Cotton transcription factor GhMYB14 gene and application thereof

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a cotton transcription factor GhMYB14 gene and application thereof. The nucleotide sequence of the cotton transcription factor GhMYB14 gene provided by the invention is as shown in SEQ ID NO. 1. According to the invention, a cotton transcription factor GhMYB14 gene is cloned, and transgenic arabidopsis thaliana over-expressing the GhMYB14 gene is constructed by using a transgenic technology. Meanwhile, the GhMYB14 gene is silenced in cotton by using a VIGS technology, and it is clear that the GhMYB14 gene can improve the salt tolerance of arabidopsis thaliana and cotton. In addition, the GhMYB14 gene responds to various plant hormones at the transcriptional level. A foundation is laid for deeply researching a molecular mechanism of the GhMYB14 gene for resisting abiotic stress, and an excellent target is provided for salt-tolerant molecular breeding of cotton.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to cotton transcription factors. GhMYB14 Genes and their applications. Background Technology

[0002] Salt stress is one of the major abiotic stresses, and improving the salt and alkali tolerance of crops will become an effective way to achieve sustainable development.

[0003] Cotton is an important fiber and oilseed crop worldwide. In international trade, cotton is a major agricultural commodity, and cotton products are closely related to human life. Cotton is relatively salt-tolerant, but its tolerance is limited. With the expansion of saline-alkali land area and the increase in salinity globally, improving cotton's salt tolerance is of great significance for fully utilizing saline-alkali land resources and promoting cotton production.

[0004] Transcription factors are proteins encoded by genes that specifically bind to cis-regulatory elements in gene promoter regions to activate gene expression. Based on the specificity of their DNA-binding regions, transcription factors are classified into many different families. Four main families are associated with plant resistance to abiotic stress: BZIP, WRKY, AP2 / ERF, and MYB. The MYB transcription factor family is widely distributed in higher plants, ranking first in both quantity and functional diversity. Therefore, MYB transcription factors have significant research value. The MYB family can be further divided into four subfamilies based on the number of repeating R sequences in the MYB domain: 1R-MYB, R2R3-MYB, 3R-MYB, and 4R-MYB. The R2R3-MYB subfamily is the most numerous and functionally diverse subfamily within the MYB transcription factor family.

[0005] Currently, the MYB transcription factors involved in the salt stress response in cotton include GhMYB73 , GhMYB36, GhMYB108 and GhMYB113 The MYB family in cotton contains numerous members, leading to incomplete functional identification of MYB genes. Cloning and functional analysis of new MYB transcription factors not only enriches the theoretical framework of plant stress resistance regulatory networks but also has potential application value for cotton variety improvement. Therefore, continued development and research on MYB genes in cotton are needed to enrich the MYB family gene resources for cotton stress resistance responses. Summary of the Invention

[0006] To further develop and study MYB genes in cotton and enrich the MYB family gene resources for cotton stress resistance response, this invention clones transcription factors with promising applications in cotton stress resistance. GhMYB14 Genes provide cotton transcription factors GhMYB14 Genes and their applications. To achieve the above objectives, the present invention adopts the following technical solution.

[0007] To further develop and study the MYB gene in cotton and enrich the MYB family gene resources for cotton stress resistance response, this invention has developed the cotton MYB gene and provided a novel transcription factor with promising applications in cotton stress resistance. GhMYB14 Genes. Specifically, this invention provides a cotton transcription factor for regulating cotton stress resistance. GhMYB14 Genes, the cotton transcription factor GhMYB14 The nucleotide sequence of the gene is shown in SEQ ID NO.1. This cotton transcription factor... GhMYB14 The transcriptional level of the gene increases under salt stress, and silencing the gene in cotton makes it more sensitive to salt stress, indicating that... GhMYB14 This gene plays an active role in cotton salt tolerance. The successful cloning and functional analysis of this gene expands the resource of MYB genes related to cotton stress response and provides a key candidate gene for salt tolerance genetic improvement.

[0008] Furthermore, the cotton transcription factor GhMYB14 The amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO.2.

[0009] The present invention also provides a cotton transcription factor containing the aforementioned cotton transcription factor. GhMYB14 Recombinant gene expression vectors.

[0010] Furthermore, the recombinant expression vector is used to express the cotton transcription factor. GhMYB14 Gene insertion pCambia2300 carrier BamH I and Stu Obtained from the I restriction site.

[0011] Furthermore, the method for constructing the recombinant expression vector includes the following steps: Amplification was performed using the correctly sequenced pMD18-T plasmid as a template.

[0012] The obtained amplification product was digested with two enzymes and then combined with... BamH I and Stu I double enzyme digestion pCambia2300 The vector is ligated and transformed, and the plasmids of the verified positive clones are extracted to obtain the recombinant expression vector.

[0013] The present invention also provides the cotton transcription factor. GhMYB14 Application of the gene or the recombinant expression vector in improving the stress resistance of cotton.

[0014] Furthermore, improving the stress resistance of cotton includes improving its salt tolerance.

[0015] Furthermore, the cotton transcription factor was overexpressed in plants. GhMYB14 Genes were developed to improve the salt tolerance of cotton.

