Sycmyb6 and application thereof in regulating drought stress tolerance of plants

Abstract

The application belongs to the technical field of biological breeding, and specifically discloses a branch fungus transcription factor SycMYB6 and application thereof in regulating drought stress tolerance of plants, and specifically provides application of overexpressed transcription factor SycMYB6 or a protein coded by the transcription factor SycMYB6 in breeding drought-resistant crop germplasm, and a nucleotide sequence of the transcription factor SycMYB6 is shown as SEQ ID NO. 1. The application can improve drought resistance of crops and breed excellent germplasm resources by overexpressing the transcription factor SycMYB6 gene in crops through genetic engineering, and compared with traditional breeding, the technology has short cycle, clear target, is not limited by intergeneric cross, and greatly improves the efficiency of breeding drought-resistant crop germplasm.

Description

Technical Field

[0001] This invention belongs to the field of biological breeding technology, specifically relating to a mycorrhizal transcription factor SycMYB6 and its application in regulating plant drought stress tolerance. Background Technology

[0002] With the intensification of global climate change, drought has become one of the major abiotic stress factors restricting agricultural production. Drought leads to reduced crop yields and seriously threatens crop safety. Traditional drought-resistant crop breeding has a long cycle and low efficiency, making it difficult to quickly respond to frequent extreme drought events. In recent years, with the rapid development of molecular biology and genetic engineering technologies, discovering and regulating key drought-resistant genes has become an effective strategy to improve crop drought resistance.

[0003] Plants accumulate large amounts of reactive oxygen species (ROS) under drought stress. The main forms of ROS within cells are hydrogen peroxide (H2O2) and superoxide (O2). .- Monoclinic oxygen ( 1O2), hydroxyl radicals (HO·), and various forms of organic and inorganic peroxides. When ROS concentrations are too high, they cause oxidative damage to cell membranes (lipid peroxidation), proteins, RNA, and DNA molecules, and can even lead to oxidative destruction of cells; this process is called oxidative stress. Maintaining low ROS levels is achieved by regulating ROS production, scavenging, and transport. The accumulation of reactive oxygen species affects the redox state of various proteins, including enzymes, receptors, and small molecules. Furthermore, it alters gene expression and enhances plant recovery from stress by activating, modifying, or integrating multiple stress-responsive signaling pathways. Numerous ROS detoxification proteins involved in this process include superoxide dismutase (SOD), peroxidase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX), and peroxide reductase (PRX). During abiotic stress, the two main sources of ROS include ROS generated due to metabolic disturbances and ROS generated through signal transduction. Metabolic reactive oxygen species (ROS) can directly alter the redox state of rate-limiting enzymes and control intracellular metabolic flux, thereby affecting transcription and translation. They can be mediated by signaling ROS generated through phosphorylation activation of NADPH oxidase (RBOH) on the plasma membrane (PM). Complex interactions also exist between plant hormones and signaling molecules NO and ROS. ROS influence the levels and functions of different plant hormones, and plant hormones induce the production of different types of ROS in plants. Different levels of ROS in different cellular compartments collectively constitute overall ROS homeostasis. ROS oxidize proteins to alter protein structure and function, thereby regulating the binding of transcription factors (TFs) to DNA and affecting transcription. TFs can also act as upstream signaling genes, influencing the expression of enzymes related to ROS production, translocation, and clearance, thus regulating ROS homeostasis. Therefore, identifying highly efficient transcription factors can provide new solutions for plant stress resistance regulation and offer a theoretical basis for plant stress resistance control. Summary of the Invention

[0004] This invention aims to provide a mycorrhizal transcription factor, SycMYB6, and its application in cultivating drought-resistant crop germplasm. Specifically, this invention provides the following technical solution:

[0005] On the one hand, the present invention provides the application of overexpression of transcription factor SycMYB6, or the protein encoded thereon, in the cultivation of drought-resistant crop germplasm.

[0006] In another aspect, the present invention provides a transcription factor SycMYB6, the full-length CDS sequence of which is shown in SEQ ID NO.1.

[0007] In another aspect, the present invention provides a protein encoded by the transcription factor SycMYB6, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] In another aspect, the present invention provides a recombinant vector, wherein the recombinant vector is linked with a sequence expressing the protein shown in SEQ ID NO.2.

[0009] In another aspect, the present invention provides a delivery vector comprising Agrobacterium, the delivery vector delivering the recombinant vector of claim 4.

