV2-pYL156, and the restriction sites are EcoRI and BamHI restriction sites.

[0017] Preferably, the Agrobacterium in step (2) is strain GV3101.

[0018] Preferably, the method used to infect cotton plants in step (3) is injection.

[0019] Preferably, the injection site in step (3) is cotton cotyledons.

[0020] Preferably, the inhibiting of cotton gland development and reducing the cottonpol content in cotton is inhibiting the gland development of cotton plant stems and reducing the cottonpol content in cotton stems.

[0021] Based on comparative analysis of leaf transcriptome data of two sets of cotton pigment gland near-isogenic lines (Zhong 12 and Zhong 12 are obviously absent, Liao 7 and Liao 7 are obviously absent), a gene (GH_D13G0533) with high expression in cotton glandular varieties and low expression in glandless varieties was obtained. Functional analysis was performed using real-time fluorescence quantitative PCR (RT-qPCR), virus-induced gene silencing (VIGS) technology and subcellular localization, and the results showed that the GH_D13G0533 gene is involved in the glandular development of glandular stems of glandular materials of upland cotton. The present invention provides gene resources for breeding glandular varieties of glandless seeds and glandless plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0023] Figure 1 The results of agarose gel electrophoresis in Example 2 are as follows;

[0024] Figure 2 The relative expression level of GH_D13G0533 gene in Zhong12, Zhong12XN, Liao7 and Liao7XN in Example 3 is measured, where ** indicates P<0.01;

[0025] Figure 3The relative expression level of GH_D13G0533 gene in the true leaves and stems of T582 material in Example 3 is measured, where ** indicates P<0.01;

[0026] Figure 4 Schematic diagram of the construction method of auxiliary vectors TRV1-pYL192, TRV2-pYL156 and TRV2-pYL156-GhPDS in Example 4;

[0027] Figure 5 The results of observation of the leaf and stem gland phenotypes of the plants in Example 5;

[0028] Figure 6 The statistical results of the number of pigment glands in the leaves and stems of the plants in Example 5;

[0029] Figure 7 The relative expression level of GH_D13G0533 gene in true leaves and stems of gene silenced plants in Example 5 is measured, where * indicates P<0.05, and ** indicates P<0.01;

[0030] Figure 8 The results of measuring the cottonpol content in the leaves and stems of the gene silenced plants in Example 5;

[0031] Fig. 9 The results of the regulatory relationship analysis between GH_D13G0533 and GoPGF in Example 6;

[0032] Fig.10 This is the subcellular localization result of the upland cotton GH_D13G0533 protein in Example 7. DETAILED DESCRIPTION

[0033] The technical scheme provided by the present invention is described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present invention. The glandular materials used in the following embodiments are Zhongmian Institute (abbreviated as Zhong 12) and Liaomian No. 7 (Liao 7), the glandless materials are Zhongmian Institute 12 Xianwu (abbreviated as Zhong 12 Xianwu) and Liaomian No. 7 Xianwu (Liao 7 Xianwu), and the glandular material on the leaves and glandless material on the stems is T582; the above materials are all planted at the experimental base of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences and managed according to general field management.

[0034] Example 1 GH_D13G0533 gene

[0035] The local Blast method was used to retrieve the full-length CDS sequence of the GH_D13G0533 gene from the cotton functional genomic database (https: / / cottonfgd.org / ), and the primer sequence was designed using primer 5.0 software. The complete open reading frame ORF was amplified from the leaves and stems of upland cotton 12 by PCR technology, that is, the GH_D13G0533 gene was 393 bp (SEQ ID NO.1), and the GH_D13G0533 gene encoded 130 amino acid residues (SEQ ID NO.2).

[0036] Example 2 Cloning of GH_D13G0533 gene

[0037] 1. Total RNA extraction: Take the true leaves and stems of the test materials Zhong 12, Zhong 12 Xianwu, Liao 7, Liao 7 Xianwu and T582, freeze them in liquid nitrogen quickly, and then store them in a -80℃ ultra-low temperature refrigerator for later use. Plant total RNA extraction refers to the instructions of TIANGEN's polysaccharide and polyphenol total RNA extraction kit RNAprep pure plant kit for extraction.

