Application of SbMYB113-like gene in improving low-temperature-resistant breeding of scutellaria baicalensis

Abstract

The invention discloses application of a SbMYB113-like gene to improvement of low-temperature-resistant breeding of scutellaria baicalensis, belongs to the technical field of plant biology, is used for scutellaria baicalensis, and is characterized in that the nucleotide sequence of the SbMYB113-like gene is shown as SEQ ID NO: 1, by reducing overexpression of the SbMYB113-like gene in the scutellaria baicalensis, the anti-oxidation defense system of the scutellaria baicalensis is improved, the stability of the cell structure and function is protected, and the stability of the low-temperature-resistant breeding of the scutellaria baicalensis is improved. The tolerance of the scutellaria baicalensis to low-temperature stress is improved. The invention aims at researching subcellular localization of SbMYB113-like protein, analyzing tissue specific expression and a low-temperature stress response expression mode of the SbMYB113-like gene in combination with real-time fluorescent quantitative PCR (Polymerase Chain Reaction), and verifying the cold resistance function of SbMYB113-like through genetic transformation, so that the SbMYB113-like gene has an application value in low-temperature scutellaria baicalensis breeding.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant biotechnology, and particularly relates to application of SbMYB113-like gene in improving low-temperature tolerance breeding of Scutellaria baicalensis Georgi. BACKGROUND

[0002] Temperature is a key environmental factor affecting plant growth and geographical distribution, and seriously restricts the yield and quality of crops. Cold stress is a ubiquitous abiotic stress factor, which seriously restricts the growth and development of plants. For perennial medicinal materials, cold resistance is the basis for crossing winter and realizing continuous growth. Certain cold stress treatment can make plants accumulate more secondary metabolites and improve the quality of medicinal plants, while excessive cold stress can hinder the accumulation of secondary metabolites, leading to inhibition or degradation of effective components, and ultimately reduce the quality of medicinal materials.

[0003] Scutellaria baicalensis Georgi, as a perennial medicinal material, is one of the local and major medicinal materials in Chengde, mainly distributed in the Yanshan mountainous and hilly areas of Chengde. The temperature difference between day and night is large, and the spring temperature is low, so the plants often suffer from cold damage during cultivation, which seriously restricts the growth of Scutellaria baicalensis Georgi.

[0004] In recent years, with the development of molecular biology technology, research teams have begun to focus on the stress resistance mechanism of Scutellaria baicalensis Georgi, such as the changes in physiological and biochemical indicators of Scutellaria baicalensis Georgi under drought and salt stress. It is found that salt stress has multiple effects on the physiological characteristics of Scutellaria baicalensis Georgi seedlings. Low concentration of salt stress can promote seed germination, while high concentration can inhibit seed germination. Scutellaria baicalensis Georgi can respond to heavy metal copper stress through different ways of key enzyme genes PAL and ICS in SA synthesis pathway. Under severe progressive drought stress, the photosynthetic response of Scutellaria baicalensis Georgi is inhibited, the osmotic adjustment substances increase, and the synergistic effect of soil water and antioxidant enzymes is stronger, which is more conducive to the accumulation of baicalein in Scutellaria baicalensis Georgi. Water stress leads to changes in the physiology of Scutellaria baicalensis Georgi, and rehydration can effectively reduce the physiological damage caused by water stress. However, the research on the cold resistance of Scutellaria baicalensis Georgi is relatively scarce. SUMMARY

[0005] The purpose of the present application is to provide application of SbMYB113-like gene in improving low-temperature tolerance breeding of Scutellaria baicalensis Georgi, which aims to clone SbMYB113-like gene through homologous alignment and conduct bioinformatics analysis, study the subcellular localization of SbMYB113-like protein, analyze the tissue-specific expression and low-temperature stress response expression pattern of SbMYB113-like gene through real-time fluorescent quantitative PCR, and verify the cold resistance function of SbMYB113-like through genetic transformation, so as to make it have application value in low-temperature Scutellaria baicalensis Georgi breeding.