[0016] Furthermore, the recombinant expression vector was transfected into cotton plants to be transformed, resulting in transgenic cotton plants with improved salt tolerance.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. To further develop and research the MYB gene in cotton and enrich the MYB family gene resources for cotton stress resistance response, this invention has developed the cotton MYB gene and provided a new transcription factor with promising applications in cotton stress resistance. GhMYB14 Gene. This invention provides a cotton transcription factor for regulating cotton stress resistance. GhMYB14 The cloning and functional identification of this gene enriches the MYB family gene resources for cotton stress resistance response, laying a theoretical foundation for the breeding of new salt-tolerant cotton varieties.

[0018] 2. This invention clones cotton transcription factors. GhMYB14 Gene (abbreviated as) GhMYB14 Gene or GhMYB14 ), and used transgenic technology to construct an overexpression GhMYB14 Transgenic Arabidopsis thaliana. Simultaneously, VIGS technology was used to silence [the virus] in cotton. GhMYB14 It was clarified GhMYB14 It can improve the salt tolerance of Arabidopsis thaliana and cotton. Furthermore, GhMYB14 It responds to multiple plant hormones at the transcriptional level. For further research... GhMYB14 This study lays the foundation for understanding the molecular mechanisms of resisting abiotic stresses and provides an excellent target for molecular breeding of cotton with salt tolerance. Attached Figure Description

[0019] Figure 1 This invention relates to cotton transcription factors. GhMYB14 Agarose gel electrophoresis image of gene amplification products.

[0020] Figure 2 This is the result of multiple sequence alignment of the amino acid sequence (positions 1-139) of the cotton transcription factor GhMYB14 in this invention with amino acid sequences of other species that have high homology.

[0021] Figure 3 The cotton transcription factor in this invention GhMYB14 A diagram illustrating tissue-specific gene expression analysis.

[0022] Figure 4 This is a subcellular localization map of GhMYB14 in this invention; wherein, Figure 4 A in the equation is 35S:: GhMYB14 - Schematic diagram of the structure of the GFP recombinant vector; Figure 4 B in the figure represents the subcellular localization of GhMYB14 in tobacco leaves.

[0023] Figure 5 The cotton transcription factor in this invention GhMYB14 Agarose gel electrophoresis image of the amplification products from positions 484-774 of the gene.

[0024] Figure 6 The cotton transcription factor in this invention GhMYB14 Identification of gene silencing efficiency.

[0025] Figure 7 The cotton transcription factor in this invention GhMYB14 Verification of salt tolerance in gene-silenced cotton plants; among which... Figure 7 A in the text refers to cotton transcription factor. GhMYB14 Salt-tolerant phenotype of gene-silenced plants, with multiple samples being parallel samples; Figure 7 B in the text refers to cotton transcription factor. GhMYB14 Fresh weight of gene-silenced plants; Figure 7 C in the text refers to cotton transcription factor. GhMYB14 Superoxide anion content in gene-silenced plants; Figure 7 D in the text represents cotton transcription factor. GhMYB14 Peroxidase activity in gene-silenced plants; Figure 7 E in the text refers to cotton transcription factor. GhMYB14 Malondialdehyde (MDA) content in gene-silenced plants.

[0026] Figure 8 The cotton transcription factor in this invention GhMYB14 Changes in gene expression levels under various treatment conditions; among them, Figure 8 A in the text refers to treatment with 200 mM NaCl; Figure 8 B in the text refers to 100 μM ABA treatment; Figure 8 C in the text refers to 100 μM IAA treatment; Figure 8 D in the text refers to 100 μM GA3 treatment; Figure 8 E in the text refers to 10 μM SA treatment; Figure 8 F in the figure represents 10 μM MeJA treatment.

[0027] Figure 9 This is a diagram of the vector used to overexpress transgenic Arabidopsis thaliana in this invention and a Western blot identification diagram; wherein, Figure 9 A in the text is 35S::GhMYB14- A simplified structural diagram of the HA recombinant vector; Figure 9 B in the figure represents the Western Blot identification of the homozygous overexpressing transgenic Arabidopsis thaliana lines OE1 and OE2.

[0028] Figure 10 Overexpression in this invention GhMYB14 Germination rate of Arabidopsis thaliana lines under salt stress; among which, Figure 10 In this context, A represents the germination phenology of the Arabidopsis thaliana strain; Figure 10 In this context, B represents the germination rate statistics.

[0029] Figure 11 Overexpression in this invention GhMYB14 The taproot length of Arabidopsis thaliana strains under salt stress conditions; among them, Figure 11 In the figure, A represents the root length phenotype of Arabidopsis thaliana strains, and multiple samples are parallel samples. Figure 11 The statistics of the principal root length of B in the figure. Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0031] Example 1: GhMYB14 Gene cloning 1. Take uniform TM-1 cotton seeds, soak them in a 28℃ incubator for 16 hours, and then sow the sprouting cotton seeds in nutrient soil. Cultivate normally for three weeks to obtain cotton seedlings.

[0032] The TM-1 cotton seeds came from Li Zhaohu's research group at China Agricultural University.

[0033] The potting soil is imported peat moss (PINDSTRUP).

[0034] 2. Take the second true leaf of a cotton seedling, flash-freeze it with liquid nitrogen, and store it at -80℃ for later use. When extracting RNA, place the cotton leaf in a 1.5mL RNase-free EP tube, grind it using an automatic sample grinder, and then follow the instructions in the Hi-Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Dual Column Type) (Cat.TSP0202) to obtain RNA.

[0035] 3. After the integrity, concentration and purity of the obtained RNA were detected by agarose gel electrophoresis, first-strand cDNA was synthesized using Adley's reverse transcription kit (PC1803).