[0038] 2. cDNA synthesis: 1.5 μg RNA was reverse transcribed into cDNA, and then the reverse transcription product cDNA solution was diluted 4 times as a PCR reaction template. It was reverse transcribed into cDNA using the PrimeSeript1stStrand cDNA Synthesis Kit from Takara Biotech. The reverse transcription reaction system I is shown in Table 1, and the reverse transcription reaction system II is shown in Table 1.

[0039] Table 1 Reverse transcription reaction system Ⅰ

[0040] Reagents Usage OligodT 1.0μL dNTP 1.0μL Total RNA 1.5 μg <![CDATA[l×gRNasefreeddH2O]]> up to 10 μL

[0041] Place 10 μL of the reverse transcription reaction system I at 65°C for 5 min.

[0042] Table 2 Reverse transcription reaction system II

[0043]

[0044]

[0045] 20 μL of the mixed solution of reverse transcription reaction system II was placed in 30°C for 10 min, 42°C for 60 min, and 95°C for 5 min.

[0046] After the reaction, 1 μL cDNA was taken as template, and Actin (GenBank: AY305733) was used as reference gene for PCR amplification, followed by agarose gel (1%) electrophoresis. The quality of cDNA was verified according to the size and brightness of the bands, and the synthesized cDNA was stored at -20°C or 4°C for future use.

[0047] 3. Amplification of target gene

[0048] The cDNA obtained by reverse transcription was used as a template, and GH_D13G0533-PF (as shown in SEQ ID NO.3) and GH_D13G0533-PR (as shown in SEQ ID NO.4) were used as primers to perform PCR amplification of the cotton GH_D13G0533 gene. The target gene amplification system is shown in Table 3, and the PCR reaction parameters are shown in Table 4.

[0049] SEQ ID NO.3: 5'-ATGGCATCTTTCAATTGCTTTGC-3'

[0050] SEQ ID NO.4: 5'-AGGAGTAGAATGAGAAAGTGGTGGA-3'

[0051] Table 3 Target gene amplification system

[0052]

[0053]

[0054] Table 4 PCR reaction parameters

[0055] Step 1 Predenature 98℃,5min Step 2 Denature 98℃,10s Step 3 Annealing 58℃,15s Step 4 Extension 68℃,30s Step 5 Extension 68℃,2min

[0056] Note: 30 cycles of amplification were performed from Step 2 to Step 4.

[0057] The product after the reaction was stored at 4°C and detected by 1% agarose gel electrophoresis to obtain a PCR amplification product of 393 bp ( Figure 1 ).

[0058] The PCR amplification product of the target fragment was purified and recovered using QIAquick Gel Extraction Kit. The recovered product was connected to the cloning vector pRI101 and transformed into the competent E. coli DH5α (Shanghai Weidi Biotechnology Co., Ltd.) and cultured overnight at 37°C. A single clone was picked from the kan-resistant LB medium and transferred to 500μL LB liquid medium containing kan for shaking. After culturing at 37°C for 8h, the bacterial solution was verified by PCR, and the bacterial solution containing the target gene fragment was sequenced. The positive clone with correct sequencing was named pRI101-GH_D13G0533.

[0059] Example 3 Analysis of GH_D13G0533 gene expression

[0060] According to the method of Example 2, cDNA of true leaves and stems of the test materials Zhong 12, Zhong 12 Xianwu, Liao 7, Liao 7 Xianwu and T582 were extracted respectively, and the expression of the candidate gene GH_D13G0533 in the above materials was determined by using a fluorescence quantitative kit, and the internal reference gene was set as Actin (GenBank: AY305733), and the RT-qPCR reaction was performed using an ABIPRISM@7500-Fast Real-Time PCR system. Among them, the sequences of the fluorescence quantitative primers are shown in SEQ ID NO.5 and SEQ ID NO.6, the RT-qPCR reaction system is shown in Table 5, and the RT-qPCR reaction conditions are: 95°C pre-denaturation for 5min, 1 cycle. 95°C denaturation for 15s, 55°C annealing for 30s, 72°C extension for 30s, 40 cycles; 95°C 15s, 60°C 1min, 95°C 15s, 1 cycle.