[0006] To achieve the above object, the application is realized by the following technical scheme:

[0007] The application discloses an application of the SbMYB113-like gene in improving the low-temperature tolerance of Scutellaria baicalensis Georgi, and the nucleotide sequence of the SbMYB113-like gene is shown as SEQ ID NO: 1. The SbMYB113-like gene is used to reduce the overexpression of the SbMYB113-like gene in the Scutellaria baicalensis Georgi, improve the antioxidant defense system of the Scutellaria baicalensis Georgi, protect the stability of cell structure and function, and improve the low-temperature tolerance of the Scutellaria baicalensis Georgi.

[0008] The cDNA sequence is genetically transformed into plants by using Agrobacterium GV3101.

[0009] The plant in the application is Nicotiana benthamiana.

[0010] The process of genetically transforming the cDNA sequence into plants by using Agrobacterium GV3101 is as follows: the SbMYB113-like gene is constructed into a plant expression vector and introduced into plant cells, subcellular localization homologous recombination primers are designed according to the SbMYB113-like gene sequence, the sequence is amplified, and the target sequence is constructed into a pYBA-1132 vector, Agrobacterium GV3101 containing the recombinant vector plasmid 35S::SbMYB113-like-GFP is resuspended in an infiltration buffer, then injected into the lower epidermis of the plant, and the plant is placed in a plant culture medium room with a temperature of 22±2 DEG C and a light cycle of 16 hours of light / 8 hours of darkness for 48 hours.

[0011] The cDNA sequence of the Scutellaria baicalensis Georgi SbMYB113-like gene is one of the following nucleotide sequences: 1) the sequence shown in SEQ ID NO: 1; and 2) a DNA sequence hybridized with the cDNA sequence of SEQ ID NO: 1 and having the SbMYB113-like transcription factor activity protein.

[0012] The primer for amplifying the cDNA sequence of the Scutellaria baicalensis Georgi SbMYB113-like gene has the sequence shown in SEQ ID NO: 2 to SEQ ID NO: 9.

[0013] A recombinant expression vector, wherein the vector adopts a pYBA-1132 vector and comprises the SbMYB113-like gene.

[0014] The SbMYB113-like protein has the molecular formula of C 428 H 691 N 137 O 119S7, with a relative molecular mass of 9.88 kD, a theoretical isoelectric point of 9.59, an instability coefficient of 30.78, and a total hydrophilicity coefficient of -0.964.

[0015] The SbMYB113-like protein described in the present application is located on the cell membrane.

[0016] Compared with the prior art, the present application has the beneficial effects that:

[0017] In the present application, the SbMYB113-like gene is cloned by homologous alignment and subjected to bioinformatics analysis, and the cold resistance function of the SbMYB113-like gene is verified by genetic transformation, filling the blank of its application in low-temperature Scutellaria baicalensis breeding. The transgenic plants exhibit higher membrane lipid peroxidation level and lower antioxidant enzyme activity (SOD, CAT, POD), and overexpression of the SbMYB113-like gene significantly reduces the tolerance of the plants to low-temperature stress, providing a new gene target for the study of Scutellaria baicalensis resistance breeding. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is the localization and domain analysis of the SbMYB113-like gene.

[0019] Figure 2 is the phylogenetic tree of the SbMYB113-like protein.

[0020] Figure 3 is the physicochemical property analysis of the SbMYB113-like protein.

[0021] Figure 4 is the transcription factor binding site and cis-acting element analysis of the SbMYB113-like gene.

[0022] Figure 5 is the expression analysis of the SbMYB113-like gene.

[0023] Figure 6 is the subcellular localization of the 35S::SbMYB113-like-GFP fusion protein in tobacco leaves (from left to right are bright field, green fluorescence, and superimposed field; scale = 20 µm).

[0024] Figure 7 is the effect of low-temperature stress on the phenotype and physiological indicators of transgenic tobacco. DETAILED DESCRIPTION

[0025] In the following examples, the experimental methods used are conventional experimental methods unless otherwise specified, and the materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified.

[0026] The technical solutions of the present application will be described in detail below with reference to the accompanying drawings.