[0036] 4. Amplification using cotton cDNA as a template GhMYB14 The full length of the gene, according to GhMYB14Gene sequence was determined, and two specific primers (upstream and downstream primers) were designed for PCR amplification to obtain PCR products. The PCR products were then subjected to 1.5% agarose gel electrophoresis. Figure 1 ).

[0037] in, GhMYB14 Genes, specifically cotton transcription factors GhMYB14 Gene, abbreviated as GhMYB14 The nucleotide sequence is shown in SEQ ID NO.1: .

[0038] The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3: 5'-ATGGTGAGAGCTCCATGCTG-3'.

[0039] The nucleotide sequence of the downstream primer is shown in SEQ ID NO.4: 5'-TCAAAATTCAGGTAGTTCAGGT-3'.

[0040] Gene amplification was performed according to the instructions for Phanta® Max Super-Fidelity DNA Polymerase (P505) from Novizan, and the system is shown in Table 1: Table 1 PCR amplification system .

[0041] The PCR program was set as follows: pre-denaturation 95℃ 3min; denaturation 95℃ 15s; 55℃ Tm 15s; extension 72℃ 1kb / 30s; steps 2 to 4 were set to 35 cycles, re-extension 72℃ 5min; incubation 16℃.

[0042] 5. The PCR amplification product was ligated into the cloning vector pMD18-T to obtain the recombinant vector. Sequencing results showed that the full-length gene sequence was 780 bp, encoding a complete ORF reading frame of 259 amino acids.

[0043] The amino acid sequence of GhMYB14 was BLASTed in NCBI, and multiple sequence alignment was performed between the 10 most similar species sequences and the AtMYB14 sequence of Arabidopsis thaliana. The results showed that GhMYB14 had the highest amino acid sequence homology with HuMYB14 (XP_021279887.1) from Colombian mallow (Herrania umbratica), TcMYB4 (XP_007050304.2) from cacao tree (Theobroma cacao), DzMYB30 (XP_022759767.1) from durian (Durio zibethinus), and HtMYB14 (GMI66443.1) from wild watermelon seedling (Hibiscus trionum), with 76.64%, 75.18%, 73.38%, and 75.76%, respectively. GhMYB14 is an R2R3 transcription factor with conserved R2[-W-(X19)-W-(X11)-W-] and R3[-F / I-(X18)-W-] DNA-binding domains. Figure 2 ).

[0044] Among them, GhMYB14 is the cotton transcription factor GhMYB14, abbreviated as GhMYB14, and its amino acid sequence is shown in SEQ ID NO.2: MVRAPCCEKMGLKKGPWTPEEDQILINYIQLHGHGNWRALPKQAGLLRCGKSCRLRWTNYLRPDIKRGNFTREEEDTIINLHEMLGNRWSAIAARLPGRTDNEIKNVWHTHLKKRLKHSHGSNANNRQPI DPSKDIKREQQPVTVYSPVSPPQSSSDVSTSENNSNSNAFTTKTETNEDVSEIDENFWSEVLSADSSSMEANFRVVGSDQYFPSSPPPPLPALETVNGYGSNLYDTDANMDFWYILFTRAADLPELPEF.

[0045] Example 2: GhMYB14 Tissue-specific localization of genes The analysis of cotton variety "TM-1" using real-time quantitative PCR was performed. GhMYB14 Analysis of gene expression sites: The instrument used for real-time quantitative PCR was a CFX96™ Real-Time System, and the primer pairs used (including qRT) -GhMYB14 -F and qRT- GhMYB14 -R), the sample used to detect tissue-specific expression is cDNA obtained by reverse transcription of total RNA from different tissues (i.e., leaves, roots and stems) of cotton at the three-leaf stage under normal nutrient levels.

[0046] Among them, qRT- GhMYB14 The nucleotide sequence of -F is shown in SEQ ID NO.5: 5'-AAGAGAACAACAACCCGTGAC-3'.

[0047] qRT- GhMYB14 The nucleotide sequence of -R is shown in SEQ ID NO.6: 5'-TTGGTTCAGTTTTGGTGGTG-3'.

[0048] PCR program: 94℃ denaturation for 30s; 94℃ denaturation for 5s, 60℃ annealing for 35s, 40 cycles.

[0049] cotton Actin9 Genes used as a control (for identifying cotton) Actin9 Primer pairs for genes (including qRT-) GhActin9 -F and qRT- GhActin9-R), relative expression level using 2 -ΔΔCt Method calculation.

[0050] Among them, qRT- GhActin9 The nucleotide sequence of -F is shown in SEQ ID NO.7: 5'-GCCTTGGACTATGAGCAGGA-3'.

[0051] qRT- GhActin9 The nucleotide sequence of -R is shown in SEQ ID NO.8: 5'-AAGAGATGGCTGGAAGAGGA-3'.

[0052] The results showed that, under normal nutrient levels, different parts of cotton at the three-leaf stage... GhMYB14 The relative expression levels of genes are shown in [the table]. Figure 3 , GhMYB14 The gene is expressed at a certain level in different tissues of cotton, with relatively high expression in the roots and lower expression in the leaves.