[0061] SEQ ID NO.5(GH_D13G0533-QF): 5'-GCCTCAAACGCCAAGTTTGT-3'

[0062] SEQ ID NO.6(GH_D13G0533-QR): 5'-AAGCACCCCAGGCCT-3'

[0063] Table 5 RT-qPCR reaction system

[0064] Reagents Usage SYBR Premix Ex Taq Ⅱ 10μL PCRForwardPrimer (10 μM) 0.5μL PCRForwardPrimer (10 μM) 0.5μL ROXReferenceDyeII(50×) 0.4μL Template (cDNA) 1μL <![CDATA[ddH2O]]> 7.6μL Total volume 20μL

[0065] The relative expression levels of GH_D13G0533 gene in the stems of Zhong12, Zhong12Xianwu, Liao7 and Liao7Xianwu plants are shown in Figure 2 Figure 2 As shown in Figure 2, the relative expression levels of GH_D13G0533 gene in true leaves and stems of T582 material are as follows: Figure 3 As shown. Figure 2 and Figure 3It can be seen that the GH_D13G0533 gene is highly expressed in the glandular materials of Zhong12 and Liao7, but is less expressed in Zhong12 and Liao7. The GH_D13G0533 gene is highly expressed in the leaves of T582 and is less expressed in the stems. Therefore, it is speculated that the candidate gene GH_D13G0533 may be involved in the development of cotton glands.

[0066] Example 4 Construction of expression vector

[0067] 1. Plasmid extraction and enzyme digestion

[0068] The pTRV plasmid vector was preserved in this laboratory. The plasmid was extracted using an endotoxin-free plasmid extraction kit. The plasmid concentration was tested to be 500-600 ng / μL. Agarose gel electrophoresis showed no protein contamination, meeting the test requirements. The endonucleases used were EcoRI and BamHI for double digestion.

[0069] 2. Construction of VIGS vector of TRV2-pYL156-GH_D13G0533

[0070] (1) Amplification of the target gene ORF sequence: Based on the multiple cloning site on the final vector pTRV2, EcoRI-BamHI was designed as the insertion site, and primers were synthesized. The primer sequences are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0071] SEQ ID NO.7(TRV2-pYL156-GH_D13G0533-EcoRI-F):

[0072] 5'-GTGAGTAAGGTTACC GAATTC ATGGCATCTTTCAATTGCTTTGC-3' (the underlined part contains the recognition sequence of restriction endonuclease EcoRI)

[0073] SEQ ID NO.8(TRV2-pYL156-GH_D13G0533-BamHI-R):

[0074] 5'-CGTGAGCTCGGTACC GGATCC GACCGAGGGAATGCTTGGC-3' (the underlined part contains the recognition sequence of restriction endonuclease BamHI)

[0075] PrimeSTAR GXL DNA Polymerase was used to obtain the GH_D13G0533 target gene sequence containing EcoRI and BamHI restriction sites. The target gene amplification system is shown in Table 6.

[0076] Table 6 Target gene amplification system

[0077] Reagents Usage <![CDATA[ddH2O]]> 8.7μL dNTPMIX (2.5 mM) 1.2μL 5× PrimeSTARGXL Buffer 3.0μL PF (10 μM) + PR (10 μM) 0.4μL+0.4μL PrimeSTARGXLDNAPolymerase 0.3μL cDNA 1μL Total volume 15μL

[0078] The PCR reaction conditions were set as follows: pre-denaturation at 98°C for 5 min, denaturation at 98°C for 10 s, annealing at 58°C for 15 s, extension at 68°C for 30 s, 30 cycles; and extension at 68°C for 2 min.

[0079] (2) Construction of VIGS silencing vector:

[0080] 1) TRV2-pYL156 was digested with restriction endonucleases EcoRI and BamHI to recover the vector backbone.

[0081] 2) Recover and purify the GH_D13G0533 amplification product containing EcoRI and BamHI restriction sites.

[0082] 3) The vector backbone obtained in step 1) and the recovered product obtained in step 2) were connected to form the VIGS vector TRV2-pYL156-GH_D13G0533.

[0083] 4) The ligation product of step 3) was heat-shock transformed into E. coli DH5α, cultured in an incubator at 37°C overnight, and positive clones were selected for bacterial liquid sequencing; the correct clones after sequencing were named TRV2-pYL156-GH_D13G0533.