[0010] In another aspect, the present invention provides primers for amplifying the transcription factor SycMYB6, the primer sequences of which are shown in SEQ ID NO.3 and / or SEQ ID NO.4.

[0011] In another aspect, the present invention provides a reagent kit, characterized in that it contains the aforementioned primers.

[0012] In another aspect, the present invention provides the application of the transcription factor SycMYB6, the protein, the recombinant vector, the delivery vector, the primers, or the kit in the cultivation of drought-resistant crop germplasm.

[0013] In another aspect, the present invention provides a method for improving crop drought resistance by using genetic engineering techniques to increase the expression level of transcription factor SycMYB6 or its encoded protein. The nucleotide sequence of transcription factor SycMYB6 is shown in SEQ ID NO.1.

[0014] Furthermore, the crop described in this invention includes *Nicotiana benthamiana*.

[0015] The technical effects achieved by this invention are as follows:

[0016] This invention identified a transcription factor named SycMYB6 from Cladosporium, and overexpressed the SycMYB6 gene in Nicotiana benthamiana to investigate its effect on drought resistance in tobacco. A SycMYB6 plant expression vector was constructed and transformed into tobacco, and RT-qPCR was used to detect the gene expression level of SycMYB6. Drought stress was applied to the SycMYB6 transgenic tobacco, and the activities of related antioxidant enzymes were measured. The full-length CDS sequence of the SycMYB6 gene is 1911 bp (SEQ ID NO.1), encoding 636 amino acids (SEQ ID NO.2). Transgenic tobacco overexpressing SycMYB6 was obtained through genetic transformation. Analysis of drought resistance phenotype and antioxidant enzyme activity showed that the drought resistance of SycMYB6-overexpressing plants was significantly enhanced compared to wild-type tobacco. SycMYB6 positively regulates the tolerance of tobacco to drought stress. Heterologous overexpression of SycMYB6 in tobacco improved the drought resistance of transgenic tobacco.

[0017] This invention uses genetic engineering to overexpress the transcription factor SycMYB6 gene in crops, which can improve crop drought resistance and cultivate superior germplasm resources. Compared with traditional breeding, this technology has a shorter cycle, clearer objectives, and is not limited by interspecific hybridization, which greatly improves the efficiency of cultivating drought-resistant crop germplasm. Attached Figure Description

[0018] Figure 1 .Diagram of SycMYB6 plant overexpression vector construction;

[0019] Figure 2 Identification diagram of SycMYB6 transgenic tobacco; A: PCR detection of SycMYB6 transgenic tobacco genomic DNA; B: RT-qPCR detection of SycMYB6 transgenic tobacco; M: DL1000 molecular weight marker; +: positive control; –: negative control; H2O: water control; 1‒3: SycMYB6 transgenic tobacco; OE: SycMYB6 transgenic tobacco. Note: *: P<0.05; **: P<0.01.

[0020] Figure 3 Comparison of growth phenotypes of SycMYB6 overexpression (OE) and wild-type (WT) plants at 1–5 weeks.

[0021] Figure 4 Comparison of SycMYB6 overexpression (OE) and wild-type (WT) plants after 14 days of drought treatment; A is a comparison of growth phenotypes, and B is a comparison of SycMYB6 expression levels.

[0022] Figure 5Statistical graphs of POD, CAT, and SOD activities in wild-type and transgenic tobacco under drought stress; A is the POD statistical graph; B is the CAT statistical graph; C is the SOD statistical graph; Note *: P<0.05; **: P<0.01. Detailed Implementation

[0023] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. For the testing methods, purchased goods, unless otherwise specified, shall be used under conventional conditions or conditions recommended by the manufacturer. Unless otherwise defined herein, the scientific and technical terms used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, but similar or equivalent methods and materials described herein may also be used in the practice and testing of this disclosure.

[0024] The full-length CDS sequence of the transcription factor SycMYB6 gene provided by this invention is 1911 bp, as shown in SEQ ID NO.1, and the sequence encoding 636 amino acids is shown in SEQ ID NO.2.