[0053] Example 3: Subcellular localization of GhMYB14 protein Subcellular localization of GhMYB14 protein was studied using Nicotiana benthamiana. This was based on a plant binary transformation vector. super1300-GFP (The recombinant vector linking the GFP gene, described in the doctoral dissertation "Metabolic Characteristics of Cuscuta during In Vitro Growth and Study of Agrobacterium mis Gene in its Genome," by Zhang Yuexia, *China Agricultural University*, 2020, may be obtained from the research group of Li Zhaohu at China Agricultural University with the author's consent for the purpose of replicating this experiment. It may not be used for any other purpose and is hereinafter referred to as "the vector.") super1300 Multiple cloning sites and GhMYB14 Designing amplification of the coding region sequence of the gene GhMYB14 Forward and reverse primers for the entire coding region of the gene were obtained. 35S:: GhMYB14 -GFP recombinant vector, the specific method is as follows:

[0054] by GhMYB14 Using the gene as a template, PCR amplification was performed using forward and reverse primers to obtain a sample containing... GhMYB14 The product of genes; utilization Sma I and Kpn I digested with enzymes respectively, containing GhMYB14 Gene products and super1300 The enzyme digestion products and vector frameworks were obtained and recovered separately; the enzyme digestion products and vector frameworks were then ligated to obtain... 35S:: GhMYB14 -GFP recombinant vector (structural diagram shown) Figure 4As shown in A), it is about to super1300 of Sma I and Kpn The DNA fragment between the I restriction sites is replaced with the one shown in SEQ ID NO.1. GhMYB14 After the nucleotides of the gene, and maintain super1300 The vector obtained by keeping the other sequences unchanged.

[0055] The primers are as follows: The nucleotide sequence of the forward primer is shown in SEQ ID NO.9: 5'- TCC CCCGGG ATGGTGAGAG CTCCATGCTG -3', underscore is Sma I restriction enzyme site.

[0056] The nucleotide sequence of the reverse primer is shown in SEQ ID NO.10: 5'- GG GGTACC AAATTCAGGTAGTTCAGGTAAA -3', underscores are Kpn I restriction enzyme site.

[0057] Will 35S:: GhMYB14 -GFP was transformed into Agrobacterium GV3101 to obtain recombinant bacteria, which then infected tobacco leaves. After 48 hours, the injected leaves were placed on slides, and GFP expression was observed under a laser confocal microscope. The results showed that GhMYB14 was located in the cell nucleus (…). Figure 4 (B in the middle).

[0058] Agrobacterium GV3101 was purchased from Daling Biotechnology.

[0059] Example 4: VIGS Silent Plant Stress Phenotype I. VIGS GhMYB14 Construction of silent carriers 1. Total RNA was extracted from the leaves of cotton variety “TM-1” and reverse transcribed into cDNA.

[0060] 2. Using the cDNA obtained in step 1 as a template, perform PCR amplification using a primer pair consisting of F1 and R1 (including F1 and R1) to obtain the PCR amplification product ( Figure 5 ).

[0061] The nucleotide sequence of F1 is shown in SEQ ID NO.11: 5'- G GAATTC GAAAACAACAGCAACAGCA -3', underscores are EcoR I restriction enzyme site.

[0062] The nucleotide sequence of R1 is shown in SEQ ID NO.12: 5'-CG GGATCC TTCAGGTAGTTCAGGTAAATCC-3', underscore is BamH I restriction enzyme site.

[0063] 3. Using restriction endonucleases EcoR I and BamH The PCR amplification product obtained from step 2 of double enzyme digestion was recovered.

[0064] 4. Using restriction endonucleases EcoR I and BamH I double enzyme digestion pYL156(pTRV2:RNA2) The vector (described in the non-patent literature "Gao X, 2013, Functional genomic analysis of cotton genes with agrobacterium-mediated virus-induced gene silencing.", which may be obtained by the public from Li Zhaohu's research group at China Agricultural University with the author's consent, for the purpose of replicating this experiment, but may not be used for other purposes), and the vector backbone was recovered.

[0065] 5. Ligate the enzyme digestion product from step 3 with the vector backbone from step 4 to obtain the recombinant plasmid. pYL156-GhMYB14 .

[0066] For recombinant plasmids pYL156- GhMYB14 Sequencing verification showed that the recombinant plasmid... pYL156-GhMYB14 To be pYL156 carrier EcoR I and BamH The DNA fragment between the I restriction sites is replaced with the portion shown in positions 484-774 of SEQ ID NO.1. GhMYB14 After the gene fragment, and maintain pYL156 The vector is obtained by keeping the other sequences of the vector unchanged.

[0067] II. VIGS- GhMYB14 Obtaining Silent Plants 1. pYL156-GhMYB14 , pYL156-GFP , pTRV-RNA1 and pYL156-GhCLA1 ( pYL156-GFP , pTRV1(pTRV-RNA1 )and pYL156-GhCLA1The method described in the non-patent literature "Gao X, 2013, Functional genomic analysis of cotton genes with agrobacterium-mediated virus-induced geneilencing." (With the author's consent, the public can obtain this information from the research group of Li Zhaohu at China Agricultural University for the purpose of replicating this experiment, but it cannot be used for other purposes.) Recombinant bacteria were obtained by electroporating Agrobacterium GV3101. pYL156- GhMYB14 / GV3101, recombinant bacteria pYL156-GFP / GV3101, recombinant bacteria pTRV1 / GV3101 and recombinant bacteria pYL156-GhCLA1 / GV3101 was collected by shaking in LB liquid medium (50 μg / mL kanamycin, 25 μg / mL gentamicin, 10 mM MES pH 5.7, 20 μM acetylsuccione, water) at 28℃ for 14 h. pYL156- GhMYB14 / GV3101, recombinant bacteria pYL156-GFP / GV3101, recombinant bacteria pTRV1 / GV3101 and recombinant bacteria pYL156-GhCLA1 / GV3101.

