GhBGH2 gene of upland cotton BR signal positive regulation factor as well as encoding protein and application of GhBGH2 gene

Abstract

The invention relates to the technical field of plant genetic engineering, and particularly provides an upland cotton BR signal positive regulation factor GhBGH2 gene as well as an encoding protein and application thereof. The coding sequence of the GhBGH2 gene is shown as SEQ ID NO: 1, a cotton material bgh2 of which the gene is knocked out is created through a gene editing technology, and under salt stress treatment, the bgh2 shows better salt tolerance compared with a control group, and the root system is more developed. In addition, it is found that bgh2 is insensitive to brassinolide reaction, after BR is externally applied, the root system of a control material is obviously inhibited, and the influence degree of the root system of bgh2 is small. Therefore, the GhBGH2 gene can positively regulate and control the BR signal and negatively regulate and control the salt tolerance of the cotton, enriches and perfects the BR signal transduction pathway of the cotton and the gene resource of salt-tolerant breeding of the cotton, and has important theoretical significance and application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a GhBGH2 gene, a positive regulatory factor of BR signal in upland cotton, and its encoded protein and application. Background Art

[0002] cotton( Gossypium spp.) is one of the world's most important cash crops, playing a crucial role in my country's and the world's economic development. Cotton, an oilseed and cash crop, is a major source of natural fiber, boasting high economic value and widespread cultivation, found in over one-third of the world's countries and regions. Therefore, efforts must be made to increase cotton production, promote comprehensive utilization, and increase production and value, thereby boosting farmers' incomes and meeting the diverse needs of national economic development.

[0003] Different crops have varying tolerances to salt stress. Although cotton is a salt-tolerant crop, high salinity can still severely impact seed germination and plant growth. With drastic environmental changes leading to a continuous reduction in arable land, cotton cultivation is shifting to salinized and arid regions. A plant's salt tolerance varies with its growth stage. Cotton during the germination stage is more sensitive to salt stress, with the presence of salt reducing seed germination rates. High salt stress in the growing cotton plant can lead to reduced evaporation, decreased photosynthesis and water uptake efficiency, and increased respiration. Phenotypically, plant height decreases, leaf growth stops, and stem diameter, bud, and root growth are severely affected. Furthermore, long-term salt stress can lead to delayed fruit development and reduced fiber quality.

[0004] Brassinosteroids (BRs) are a class of sterol hormones widely found in plants. They have been widely reported to have multiple physiological functions, including promoting cell division and elongation, enhancing abiotic stress and disease resistance, and regulating chloroplast development. In response to salt stress, existing studies have confirmed that BRs positively promote plant salt tolerance through multiple pathways. For example, BRs can regulate plant ion balance, promoting the absorption of beneficial ions such as potassium and inhibiting the accumulation of sodium ions, thereby reducing ion toxicity; protect cell membrane structure and function, maintaining fluidity and selective permeability, and preventing salt damage to cell membranes; and inhibit the production of reactive oxygen species (ROS), reducing oxidative damage, and maintaining intracellular redox balance, thereby enhancing plant salt tolerance. In crops such as barley and wheat, exogenous BR treatment significantly improves seed germination and growth under salt stress, further validating the role of BRs in promoting salt tolerance.

[0005] However, the previous study of this invention found that a new salt tolerance related gene in upland cotton GhBGH2The function of Brz-insensitive-pale green homologous is in stark contrast to the above-mentioned known mechanisms. GhBGH2 The gene was down-regulated in both salt-tolerant and salt-intolerant cotton germplasms, and after silencing the gene using VIGS technology, the salt tolerance of the plants was significantly enhanced. bgh2 The mutant showed a better salt tolerance phenotype under salt stress and a more developed root system. More importantly, the study found bgh2 The mutant was significantly insensitive to BR: after exogenous application of BR, the root growth of the control material was significantly inhibited, while bgh2 The root system of the mutant was significantly less affected, and the marker genes related to the BR signaling pathway (such as GhDWF1 、 GhBA1 、 GhCPD ) expression level was significantly downregulated in the mutant. These results indicate that GhBGH2 Gene silencing may enhance cotton salt tolerance by inhibiting the BR signaling pathway. This mechanism is completely contrary to the existing common understanding that "BR promotes salt tolerance", suggesting that it may be a new and unreported salt tolerance regulatory pathway.