[0084] 3. Transformation of silencing vector plasmid TRV2-pYL156-GH_D13G0533 into Agrobacterium

[0085] (1) Take 100 μL of GV3101 competent cells melted on ice, add 4 μL (about 1 μg) of silencing vector plasmid to the competent cells under sterile conditions, mix gently, and let stand in an ice water bath for 30 min;

[0086] (2) The centrifuge tubes were sequentially placed in liquid nitrogen for quick freezing for 2 min, incubated in a 37°C water bath for 4 min, and placed in an ice water bath for 2 min;

[0087] (3) Repeat step (2);

[0088] (4) Add 1 mL of antibiotic-free LB liquid medium and culture at 28°C, 175 rpm, with shaking for 5 h;

[0089] (5) Take 100 μL of bacterial solution and spread it on LB selection medium containing kanamycin, gentamicin and rifampicin, and culture it upside down at 28°C for 3 days;

[0090] (6) Select positive clones and culture single colonies on LB liquid medium at 28°C for 72 h for bacterial expansion and PCR testing. The ratio of bacterial liquid to glycerol is 1:1 and stored at -20°C for future use.

[0091] 4. Simultaneously construct auxiliary vectors TRV1-pYL192, pTRV1 / TRV2-pYL156 (TRV2-pYL156) and TRV2-pYL156-GhPDS. The construction method is shown in the following figure. Figure 4 As shown, MCS (multiple cloning site) refers to the multiple cloning site, and a partial fragment of GhPDS is inserted between EcoRI and BamHI in the TRV2-pYL156-GhPDS vector MCS.

[0092] Example 5

[0093] Using 12 from the glandular material as the receptor, the pTRV1 / TRV2-pYL156 (TRV: PDS), pTRV1 / TRV2-pYL156 (TRV: 00) and pTRV1 / TRV2-pYL156: GH_D13G0533 (TRV: GH_D13G0533) bacterial solutions were injected into the flat back of the cotyledons of the plants using the VIGS (virus-induced gene silencing) method. After 21 days, the phenotypes of the glands in the leaves and stems of the plants and the number of glands were observed and counted. The observation results of the phenotypes of the glands in the leaves and stems of the plants are shown in the figure. Figure 5 The results of gland number statistics are shown in Figure 6 As shown. Among them, Figure 5 a and d are Zhong 12 plants inoculated with pTRV1 / TRV2-pYL156 vector, b and e are Zhong 12 plants inoculated with pTRV1 / TRV2-pYL156 vector, c and f are Zhong 12 plants inoculated with pTRV1 / TRV2-pYL156:GH_D13G0533 vector; Figure 6 ns means the number of glands is not significant, ** means P < 0.01.

[0094] Depend on Figure 5 and Figure 6 It can be seen that compared with the leaf glands of Zhong12 plants injected with pTRV1 / TRV2-pYL156, the number of newly formed stem glands of Zhong12 plants injected with pTRV1 / pTRV2:GH_D13G0533 was significantly reduced; while the number of new leaf glands of Zhong12 plants injected with pTRV1 / pTRV2:GH_D13G0533 did not change significantly compared with the number of leaf glands of Zhong12 plants injected with pTRV1 / TRV2-pYL156.

[0095] RNA was extracted from the true leaves and stems of the silenced pTRV1 / TRV2-pYL156:GH_D13G0533 (TRV:GH_D13G0533) plants and the true leaves and stems of the negative control (pTRV1 / TRV2-pYL156, TRV:00) plants, and reverse transcribed into cDNA, and then fluorescent quantitative PCR analysis was performed using the cDNA of the true leaves and stems of the silenced plants and the true leaves and stems of the negative control plants as templates and the internal reference gene primers QRactin-PF: ATCCTCCGTCTTGACCTTG (SEQ ID NO.9) and QRactin-PR: TGTCCGTCAGGCAACTCAT (SEQ ID NO.10). The results are shown in Figure 1. Figure 7 As shown, it can be seen that compared with the expression level of GH_D13G0533 gene in the stems of 12 plants in the negative control, the expression level of GH_D13G0533 gene in the leaves of plants injected with pTRV1 / pTRV2:GH_D13G0533 was significantly decreased, and it is speculated that the GH_D13G0533 gene is involved in the development of stem glands of upland cotton plants.

[0096] The cottonpol content in the leaves and stems of the negative control and the GH_D13G0533-silenced Zhong12 plants was analyzed using a high performance liquid chromatography (HPLC). Figure 8 As shown, ns indicates no significant difference, ** indicates P < 0.01. It can be seen that compared with the negative control, the cottonpol content in the stems of TRV2-pYL156:GH_D13G0533 silenced plants was significantly decreased, indicating that the GH_D13G0533 gene may affect the cottonpol content in the stems of cotton plants by regulating the glandular development of the cotton plant stems.