[0027] I. Test materials, main reagents and instruments and equipment

[0028] 1.1. Materials

[0029] The Scutellaria baicalensis Georgi material used in the present application is Jiqin No. 1, which is planted in an artificial climate incubator (25 ℃ / 22 ℃, light cycle 16 h / 8 h, relative humidity 60%) of the Institute of Traditional Chinese Medicine of Chengde Medical College. After growing for 30 days, seedlings with consistent growth vigor are selected for 4 ℃ low temperature treatment. After 0, 3, 6, 12 and 24 h of treatment, the root tips and leaves are cut and rapidly frozen in liquid nitrogen. The remaining plants are cultured under normal conditions until flowering, and samples of different tissue parts are collected. All samples are stored at -80 ℃ for qRT-PCR detection.

[0030] The Nicotiana benthamiana L. used in the present application is also planted in the above incubator.

[0031] 1.2. Main reagents and instruments and equipment

[0032] Hi-Pure polysaccharide and polyphenol plant total RNA extraction kit (double column type), cDNA reverse transcription kit SynScript® III RT SuperMix for qPCR (+gDNA Remover), rapid fluorescent quantitative PCR premix, GelRed nucleic acid dye, TAE solution, agarose are purchased from Genview Biologic, PrimeSTAR® Max DNA Polymerase is purchased from Baoribio, Nanodrop accounting instrument, gel electrophoresis image analysis system, fluorescent quantitative PCR instrument, test tube, etc.

[0033] II. Test method

[0034] 2.1. Total RNA extraction and cDNA synthesis

[0035] The Scutellaria baicalensis leaves stored at -80 ℃ were rapidly ground into powder in liquid nitrogen. Total RNA was extracted from Scutellaria baicalensis using Hi-Pure polysaccharide and polyphenol plant total RNA extraction kit (Beijing Engenco Biotech Co., Ltd., SP0202-50), and then the purity and concentration of the RNA were detected using a nucleic acid detector. The integrity of the RNA was detected using 1.0% agarose gel electrophoresis, and the RNA that met the quality detection standard was selected for reverse transcription. Reverse transcription was performed using SynScript® III RT SuperMix for qPCR (+gDNA Remover) kit (Beijing Engenco Biotech Co., Ltd., TSK314S), 1 μg of total RNA was added to a sterile PCR tube, 5×gDNA Remover Mix 2 μL was added, and finally ddH2O was added to 10 μL. The mixture was incubated at 60 ℃ for 1 min to denature the RNA, and then immediately cooled on ice to anneal the primers to the RNA template. Next, 4 μL of 5×SynScript® III RT SuperMix was added, ddH2O was added to 20 μL, and then mixed gently and centrifuged briefly. The reaction system was incubated at 50 ℃ for 15 min for reverse transcription, and then heated at 85 ℃ for 5 s to inactivate the reverse transcriptase. The final cDNA product was stored at -20 ℃ for later use.

[0036] 2.2, Cloning and sequence analysis of SbMYB113-like gene

[0037] The CDS sequence of Scutellaria baicalensis SbMYB113-like gene was obtained from the NCBI database, and homologous recombination specific primers were designed (Table 1). PCR amplification was performed using Scutellaria baicalensis cDNA as the template, and the reaction system (50 μL) was as follows: cDNA template 200 ng, forward and reverse primers each 1 μL, PrimeSTAR Max Premix (2×) 25 μL, and ddH2O to 50 μL. The PCR program was as follows: 98 ℃ pre-denaturation for 5 min; 98 ℃ denaturation for 10 s, annealing temperature 56 ℃ annealing for 5 s, 72 ℃ extension for 1 min, a total of 35 cycles; 72 ℃ final extension for 5 min. After the PCR product was detected by 1.0% agarose gel electrophoresis, the gel recovery kit was used to purify the target band. The overexpression vector pMDC43 was digested with Kpn I and Sac I restriction endonucleases, and after 37 ℃ digestion for 1 h, it was inactivated at 80 ℃ for 10 min, and the linearized vector was recovered by gel cutting. The target band was connected to the pMDC43 vector and transformed into DH5α competent cells, and positive clones were selected for sequencing verification by Engenco Biotech Co., Ltd. The nucleotide sequence of the vector pMDC43 is shown as SEQ ID NO: 10