[0006] Given that the positive effect of BR on plant salt tolerance has been widely confirmed, the " GhBGH2 The mechanism of "silencing improves salt tolerance by inhibiting the BR signaling pathway" provides a new perspective for analyzing the complex regulatory network of plant salt tolerance and has important theoretical innovation value and application potential. Summary of the Invention

[0007] The purpose of this invention is to break the existing research conclusion that "BR promotes salt tolerance", analyze the new mechanism in the complex regulatory network of cotton salt tolerance, and explore the key genes for cotton salt tolerance. GhBGH2 , clarifying its unconventional pathway of action in improving salt tolerance by inhibiting the BR signaling pathway, adding a new perspective to the theory of plant salt tolerance regulation.

[0008] In order to achieve the above object, the present invention provides the following technical solutions: Upland cotton GhBGH2 Application of genes in positively regulating BR signals in upland cotton, the GhBGH2 The coding sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2.

[0009] In the above application, the positive regulation of upland cotton BR signal is specifically manifested as GhBGH2 After gene knockout, the root development of upland cotton seedlings was promoted.

[0010] In the above application, the positive regulation of upland cotton BR signal is specifically manifested as GhBGH2 After gene knockout, the sensitivity of upland cotton seedling roots to exogenous BR treatment was reduced.

[0011] In the above application, the positive regulation of upland cotton BR signal is specifically manifested as GhBGH2 After gene knockout, the resistance of upland cotton to salt stress was enhanced.

[0012] In the above application, the GhBGH2 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: This study breaks the existing conclusion that "BR promotes salt tolerance" and analyzes the new mechanism in the complex regulatory network of cotton salt tolerance, and explores the key genes for cotton salt tolerance. GhBGH2 , clarifying its unconventional pathway of improving salt tolerance by inhibiting the BR signaling pathway, and providing a new perspective for the theory of plant salt tolerance regulation. GhBGH2 Gene functions and mechanisms provide genetic resources and technical support for the cultivation of salt-tolerant cotton varieties. By regulating this gene, it helps solve the problem of salt stress restricting cotton growth and development, promotes salt-tolerant cotton germplasm innovation, and serves the sustainable development of agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 VIGS-mediated GhBGH2 Silencing and salt stress treatment.

[0015] Figure 2 This is the root phenotype of the mutant materials and the expression analysis of BR-related markers.

[0016] Figure 3 The effects of different concentrations of BR on the mutant bgh2 The influence of the root system.

[0017] Figure 4 These are differentially expressed genes in the roots of Z49 and bgh2.

[0018] Figure 5 GO annotations for differentially expressed genes.

[0019] Figure 6 KEGG annotations for differentially expressed genes. DETAILED DESCRIPTION

[0020] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0021] Description of the sequence listing: SEQ ID NO: 1: atgaacatggatgactcttctgcttcatacatccatatggtgcatcatctttatagaagagtgtttgatattcaacatgagcaaggaggagtgcatggaagctttgtctaagcatgcaagtattaaaccagtcattacttcaacag tttggaatgagttggaaaaagagaacaaggagttctttgaggcttacacaagaaacagagaccaaagagctacagacatggagaaaaggcaaaggatccagttcagggttaatgcatacatgagggagaaaggcaataaggactaa SEQ ID NO: 2: MDDSSASYIHMVHHLIEECLIFNMSKEECMEALSKHASIKPVITSTVWNELEKENKEFFEAYTRNRDQRATDMEKRQRIQFRVNAYMREKGNKD* Example 1 GhBGH2 Functional identification of genes 1. Materials and Methods 1.1 Plant materials The recipient variety of upland cotton is Zhongmian 49 (Z49).

[0022] 1.2 Strains and plasmids The vectors used in this experiment were pCBSG015 and pCAMBIA2301 (stored in our laboratory), the competent E. coli DH5α was purchased from Quanshijin (Beijing) Biological Company, and the Agrobacterium tumefaciens LBA4404 was stored in our laboratory.