[0025] Example 1

[0026] 1. Materials and Methods

[0027] 1.1 Test Materials

[0028] Tobacco materials: Wild-type Nicotiana benthamiana was used as the recipient material to construct a SycMYB6 gene overexpression line. Bacterial strains: Escherichia coli competent cells (DH5α), yeast competent cells (Y2HGold), and Agrobacterium tumefaciens competent cells (GV3101). Vector ( Figure 1 ): Overexpression vector (pBWA(V)BS-MYB6-linker-osgfp), subcellular localization vector (PGreenII 62-SK-GFP). Reagents: High-fidelity enzyme (2×PhantaMaxMasterMix), plant DNA extraction kit, total RNA extraction kit, reverse transcription kit, and real-time PCR reagent were provided by Novizan Biosciences Co., Ltd. (Nanjing). Antioxidant enzyme activity assay kit was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0029] 1.2 Methods

[0030] 1.2.1 Construction of SycMYB6 gene overexpression vector in plants

[0031] Cloning primers were designed based on the coding sequence (CDS) of the SycMYB6 gene (see Table 1). Using SycMYB6 cDNA as a template, PCR amplification was performed using a high-fidelity enzyme, 2×PhantaMax Master Mix. The SycMYB6 (1911bp) electrophoresis product was subjected to UV-guided digestion and gel recovery after 1% agarose gel electrophoresis at 5 V / cm for 20 minutes. The recovered DNA product was resuspended in 40 μL of double-distilled water (labeled as rDNAM1). After verification, recombination with a vector was performed. The amplified fragment was digested and recovered from the gel, then ligated into the corresponding vector via homologous recombination. The vector digest was purified using a PCR purification kit, and the purified product was labeled as pBWA(V)BS-ccdb-linker-osgfp for recombination. 10 μL of the ligation product was transformed into competent E. coli cells, plated on Kans resistant plates, and incubated at 37°C for 12 hours. Single clones were picked and cultured in 1.5 ml EP tubes for PCR identification. The bacterial cultures with positive bands were selected for sequencing, and the remaining bacterial cultures with positive bands were transferred to 10 ml LB medium. Plasmids were extracted from the selected strains with correct sequencing results for genetic transformation.

[0032] Table 1. Primers used for SycMYB6 gene cloning, subcellular localization, and functional analysis

[0033]

[0034] 1.2.2 Analysis of SycMYB6 genetically transformed tobacco and its identification

[0035] Add 1 μL of plasmid to 50 μL of HGV3101 Agrobacterium competent cell suspension, mix thoroughly, transfer to an electroporation cuvette for electroporation transformation, resuspend in 1 mL LB liquid medium, vortex to mix, and transfer to a 1.5 mL centrifuge tube. Incubate at 180 rpm and 30°C for 30 minutes on a shaker. Inoculate 50 μL of the activated Agrobacterium culture onto LB solid medium and incubate at 30°C in darkness for two days and nights. Synthesize the corresponding detection primers, prepare the PCR amplification system, and perform amplification. Observe the PCR results on a gel. When both the positive control and the detection sample show clear bands with matching molecular weights, and no band is produced in the negative control group, Agrobacterium infection is performed.

[0036] Take a sample from the prepared Agrobacterium suspension and prepare OD. 600Agrobacterium suspension in the range of 0.8-1.2 was diluted with Silwet-77 to a final concentration of 0.02%, and the entire inflorescence of the Arabidopsis plant was submerged in the bacterial solution for 2 to 3 seconds. The plant was then sealed to maintain humidity above 90% and incubated in the dark at 25°C for 24 hours. The incubation process was repeated every 7 days for a total of 3 times. After incubation, seedlings were cultured at 23°C under 16 hours of light and 8 hours of darkness until seed production. Mature pods were dried at 37°C for 24 hours, then sieved through a 60-mesh sieve. The clean seeds were stored at 4°C. Sterilize the seeds in 95% ethanol for 10 min, then disinfect them with 75% ethanol for 10 min. Wash them 2-3 times with sterile water for 1 minute each time. Spread them evenly on a selection medium containing kanamycin resistance and refrigerate them at 4℃ for 2-3 days. Place the plates in a constant temperature incubator at 23-25℃ and culture them for 10-14 days with a 16-hour light-8-hour dark cycle. After screening, the surviving seedlings are transplanted to nutrient soil and cultured at 23℃ with alternating 16-hour light-8-hour dark cycles. When the seedlings are about 20 days old, extract genomic DNA using the CTAB method and perform PCR detection.