[0097] Example 6

[0098] GoPGF is a key transcription factor that affects the synthesis of pigment glands in cotton plants. When the gene is silenced, the whole cotton plant shows gland-free characteristics. In order to study the regulatory relationship between GH_D13G0533 and GoPGF, the two genes were silenced by VIGS method, and fluorescence quantitative analysis was performed by qRT-PCR.

[0099] The analysis results are as follows Fig. 9As shown, a is the relative expression level of GoPGF gene in the stem of plants with silenced GH_D13G0533 gene, b is the relative expression level of GH_D13G0533 gene in plants with silenced GoPGF gene, c is the relative expression level of GH_D13G0533 gene and GoPGF gene in the stem of glandular variety (CCRI12) and glandless variety (CCRI12XW), d is the relative expression level of GH_D13G0533 gene and GoPGF gene in the stem of glandular variety (CCRI12) after ethylene treatment. It can be seen that the expression level of GH_D13G0533 gene in the stem of TRV-GoPGF silenced plants did not change or changed slightly, while the expression level of GoPGF decreased significantly. GH_D13G0533 and GoPGF are highly expressed in cotton glandular varieties and lowly expressed in glandless varieties. GH_D13G0533 and GoPGF were expressed consistently in cotton glandular varieties under ethylene treatment. Therefore, we speculated that GH_D13G0533 was located upstream of GoPGF, regulating the expression of the GoPGF gene, and thus affecting the synthesis of the pigment glands in the stems of cotton plants.

[0100] Example 7 Subcellular localization of upland cotton GH_D13G0533 protein

[0101] 1. Plasmid extraction and enzyme digestion

[0102] The pRI101-GFP plasmid vector was preserved in this laboratory. The plasmid concentration was detected to be 500-650 ng / μL. Agarose gel electrophoresis showed no protein contamination and met the test requirements. EcoRI and BamHI were used for double enzyme digestion.

[0103] 2. Construction of subcellular fusion vector of pRI101-GH_D13G0533-GFP

[0104] (1) Amplification of the target gene ORF sequence: According to the multiple cloning site on the vector pRI101-GFP, SalI-BamHI was designed as the insertion site, and primers were synthesized. The primer sequences are shown in SEQ ID NO.11 and SEQ ID NO.12.

[0105] SEQ ID NO.11(pRI101-GH_D13G0533-GFP-EcoRI-F):

[0106] 5'-TTGATACATATGCCC GTCGAC ATGGCATGGCCTCTTTCAATTGCT TTGC-3' (the underlined part contains the recognition sequence of restriction endonuclease SalI)

[0107] SEQ ID NO.12(pRI101-GH_D13G0533-GFP-BamHI-R):

[0108] 5'-CTGAACCGCCTCCACC GGATCC AGGAGTAGAATGAGAAAGTGG TGGA-3' (the underlined part contains the recognition sequence of restriction endonuclease BamHI)

[0109] PrimeSTAR GXL DNA Polymerase was used to obtain the GH_D13G0533 target gene sequence containing SalI and BamHI restriction sites. The reaction system was the same as in Table 6.

[0110] (2) Construction of fusion expression vector:

[0111] 1) pRI101-GH_D13G0533-GFP was digested with restriction endonucleases SalI and BamHI to recover the vector backbone.

[0112] 2) Recover and purify the GH_D13G0533 amplification product containing SalI and BamHI restriction sites.

[0113] 3) The vector backbone obtained in step 1) and the recovered product obtained in step 2) were connected to form a fusion expression vector pRI101-GH_D13G0533-GFP.

[0114] 4) The ligation product of step 3) was heat-shock transformed into E. coli DH5α, cultured inverted in a 37°C incubator overnight, and positive clones were selected for bacterial liquid sequencing; the correct clones after sequencing were named pRI101-GH_D13G0533-GFP.

[0115] 3. The vector plasmid pRI101-GH_D13G0533-GFP was transformed into Agrobacterium GV3101. The steps were similar to the transformation of Agrobacterium with the silencing vector plasmid TRV2-pYL156-GH_D13G0533 in Example 4.