[0068] 2. Recombinant bacteria pYL156-GhMYB14 / GV3101, recombinant bacteria pYL156-GFP / GV3101, recombinant bacteria pTRV1 / GV3101 and recombinant bacteria pYL156-GhCLA1 / GV3101 cells were resuspended in VIGS solution (10mM MES pH 5.6, 10mM MgCl2, 200μM acetylsalicylic acid, water as solvent) and the bacterial concentration was adjusted to OD. 600 =1.5, recombinant bacteria pYL156- GhMYB14 / GV3101、 pYL156-GFP / GV3101 and pYL156-GhCLA1 / GV3101 and recombinant bacteria pTRV1 The bacterial culture of / GV3101 was mixed in a volume ratio of 1:1 to obtain mixture 1, mixture 2 and mixture 3.

[0069] 3. Using a 1mL needleless syringe, spray the mixture 1 onto the lower surface of the cotyledons of different cotton plants ("Xinshi 17") to obtain VIGS- GhMYB14 Silent plants. Among them, the seeds of cotton variety "Xinshi 17" were provided by the research group of Li Zhaohu at China Agricultural University.

[0070] Using a 1mL needleless syringe, spray the mixture 2 onto the lower surface of the cotyledons of different cotton plants ("Xinshi 17") to obtain VIGS-GFP Control plants.

[0071] Using a 1mL needleless syringe, mix solution 3 was applied to the lower surface of the cotyledons of different cotton plants, "Xinshi 17". After two weeks of cultivation, VIGS- was obtained. GhCLA1 Indicator plants.

[0072] Approximately two weeks after the plants injected with mixture 3 exhibited the albino phenotype, RNA was extracted from the leaves of plants injected with mixture 1 and mixture 2 (using the Hi-Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Dual Column Type) (Cat.TSP0202) from Qingke Biotechnology). cDNA was then reverse transcribed using the Adley Reverse Transcription Kit (PC1803) according to the kit instructions. Gene silencing efficiency was analyzed by real-time quantitative PCR. The primer pairs used (including qRT) were analyzed. -GhMYB14 -F and qRT- GhMYB14 -R).

[0073] PCR program: 94℃ denaturation for 30s; 94℃ denaturation for 5s, 60℃ annealing for 35s, 40 cycles.

[0074] cotton Actin9 Genes used as a control (for identifying cotton) Actin9 Primer pairs for genes (including qRT-) GhActin9 -F and qRT- GhActin9 -R), relative expression level using 2 -ΔΔCt Method calculation.

[0075] The results showed that the injection mixture 1 (containing pYL156-GhMYB14 / GV3101 and pTRV1 Plants with / GV3101 bacterial solution GhMYB14 Gene expression levels were significantly lower than those in injection mixture 2 (containing pYL156-GFP / GV3101 and pTRV1 Plants (GV3101 bacterial solution) Figure 6 As shown in the figure, silence was achieved through the above method. GhMYB14 VIGS of genes GhMYB14 Silent plants (plants injected with mixture 1) and VIGS- GFP Control plants (plants injected with mixture 2).

[0076] III. VIGS GhMYB14 Salt tolerance phenotype of silent plants Through the silence obtained in step two GhMYB14 VIGS of genes GhMYB14 Silent plants and VIGS- GFPControl plants were treated with 250 mM NaCl stress. Under salt stress, VIGS- GhMYB14 Silent plants and VIGS- GFP Compared to the control plants, the plant growth inhibition was more pronounced, and salt damage symptoms such as leaf wilting, yellowing, and shedding were more significant, indicating greater sensitivity to salt stress. In the control group without NaCl treatment, VIGS- GhMYB14 Silent plants and VIGS- GFP There was no difference in growth characteristics between the control and control plants. Figure 7 (As shown in A).

[0077] IV. VIGS GhMYB14 Fresh weight determination of silent plants The silence obtained in step two GhMYB14 VIGS of genes GhMYB14 Silent plants and VIGS- GFP Weigh the control plant to obtain the fresh weight. Figure 7 (As shown in B). VIGS- GhMYB14 Silent plants and VIGS- GFP Under normal treatment conditions, the fresh weight of control plants showed no significant difference. After salt treatment, the VIGS- GhMYB14 The fresh weight of silent plants was significantly lower than that of VIGS- GFP Control plants.

[0078] V. VIGS GhMYB14 Determination of superoxide anion content and peroxidase (POD) activity in silent plants Take the second true leaf from cotton plants before and after salt treatment. Add 1g of leaf sample to 5mL of 50mM phosphate buffer solution (PBS, pH 7.8) and grind thoroughly. Centrifuge at 8000rpm and 4℃ for 20 minutes, and collect the supernatant as the crude enzyme solution, which is used for POD activity detection and superoxide anion content determination.

[0079] 1. Determination of superoxide anion content Prepare 10 nM, 15 nM, 20 nM, 30 nM, 40 nM, and 50 nM NaNO₂ solutions. Take more than 1 mL of each solution (using distilled water as a control) and mix thoroughly with 1 mL of 50 mM phosphate buffer solution (pH 7.8), 1 mL of 17 mM p-aminobenzenesulfonic acid, and 1 mL of 7 mM α-naphthylamine. React at 25 °C for 20 min. [The remaining text appears to be incomplete and requires further context.] 530nm The absorbance value at the specified wavelength was used as a control for zeroing. A standard curve was plotted with nitrite concentration on the x-axis and absorbance on the y-axis.