[0023] 1.3 Main Reagents The high-fidelity 2× Phanta Max Master Mix used in the experiment was purchased from Nanjing Novozymes Biotechnology Co., Ltd.; KpnⅠ enzyme, SalⅠ enzyme, EcoRⅠ enzyme and T4 ligase were purchased from New England Biolabs, and pEASY-T1 simple cloning vector was purchased from Quanshijin (Beijing) Biotechnology Co., Ltd.

[0024] 1.4 VIGS silencing experiment 1.4.1 Construction of pTRV2-GhBGH2 plasmid The multiple cloning sites of the pTRV2 vector were analyzed, and EcoR I and BamH I were selected as restriction sites to perform enzyme digestion on the pTRV2 body. After enzyme digestion, the enzyme digestion products were purified and recovered after identification. GhBGH2 The VIGS fragments of the gene were ligated using the Uniclone One Step SeamLess Cloning Kit.

[0025] 1.4.2 Injection cotton (1) Activation of strains: Add the Agrobacterium culture containing pTRV2 (empty), pTRV2-GhBGH2, pTRV2-CLA1 and 192 stored at -80 °C to LB liquid culture medium containing 50 μg / mL Kan and 50 μg / mL Rif, and activate the strains in a shaker (28 °C, 200 rpm).

[0026] (2) The activated bacterial solution was aspirated and added to LB liquid medium (this medium contains 10 mM MES pH = 6.5 and 20 μM AS) containing 50 μg / mL Kan and 50 μg / mL Rif. The culture was expanded at 28 °C and 200 rpm until OD600 = 1.2.

[0027] (3) Centrifuge at 5500 rpm for 10 min at 4°C.

[0028] (4) Discard the supernatant and retain the precipitated bacteria, resuspend them in sterile water, and centrifuge and wash them twice.

[0029] (5) Resuspend the Agrobacterium cells in infiltration buffer (containing 10 mM MgCl2, 10 mM MES pH = 5.6, 20 μM AS) to OD600 = 1.2.

[0030] (6) Let stand at room temperature for 3 h.

[0031] (7) Before infection, the pTRV2 (empty), pTRV2-GhBGH2, and pTRV2-CLA1 strain suspensions resuspended in buffer were mixed evenly with the pTRV1 (192) strain suspension in equal volumes.

[0032] (8) Inoculation of Agrobacterium: Use the leaf syringe infiltration method to select cotton seedlings that are in good growth condition and have only two cotyledons. First, use the syringe needle to gently puncture the back of the cotyledon to create a micro-wound, but do not pierce the leaf. Then use a syringe without the needle to inject the bacterial solution from the wound on the back, so that both cotyledons are completely soaked. Plants without treatment serve as controls. The types of plants to be injected are as follows: pTRV1+pTRV2 (empty): negative control for the experiment.

[0033] pTRV1+CLA1: The appearance of an albino phenotype indicates that the VIGS results are reliable: this serves as a positive control for the experiment.

[0034] pTRV1+ pTRV2-GhBGH2: experimental group materials.

[0035] (9) Cultivation of plants after gene silencing: After gene silencing inoculation, the plants were first cultured in the dark at 23 °C for 24 h, and then cultured at 23 °C with a 16 h / 8 h light / dark cycle for 3-4 weeks to observe the phenotype and detect the gene silencing efficiency.

[0036] 1.5 Agrobacterium-mediated genetic transformation of cotton (1) Select mature and plump seeds of the upland cotton variety Zhongmianso 49, remove the shells, soak them in 50% 84 disinfectant for 30 min, and rinse them with sterile water several times to remove the residual 84 disinfectant on the seeds. Incubate the sterilized cotton seeds for 7 days (28°C) to obtain sterile seedlings.

[0037] (2) Activate the Agrobacterium that has been transformed with the target plasmid at -80 °C, expand the culture, collect the bacteria, add 1 / 2 MS solution to resuspend the bacteria, and adjust the OD600 to 0.4-0.5.

[0038] (3) Cut the hypocotyls of sterile seedlings into small segments of 0.5-0.7 cm and place them in the bacterial infection solution and shake for 5 minutes. Transfer the infected hypocotyl segments to sterile filter paper and blow dry them. Then transfer them to the symbiotic culture medium and culture them in the dark for 2 days (28 ℃).

[0039] (4) Transfer the hypocotyl segments to the resistant callus culture medium and continue to subculture for three weeks each round. After 3-4 rounds of culture, induce callus formation and screen for successfully infected resistant calli.