[0037] 1.2.3 Real-time quantitative PCR of SycMYB6 gene

[0038] Healthy transgenic and wild-type tobacco plants with consistent growth conditions were selected, and leaf tissue samples were taken and immediately flash-frozen in liquid nitrogen. Total RNA was extracted from the samples, and cDNA was reverse transcribed using a reverse transcription reagent. Actin-F and Actin-R primers were used as internal reference genes for quantitative real-time qPCR analysis. The obtained data were analyzed using a 2... -ΔΔCT The method is used for processing.

[0039] 1.2.4 Phenotypic observation and physiological and biochemical index determination of transgenic tobacco overexpressing SycMYB6

[0040] Wild-type and transgenic tobacco seeds were sown on a culture medium and cultured for two weeks. Afterward, they were transferred to a mixed substrate of vermiculite and nutrient soil at a volume ratio of 1:2. Under suitable conditions, they were cultured for one month, during which the differences in growth phenotypes between wild-type and transgenic tobacco were observed and recorded regularly, and plant morphology photographs were collected simultaneously. One-month-old seedlings of both lines were selected for a short-term drought stress experiment. This treatment is suitable for rapidly assessing the effects of drought on seedling physiological indicators and studying the plant's adaptability to mild stress. The total drought stress period was set at 7 days, with time points at 3d, 5d, and 7d. After the treatment, the samples were pretreated according to the instructions of the Novizan antioxidant enzyme activity assay kit. The absorbance values ​​were measured using a microplate reader, and the activity parameters of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were calculated using the formulas provided in the kit.

[0041] 1.2.5 Data Statistics and Analysis

[0042] One-way ANOVA was performed using SPSS 21.0 software (IBM, Chicago, IL, USA). All data were obtained after three repeated trials, and the standard deviation (SD) was measured separately. Statistically significant differences were considered to be (* p ≤ 0.05, ** p ≤ 0.01).

[0043] 2 Results and Analysis

[0044] 2.1 Construction of SycMYB6 gene overexpression vector

[0045] Electrophoresis was performed on a 1% agarose gel at 5 V / cm for 20 minutes. The 1911 bp SycMYB6 fragment was excised under UV light. The excised gel fragments were placed in the same reaction system, and sol-gel recovery was performed according to the kit instructions. The DNA was finally recovered by dissolving it in 40 μL of sterile water. The product was labeled as rDNAM1 and was used after passing the test. The vector digestion product was purified using a PCR purification kit. The purified product was labeled as pBWA (V) BS-ccdb-linker-osgfp (D) and used for subsequent recombination reactions. rDNAM1 and pBWA (V) BS-ccdb-linker-osgfp (D) were mixed in a certain ratio for ligation. 10 μL of the ligation product was transformed into E. coli competent cells (following the standard procedure for E. coli competent cell transformation). The transformation product was plated on LB solid medium plates containing Kan resistance and incubated at 37°C for 12 hours. Several single colonies were randomly selected and cultured in 1.5 mL EP tubes for liquid culture and PCR identification. A small amount of the PCR-positive bacterial culture was sent to a biotechnology company for sequencing. The remaining bacterial culture was inoculated into 10 mL of Kan-resistant LB broth and cultured on a shaker. After receiving the sequencing results, the correctly sequenced strains were selected, plasmids were extracted, and the strain and plasmid were stored long-term.

[0046] 2.2 Analysis of SycMYB6 genetically transformed tobacco and its identification

[0047] The constructed overexpression vector pBWA(V)BS-SycMYB6 was transformed into Nicotiana benthamiana to obtain T0 generation transgenic tobacco seeds. After kanamycin selection, resistant plants were obtained. PCR detection showed that all tobacco plants overexpressing SycMYB6 contained the SycMYB6 target gene fragment. The band size of the target fragment matched that of the positive control wells, and no band was observed in the negative control and water control lanes. Single plants that amplified the target band were selected. Figure 2 (A) was identified as a positive transgenic plant. Positive T3 generation transgenic plants were screened using the same method. Leaves from independent transgenic plants and WT plants were subjected to qPCR detection, and the results showed ( Figure 2 Compared with WT plants, the expression level of SycMYB6 in transgenic plants was higher than that in wild-type plants, further proving that the SycMYB6 sequence has been successfully integrated into the tobacco genome and overexpressed.