[0080] Mix 0.5 mL of crude enzyme solution (with 0.5 mL of distilled water added to the blank control), 1 mL of 50 mM phosphate buffer (pH 7.8), and 1 mL of 1 mM hydroxylamine hydrochloride, and react at 25 °C for 1 h. Add 1 mL of 17 mM p-aminobenzenesulfonic acid and 1 mL of 7 mM α-naphthylamine, mix thoroughly, and react at 25 °C for 20 min. After zeroing with the blank control, measure the OD of the reaction solution. 530nm The absorbance value at a given wavelength. The calculation formula is as follows:

[0081] Superoxide anion content = NO2 in the standard curve - × Total amount of extract × 2 / Sample weight / Amount of extract used during the test; In the above formula, the unit of superoxide anion content is nmol·g. -1 FW; Standard track NO2 - The unit is nmol; the unit for total extract volume is mL; the unit for sample weight is g; and the unit for extract volume used in the determination is mL.

[0082] Figure 7 The results in C show that VIGS- GhMYB14 Silent plants and VIGS- GFP Under normal treatment conditions, the superoxide anion content of control plants showed no significant difference. After salt treatment, the VIGS- GhMYB14 The superoxide anion content of silent plants was significantly higher than that of VIGS- GFP Control plants.

[0083] 2. Determination of POD activity The reaction solution (0.038 mL 0.2% (v / v) guaiacol solution, 0.56 mL 3% (v / v) H2O2, and 100 mL 0.2 M pH 6.0 PBS) was reacted with 200 μL of crude enzyme solution. PBS was used as a control for zeroing. The OD was measured within one minute using a UV-2802S visible spectrophotometer (Unic). 470nm Wavelength variation. Enzyme activity calculation: based on OD. 470nm A change (increase) of 0.01 per minute is calculated as one unit of enzyme activity (U). The calculation formula is as follows:

[0084] POD activity = (ΔA) 470 (×Total volume of enzyme extract) / (Sample weight × Volume of enzyme solution to be measured × 0.01 × Reaction time); In the above formula, the unit of POD activity is U / g min; the unit of total enzyme extraction volume is mL; the unit of sample weight is g; the unit of enzyme volume measurement is mL; and the unit of reaction time is min.

[0085] Figure 7The results of the D-plot show that VIGS- GhMYB14 Silent plants and VIGS- GFP Under normal treatment conditions, there was no significant difference in POD activity among control plants. After salt treatment, VIGS- GhMYB14 The POD activity of silent plants was significantly lower than that of VIGS- GFP Control plants.

[0086] VI. VIGS GhMYB14 Determination of malondialdehyde (MDA) content in silent plants Weigh approximately 1g of the second true leaf from a cotton seedling, place the leaf in a mortar, add 10mL of 5% (m / v) trichloroacetic acid (TCA) and a small amount of quartz sand, and grind. Transfer the homogenate to a 10mL centrifuge tube. Place the centrifuge tube in a centrifuge and centrifuge at 4000rpm. -1 Centrifuge for 10 min, and the supernatant is the MDA extract. Take 2 mL of the MDA extract (using 2 mL of distilled water as a control), add 2 mL of 0.6% (m / v) thiobarbituric acid (TBA), shake well, incubate in a boiling water bath for 10 min, then cool to room temperature and incubate at 3000 rpm. -1 Centrifuge for 15 min, and measure the OD values ​​of the supernatant at three wavelengths: 532 nm, 600 nm, and 450 nm. The calculation formula is as follows:

[0087] MDA content = [6.452 × (OD)] 532 -OD 600 -0.559×OD 450 × Total volume of extract / Volume of extract to be measured / Fresh weight of sample; In the above formula, the unit for MDA content is mmol·g. -1 FW; the unit for total extract volume is mL; the unit for measured extract volume is mL; the unit for sample fresh weight is g.

[0088] Figure 7 The results in E showed that VIGS- GhMYB14 Silent plants and VIGS- GFP Under normal treatment conditions, the MDA content of control plants showed no significant difference. After salt treatment, the VIGS- GhMYB14 Silent plants had significantly higher MDA content than VIGS- GFP Control plants.

[0089] Example 5: GhMYB14 Changes in expression levels under salt and various hormone treatments Cotton plants of the 'TM-1' variety at the three-leaf stage under normal growth conditions were treated with 200 mM NaCl. The second true leaf was collected at 0 h, 12 h, 24 h, 48 h, and 72 h and stored at -80℃. The cotton leaves were then sprayed with 100 μM ABA, 100 μM IAA, 100 μM GA3, 10 μM SA, and 10 μM MeJA, respectively, and samples were collected at 0 h, 0.5 h, 1 h, 3 h, 6 h, 12 h, and 24 h. RNA was extracted from these samples and reverse transcribed into cDNA, which was then detected by real-time quantitative PCR. GhMYB14 Gene expression levels, primer pairs used (including qRT) -GhMYB14 -F and qRT- GhMYB14 -R).

[0090] PCR program: 94℃ denaturation for 30s; 94℃ denaturation for 5s, 60℃ annealing for 35s, 40 cycles.

[0091] cotton Actin9 Genes used as a control (for identifying cotton) Actin9 Primer pairs for genes (including qRT-) GhActin9 -F and qRT- GhActin9 -R) is: and, relative expression level is 2. -ΔΔCt The method was used for calculation. Results showed that each treatment had varying degrees of effect. GhMYB14 The expression ( Figure 8 ).