[0040] (5) The resistant calli obtained are transferred to differentiation medium and continue to grow for 4-5 months. After that, they are transferred to embryonic callus induction medium again and subcultured once every 30 days until adventitious buds appear.

[0041] (6) Cut off the adventitious buds and transfer them to rooting medium for cultivation until cotton seedlings are obtained.

[0042] 1.6 Plant stress treatment Simulated plant stress experiments were conducted in a greenhouse. Plants at the 3-4 true leaf stage were divided into two groups: a control group (irrigated with 2 L of water every four days) and a salt (NaCl)-treated group (irrigated with 2 L of 400 mM NaCl solution every four days). Each group included two strains, the wild-type and the bgh2 mutant, with 12 seedlings per strain. Phenotypic photographs and physiological parameters were measured after one month of continuous treatment.

[0043] 1.7 Plant BR hormone treatment Seedling culture media with varying BR concentration gradients were prepared: 0.05, 0.1, 0.5, and 1 mg / L. Mature, plump seeds from the cultivar Zhongmianso 49 were shelled and soaked in 3.5% H₂O₂ for 8 hours. Residual H₂O₂ was then rinsed multiple times with sterile water. Sterilized cotton seeds were then planted in the culture media containing varying BR concentration gradients and incubated in the dark at 28°C for 7 days. Root growth of the sterilized cotton seedlings was observed, and root length and weight were measured using a ruler and a micrometer scale.

[0044] 1.8 Antioxidant enzyme activity assay First, prepare the plant tissue to be tested into a tissue homogenate. Accurately weigh the plant tissue and add phosphate buffer (0.1 mol / L, pH = 7.0-7.4) at a ratio of weight (g) / volume (mL) = 1 / 9. Mechanically homogenize in an ice-water bath to prepare a 10% plant tissue homogenate. Centrifuge for 10 minutes (3500 rpm) and collect the supernatant for subsequent determination.

[0045] 1.8.1 SOD enzyme activity determination The xanthine and xanthine oxidase reaction system produces superoxide anion free radicals (O2 - ), which oxidizes hydroxylamine to form nitrite, which develops a purple-red color under the action of a color developer. Its absorbance is measured using a visible light spectrophotometer. When the sample being tested contains SOD, it specifically inhibits superoxide anion free radicals, reducing the formation of nitrite. During colorimetry, the absorbance of the test tube is lower than that of the control tube. The SOD activity in the sample being tested can be calculated using a formula.

[0046]

[0047] Vortex to mix thoroughly and incubate in a water bath for 40 min (37°C).

[0048] Add 2 mL of color developer to the test tube and control tube respectively, mix well, and place at room temperature for 10 minutes. At 550 nm, use distilled water to adjust the light path to zero in a 1 cm cuvette, and measure the OD value of each tube sample.

[0049] Calculate the sample protein concentration using the following formula.

[0050]

[0051] The total activity is the SOD amount corresponding to the SOD inhibition rate of 50% per milligram of tissue protein in 1 mL of reaction solution, which is one SOD activity unit (U).

[0052] 1.8.2 CAT enzyme activity determination The reaction of catalase decomposing H2O2 can be quickly terminated by adding ammonium molybdate. The remaining H2O2 reacts with ammonium molybdate to produce a light yellow complex. The change in the complex is measured at 405nm, and the activity of CAT can be calculated.

[0053] Preheat reagents 1 and 2 at 37°C

[0054] Mix thoroughly, and measure the OD value of each tube of sample at 405 nm using a 0.5 cm optical path cuvette, adjusted to zero with distilled water.

[0055]

[0056] Note: * 271 is the reciprocal of the slope 2. Results Analysis 2.1 VIGS-mediated GhBGH2 silencing and salt stress treatment Based on the VIGS-mediated gene silencing experiment, a 200 mM salt stress experiment was conducted 7 days after injection, and after 12 days of treatment. The expression level was detected by RT-qPCR, and the expression level of GhBGH2 in TRV::GhBGH2 plants was significantly reduced. TRV::00 plants showed a more severe wilting phenotype in TRV::GhBGH2, and the leaves were obviously wrinkled. The determination of relevant physiological indicators found that the relative water content, SOD and CAT of TRV::GhBGH2 plants were also significantly higher than those of TRV::00. In addition, H2O2 showed that they had better salt tolerance ( Figure 1 ).