[0048] 2.3 Phenotypic and physiological index analysis of transgenic tobacco plants

[0049] Wild-type Nicotiana benthamiana seeds and genetically modified Nicotiana seeds were simultaneously vernalized and sown, and... Figure 3 It was found that overexpressing plants in *Nicotiana benthamiana* exhibited significantly higher leaf length, leaf width, and plant height than wild-type plants during the seedling stage. Furthermore, during the growth process, overexpressing plants sown and transplanted simultaneously showed significantly faster growth rates and larger plant sizes than wild-type plants. These results indicate that the SycMYB6 transcription factor has a significant positive impact on the seedling growth of *Nicotiana benthamiana* plants.

[0050] Drought treatment was applied to T3 generation tobacco plants overexpressing SycMYB6 until the plants wilted. The phenotypes of the two plants were observed and the expression level of SycMYB6 was measured. Figure 4 As can be seen from Figure A, the wilting degree of wild-type tobacco after drought stress was significantly more severe than that of the overexpression-type tobacco, suggesting that the overexpression-type tobacco has stronger drought resistance than the wild-type tobacco. The activities of CAT, SOD, and POD in the plants were then measured. The results of antioxidant enzyme activity detection showed that under drought stress, the activities of all three antioxidant enzymes in the transgenic lines were increased and significantly higher than those in the WT plants. Figure 5 This indicates that heterologous overexpression of SycMYB6 in tobacco promotes the increase of antioxidant enzyme activity in transgenic tobacco under drought stress, enhances the reactive oxygen species scavenging capacity of transgenic tobacco plants under drought stress, reduces the accumulation of peroxides, and thus improves the drought resistance of tobacco.

[0051] This invention demonstrates that heterologous overexpression of SycMYB6 in transgenic tobacco significantly enhances the plant's drought resistance. This phenomenon is closely related to the protein's structural characteristics and subcellular localization. Its conserved SANT domain is a typical DNA-binding region of the MYB family, while its nuclear localization provides the structural basis for its transcriptional regulatory function. Further analysis revealed multiple cis-regulatory elements associated with abiotic stress in the SycMYB6 promoter, suggesting that its expression may be directly regulated by environmental signals such as drought. Under drought stress, the activities of antioxidant enzymes (SOD, POD, CAT) in SycMYB6-overexpressing plants were significantly increased. This result indicates that this transcription factor may alleviate membrane lipid peroxidation damage by activating the antioxidant system to scavenge excess reactive oxygen species (ROS). This regulatory pattern is consistent with common stress-resistance gene pathways: when plants encounter drought, transcription factors maintain ROS homeostasis by initiating the expression of antioxidant enzyme genes, thereby enhancing cellular tolerance. This invention provides new evidence for the function of fungal-derived MYB transcription factors in plant stress resistance. Under drought stress, heterologous overexpression of SycMYB6 in transgenic tobacco promotes the accumulation of osmotic regulatory substances, increases the activity of antioxidant enzymes, reduces cell damage in transgenic tobacco, and enhances the drought resistance of transgenic tobacco by regulating the expression of stress-related genes. This demonstrates that SycMYB6 participates in the plant's response to abiotic stress.

Claims

1. The application of overexpression of transcription factor SycMYB6, or its encoded protein, in the breeding of drought-resistant crop germplasm, characterized in that, The nucleotide sequence of transcription factor SycMYB6 is shown in SEQ ID NO.

1.

2. Transcription factor SycMYB6, characterized in that, The sequence of transcription factor SycMYB6 is shown in SEQ ID NO.

1.

3. A protein encoded by the transcription factor SycMYB6, characterized in that, The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

4. A recombinant vector, characterized in that, The recombinant vector is linked to a sequence expressing the protein shown in SEQ ID NO.

2.

5. A delivery vector comprising Agrobacterium, characterized in that, The delivery carrier delivers the recombinant carrier of claim 4.

6. A primer for amplifying the transcription factor SycMYB6 of claim 2, characterized in that, The primer sequences are shown in SEQ ID NO.3 and / or SEQ ID NO.

4.

7. A reagent kit, characterized in that, Contains the primers as described in claim 6.

8. The use of the transcription factor SycMYB6 of claim 2, the protein of claim 3, the recombinant vector of claim 4, the delivery vector of claim 5, the primer of claim 6, or the kit of claim 7 in the cultivation of drought-resistant crop germplasm.

9. A method for improving crop drought resistance, characterized in that, The expression level of transcription factor SycMYB6 or its encoded protein was increased by means of genetic engineering technology. The full-length CDS sequence of transcription factor SycMYB6 is shown in SEQ ID NO.

1.

10. The application according to claim 1 or claim 2, characterized in that, The crop includes tobacco.

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