[0092] Example 6: Obtaining Transgenic Plants 1. With GhMYB14 Using the gene as a template, PCR amplification was performed using forward and reverse primers to obtain a sample containing... s The product of genes; utilization GhMYB14 I and BamH I digest the vector with enzymes respectively. Stu The gene amplification product and enzyme digestion product were recovered and ligated to obtain... pCambia2300 -HA recombinant vector (structural diagram shown) 35S:: GhMYB14 As shown in A), it is about to Figure 9 of pCambia2300 I and BamH The DNA fragments between the I restriction sites are replaced with Stu After the gene, and maintain GhMYB14 The vector obtained by keeping the other sequences unchanged.

[0093] The primers are as follows: The nucleotide sequence of forward primer 1 is shown in SEQ ID NO. 13: 5'-CTCCCCTTGCTCCGT pCambia2300ATGGTGAGAGCTCCATGCTG-3', underscore is GGATCC I restriction enzyme site.

[0094] The nucleotide sequence of reverse primer 2 is shown in SEQ ID NO.14: 5'-AACGTCGTATGGGTA BamH AAATTCAGGTAGTTCAGGTAAA-3', underscore AGGCCT I restriction enzyme site.

[0095] Will Stu -HA was transferred into Agrobacterium GV3101 to obtain recombinant Agrobacterium.

[0096] 2. The recombinant Agrobacterium obtained in step 1 was cultured at 28°C in LB liquid medium (50 μg / mL kanamycin, 25 μg / mL gentamicin) for 24 h. The recombinant Agrobacterium was collected by centrifugation at 4000 rpm for 10 min and resuspended (resuspending solution: 50 mM MES, pH 5.6, 5% (m / v) sucrose, solvent: water). The concentration of the resuspended bacterial solution was adjusted to OD. 600 =0.8, add 500 μl of silwetL-77 -1 Wet the Arabidopsis thaliana inflorescences that have not yet shown white with the bacterial suspension, then wrap the Arabidopsis thaliana in a black plastic bag to maintain humidity, lay it flat, and incubate it in the dark at 20˚C for 24 hours. After that, remove the plastic bag, restore light, and cultivate the plants according to the conventional method until they bear fruit. Harvest mature T0 generation seeds.

[0097] 3. T0 generation seeds were cultured in 1 / 2 MS medium containing 50 μg / mL kanamycin and positive plants were selected (positive plants are characterized by healthy, dark green true leaves and roots extending into the medium).

[0098] 4. Self-pollinate the positive plants obtained in step 4 to obtain T1 generation seeds.

[0099] 5. Culture T1 generation seeds in 1 / 2 MS medium containing 50 μg / mL kanamycin and select positive plants (the selection criterion is that the proportion of positive plants is greater than 3:1).

[0100] 6. Self-pollinate the positive plants obtained in step 6 to obtain T2 generation seeds.

[0101] 7. T2 generation seeds were cultured in 1 / 2 MS medium containing 50 μg / mL kanamycin and positive plants were selected (the selection criterion was that all plants in the line were positive).

[0102] 8. Self-pollinate the positive plants obtained in step 8 to obtain T3 generation seeds. Cultivate T3 generation seeds to obtain T3 generation transgenic plants. 35S:: GhMYB14 Arabidopsis thaliana plant.

[0103] 9. For T3 conversion GhMYB14 Total protein was extracted from Arabidopsis thaliana plants and molecularly identified using Western blotting. GhMYB14 As shown in Figure B), we obtain Figure 9 Overexpression of transgenic homozygous lines.

[0104] The specific steps of Western blotting are as follows: Leaf samples were taken using a punch when the plants were in the fourth week of growth. The leaf tissue was ground with liquid nitrogen and then SDS plant total protein extraction solution (250mM Tris-HCl pH 6.8, 4% (m / v) SDS, 40% glycerol, 0.1% (m / v) bromophenol blue, 4% (v / v) β-mercaptoethanol) was added. After mixing, the mixture was denatured at 95 degrees Celsius or above for 10 minutes. The supernatant was collected by centrifugation and used as the denatured total protein for SDS-PAGE analysis. Set up the electrophoresis apparatus with a 10% SDS-PAGE gel, pour in the electrophoresis buffer, add the protein sample and pre-stained marker, and set the voltage to 90-120V for constant voltage electrophoresis separation (SDS-PAGE gel purchased from BIO-RAD, instrument used from Bio-Rad Mini-PROTEANR Tetra System, pre-made marker purchased from Thermo Fisher, 10× Tris-glycine electrophoresis buffer: 30.3g / L Tris base, 144g / L glycine, 10g / L SDS, diluted with distilled water to 1× before use). Stop electrophoresis when the dye line is 1cm from the bottom of the gel.

[0105] Remove the SDS-PAGE gel and immerse it in transfer buffer (2.9 g / L glycine, 5.8 g / L Tris base, 0.37 g / L SDS, 20% (v / v) methanol) for equilibration for 10 min. Cut the PVDF membrane to the appropriate size, wet it in methanol for 15 s, and then transfer it to transfer buffer for equilibration for 20 min. Similarly, thoroughly wet the filter paper used for transfer in transfer buffer. Place the filter paper, PVDF membrane, SDS-PAGE gel, and filter paper in the transfer apparatus (BIO-RAD TRANS-BLOT SD SEMI-DRYTRANSFER CELL) from the positive electrode to the negative electrode, and transfer at a constant current of 60 mA for 1 h.