[0057] 2.2 Root phenotypes of mutant materials and expression analysis of BR-related markers We observed bgh2 The root growth of the knockout material and the control was measured and the results showed that bgh2 The root system is more developed than the control. In addition, we also measured the expression of BR-related marker genes (GhDWF1, GhBA1, GhCPD), and found that the expression levels of the three BR-related marker genes were significantly downregulated in the knockout materials ( Figure 2 ), the results showed that GhBGH2 It may affect root development through the BR pathway.

[0058] 2.3 Effects of different concentrations of BR on mutants bgh2 Impact of roots To explore bgh2 Whether it is involved in the BR pathway, Z49 and bgh2 Different concentrations of BR were applied to the mutant materials. The results showed that the roots of Z49 and bgh2 were shortened. Compared with the control Z49, the mutant bgh2 The root system of the plant was shortened less and the root weight was reduced less. This shows that after applying BR, bgh2 Insensitive to BR ( Figure 3 These results again show that GhBGH2 It may affect root development through the BR pathway.

[0059] 2.4 Transcriptome analysis reveals the mechanism of GhBGH2 response to BR Differentially expressed genes (DEGs) were identified using DESeq2 with |Fold Change| ≥1. The obtained P values ​​were adjusted using the method of Benjamini and Hochberg. DESeq2 adjusted genes with P-value < 0.05 to differential expression, and eggNOG mapper v2 was used for gene annotation. Z49 was compared with bgh2-0, bgh2-2, and bgh2-3, and 11508, 20696, and 21482 DEGs were obtained, respectively. After screening, Veen analysis was performed to obtain 7269 differentially expressed genes in the three groups, which were subjected to KEGG and GO analysis. The clusterProfiler R package was used to perform gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on DEGs ( Figure 4 ).

[0060] GO enrichment analysis of DEGs showed that DEGs were enriched in multiple GO terms, mainly in biological process, cellular component, and molecular function. We found that a large number of hormone-related pathways were significantly enriched, mainly involving auxin (response to auxin, auxin-activated signaling pathway, auxin transport, regulation of auxin mediatedsignaling pathway, and auxin transmembranetransporter activity), jasmonic acid (response to jasmonic acid and regulation of jasmonic acid mediated signaling pathway), and brassinosteroid (response to brassinosteroid, brassinosteroid metabolic process, and brassinosteroid homeostasis). In addition, some pathways related to cell division (plant-type cell wall and cell division) and root growth (lateral root development) were also significantly enriched. The results suggest that bgh2 may affect root development by mediating the division of growing point cells through a complex hormone regulatory network. Figure 5 ).

[0061] To identify the pathways in which DEGs may be involved, we performed pathway analysis using the KEGG database. The formula used to determine the number of DEGs in each pathway was the same as that used in the GO analysis. KEGG enrichment analysis also showed that the hormone-related pathway (Plant hormone signal transduction) was significantly enriched. In addition, pathways involving photosynthesis (Photosynthesis, Photosynthesis - antenna proteins) and some amino acids (Cysteine ​​and methionine metabolism, Tryptophan metabolism and Glutathione metabolism) and secondary metabolites (Glucosinolate biosynthesis and Cutin, suberine and wax biosynthesis) were also found. Figure 6 ).

Claims

1. Upland cotton GhBGH2 The application of the gene in positively regulating BR signal of upland cotton is characterized in that: described GhBGH2 The coding sequence of the gene is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The positive regulation of BR signal of upland cotton is specifically manifested as follows: GhBGH2 After gene knockout, the root development of upland cotton seedlings was promoted.

3. The use according to claim 1, characterized in that The positive regulation of BR signal of upland cotton is specifically manifested as follows: GhBGH2 After gene knockout, the sensitivity of upland cotton seedling roots to exogenous BR treatment was reduced.

4. The use according to claim 1, characterized in that The positive regulation of BR signal of upland cotton is specifically manifested as follows: GhBGH2 After gene knockout, the resistance of upland cotton to salt stress was enhanced.

5. The use according to claim 1, characterized in that described GhBGH2 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2.

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