[0116] 4. Agrobacterium infection of tobacco

[0117] Agrobacterium GV3101 containing the pRI101-GH_D13G0533-GFP recombinant vector was cultured at 28°C for 24 hours with constant temperature shaking, and the OD600 value was determined to be 1.0. Centrifuge at 4000r / min for 5 minutes and discard the supernatant. Resuspend with infiltration solution, adjust the OD600 value of the bacterial solution to 1.0, and let the adjusted bacterial solution stand in the dark room for 3 hours. When infecting, gently make a small hole in the lower epidermis of the tobacco leaf, and use a 10mL needleless syringe to inject the mixed bacterial solution into the lower epidermis of the leaf, so as to soak most of the leaf. The injected seedlings were returned to the greenhouse for dark culture for 24 hours, and then transferred to normal light cycle growth. Set the temperature to 25°C, culture under the conditions of 16 hours of light and 8 hours of darkness, and observe fluorescence for about 3 days.

[0118] 5. Subcellular localization of GH_D13G0533 protein in Upland cotton

[0119] The GH_D13G0533 transcription factor may play a regulatory role in the cell nucleus. In order to verify the prediction results, the pRI101-GH_D13G0533-GFP fusion expression vector was constructed, and then the constructed pRI101-GH_D13G0533-GFP vector was used to infect tobacco epidermal cells with Agrobacterium and observed under a laser confocal microscope. Fig.10 As shown in the figure, N represents the nucleus. It can be seen that after the empty vector pRI101-GFP infects the tobacco epidermis, fluorescence can be seen in the entire tobacco cell nucleus and cytoplasm ( Fig.10 After the tobacco was transformed with pRI101-GH_D13G0533-GFP, the GH_D13G0533 protein only released fluorescence in the nuclei of the tobacco epidermal cells, and no fluorescence appeared in other parts of the cells ( Fig.10 e~h), indicating that GH_D13G0533 protein is localized in the cell nucleus, further proving that the protein is a transcription factor.

[0120] As can be seen from the above examples, the present invention is based on the comparative analysis of the transcriptome data of leaves of two sets of cotton pigment glands (Zhong 12 and Zhong 12 are obviously absent, Liao 7 and Liao 7 are obviously absent), and a gene (GH_D13G0533) with high expression in cotton glandular varieties and low expression in glandless varieties is obtained. Functional analysis was performed using real-time fluorescence quantitative PCR (RT-qPCR), virus-induced gene silencing (VIGS) technology and subcellular localization, and the results showed that the GH_D13G0533 gene is involved in the glandular development of the stem of glandular materials of upland cotton. The present invention provides genetic resources for breeding glandular varieties of glandless seeds and glandular plants.

[0121] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. GH_D13G0533 The use of a gene in regulating the development of plant glands is characterized in that: Said GH_D13G0533 The nucleotide sequence of the gene is shown in SEQ ID NO.1; Wherein, the plant is cotton; the application is silencing or knocking out in the plant GH_D13G0533 gene, thereby inhibiting the development of plant glands.

2. The use according to claim 1, characterized in that Said GH_D13G0533 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

3. A method for inhibiting cotton gland development and reducing cottonpol content in cotton, characterized in that: The steps include: (1) Use restriction endonuclease to cut the plasmid vector to obtain the plasmid vector skeleton; recover and purify the plasmid containing restriction endonuclease sites. GH_ D13G0533 Gene amplification products, obtaining recovery products; (2) Connecting the plasmid vector backbone with the recovered product to obtain a silencing vector plasmid and transferring the silencing vector plasmid into Agrobacterium; (3) Infecting cotton plants with the Agrobacterium obtained in step (2) to obtain gene-silenced plants that inhibit cotton gland development and reduce the cotton phenol content.

4. The method according to claim 3, characterized in that The restriction endonuclease in step (1) is Eco RI and Bam HI, the plasmid vector is TRV2-pYL156; the restriction site is Eco RI and Bam HI restriction site.

5. The method according to claim 4, characterized in that The Agrobacterium in step (2) is the GV3101 strain.

6. The method according to claim 5, characterized in that The method used in step (3) to infect cotton plants is injection.

7. The method according to claim 6, characterized in that The injection site in step (3) is the cotton cotyledon.

8. The method according to any one of claims 3 to 7, characterized in that: The method of inhibiting the glandular development of cotton and reducing the cottonpol content in cotton is to inhibit the glandular development of cotton plant stems and reduce the cottonpol content in cotton stems.

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