[0106] After the transfer is completed, the PVDF membrane is placed in a TBST solution of 2% (m / v) BSA or 5% (m / v) skim milk powder (10×TBS solution: 80g / L NaCl, 30g / L Tris-base, 2g / L KCl, adjust pH to 7.5, dilute with distilled water to 1× before use, and add Tween 20 at a ratio of 1:500-1:1000 to obtain the TBST solution) for 2 hours.

[0107] Anti-HA antibody (purchased from Abclonal) was added to the blocking buffer at a volume ratio of 1:1500. Hybridization was performed overnight at 4°C. The membrane was washed three times with TBST solution for 10 min each time. Secondary antibody anti-mouse (purchased from Abclonal, diluted 1:10000 in 2% (m / v) BSA or 5% (m / v) skim milk powder TBST solution) was added, and the membrane was incubated at room temperature for 1 h. The membrane was washed three times with TBST solution and once with TBS solution before development (developer was the Kangwei Century eECL Western Blot Kit, and imaging system was Tanon-5200). "Overnight" refers to a time ≥12 h.

[0108] After development, immerse the PVDF membrane in Ponceau S staining solution (Ponceau S solution: 0.2% m / v Ponceau S, 3% (v / v) acetic acid), shake for 5 min or longer, remove it, rinse 3 times with distilled water, and record the results after clear bands appear.

[0109] Ultimately, selected GhMYB14 Stress resistance analysis of overexpression transgenic homozygous lines OE1 and OE2 ( GhMYB14 (B in the middle).

[0110] Example 7: Analysis of stress resistance of different transgenic lines Arabidopsis Figure Germination rate analysis of overexpression transgenic homozygous lines under abiotic stress: ​ Seeds of overexpressing transgenic homozygous lines OE1, OE2, and wild-type Arabidopsis thaliana (WT) were vernalized at 4°C for 72 h in normal MS medium and media containing 75 mM NaCl and 100 mM NaCl, respectively. After vernalization, the seeds were transferred to a 20°C greenhouse with 16 h of light / 8 h of darkness at a light intensity of 60 μmol / m². 2 The plants were cultured in a 60% humidity incubator for 10 days, and the germination rate of each strain was measured. The results are as follows: ​As shown in the figure, WT, OE-1, and OE-2 all germinated on normal culture plates with germination rates exceeding 90%. Germination of WT, OE-1, and OE-2 was inhibited on MS culture plates containing 75 mM and 100 mM NaCl, but OE-1 and OE-2 showed higher salt tolerance than WT, achieving germination rates of 77% and 52% respectively on MS culture plates containing 75 mM NaCl, significantly higher than that of WT.

[0111] Seedlings of WT, OE-1, and OE-2 that germinated for 6 days on normal culture plates were transferred to MS culture plates containing 100 mM NaCl for root length observation. After 15 days, WT, OE-1, and OE-2 all grew normally on the normal culture plates with no significant difference in root length. However, on the MS culture plates containing 100 mM NaCl, the root length of WT, OE-1, and OE-2 was inhibited, but the root lengths of OE-1 and OE-2 were significantly longer than those of WT by 35% and 56%, respectively. ​ The above results indicate that... ​ Positive regulation of salt stress response.

[0112] As can be seen from the above, GhMYB14 is an R2R3 type MYB transcription factor located in the cell nucleus. ​ The transcriptional level of this substance is induced by salt stress and responds to signals from multiple plant hormones, including IAA, GA3, and SA. Silencing occurs in cotton. ​ Later, the plants became more salt-sensitive. Overexpression of this gene in Arabidopsis thaliana resulted in more salt-tolerant plants, indicating that... ​ Actively regulating salt tolerance lays a solid molecular foundation for effectively improving plant salt tolerance.

[0113] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0114] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. Cotton transcription factors used to regulate cotton stress resistance GhMYB14 Genes, characterized by, The cotton transcription factor GhMYB14 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The cotton transcription factor according to claim 1 GhMYB14 Genes, characterized by, The cotton transcription factor GhMYB14 The amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO.

2.

3. A cotton transcription factor containing the cotton transcription factor of claim 1 GhMYB14 Recombinant gene expression vectors.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector is used to express the cotton transcription factor. GhMYB14 Gene insertion pCambia2300 carrier BamH I and Stu Obtained from the I restriction site.

5. The recombinant expression vector according to claim 4, characterized in that, The method for constructing the recombinant expression vector includes the following steps: Amplification was performed using the correctly sequenced pMD18-T plasmid as a template. The obtained amplification product was digested with two enzymes and then combined with... BamH I and Stu I double enzyme digestion pCambia2300 The vector is ligated and transformed, and the plasmids of the verified positive clones are extracted to obtain the recombinant expression vector.

6. The cotton transcription factor according to claim 1 GhMYB14 The application of the gene or the recombinant expression vector as described in claim 3 in improving the stress resistance of cotton.

7. The application according to claim 6, characterized in that, Improving the stress resistance of cotton includes improving its salt tolerance.

8. The application according to claim 7, characterized in that, Overexpression of the cotton transcription factor in plants GhMYB14 Genes were developed to improve the salt tolerance of cotton.

9. The application according to claim 7, characterized in that, The recombinant expression vector was transfected into cotton plants to be transformed, resulting in transgenic cotton plants with improved salt tolerance.

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