[0038] Table 1 Primer information used in this study

[0039]

[0040] 2.3, Homology analysis of Scutellaria baicalensis SbMYB113-like protein

[0041] The amino acid sequence was subjected to BLAST comparison in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), with a screening standard of threshold E-value≤1e-10, sequence similarity>50%, and priority given to species that have completed whole genome sequencing and have partially verified functions. Seven SbMYB113-like homologous protein sequences were downloaded. MEGA 11.0 software was used to draw the phylogenetic tree.

[0042] 2.4, Bioinformatics analysis of SbMYB113-like protein sequence

[0043] After obtaining the accurate SbMYB113-like gene cDNA sequence through sequencing, the physicochemical properties of SbMYB113-like protein were analyzed using the online tool Expasy-ProtParam (https: / / web.expasy.org / protparam / ), the hydrophilic and hydrophobic characteristics of the protein were predicted by Expasy-ProtScale (https: / / swissmodel.expasy.org / interactive), its phosphorylation sites were predicted by NetPhos-3.1 (https: / / services.healthtech.dtu.dk / services / NetPhos-3.1 / ), signal peptide prediction was performed using SignalP-4.1 tool (https: / / services.healthtech.dtu.dk / services / SignalP-4.1 / ), transmembrane domain distribution was analyzed by TMHMM-2.0 (https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ), and secondary and tertiary structures of the protein were predicted using SOPMA online tool (https: / / npsa.lyon.inserm.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_sopma.html) and SWISS-MODEL (https: / / swissmodel.expasy.org / interactive), respectively.

[0044] 2.5, Prediction of SbMYB113-like binding sites and cis-elements

[0045] Scutellaria baicalensis genome data were obtained from the National Bioinformatics Center (https: / / ngdc.cncb.ac.cn / gwh / Assembly / 10411 / show). The downloaded gff file of S. baicalensis was processed using GXF Sequence Extract in TBtools to obtain the 2000 bp sequence upstream of the SbMYB113-like gene. The transcription factor binding sites were analyzed using PlantRegMap (http: / / plantregmap.gao-lab.org / index.php), and the cis-elements were obtained using the PlantCARE website (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ).

[0046] 2.6, Subcellular localization of SbMYB113-like protein

[0047] Subcellular localization homologous recombination primers were designed according to the sequence of SbMYB113-like gene (Table 1), and the sequence was amplified according to the method of 1.2.2 and constructed into the pYBA-1132 vector. The nucleotide sequence of the pYBA-1132 vector is shown in SEQ ID NO: 11. Using the method of Agrobacterium infection, Agrobacterium GV3101 containing the recombinant vector plasmid 35S::SbMYB113-like-GFP and the empty vector plasmid 35S::GFP were resuspended in the infiltration buffer, and then injected into the lower epidermis of Nicotiana benthamiana leaves. The injected tobacco plants were placed in a plant culture medium room with a temperature of 22±2 ℃ and a light cycle of 16 h / 8 h (light / dark) for 48 h. After the culture ended, the laser confocal scanning microscope (LSM880, Zeiss) was used to observe and detect the GFP fluorescence signal to determine the subcellular localization of SbMYB113-like protein.

[0048] 2.7, Expression analysis of SbMYB113-like in S. baicalensis

[0049] Using cDNA from different tissues of *Scutellaria baicalensis* and samples subjected to 4 °C low-temperature stress for a specified duration as templates, and the *Scutellaria baicalensis* Actin 7 gene as an internal reference, the relative expression level of the SbMYB113-like gene was analyzed by qRT-PCR. The qRT-PCR reaction system consisted of 10 μL: 5 μL SYBR Premix Ex Taq, 0.5 μL forward and reverse primers (10 μmol / L), and 4 μL cDNA. The reaction program was: 95 °C for 30 s; 95 °C for 5 s, 55 °C for 10 s, 72 °C for 15 s, for 40 cycles; melting curve analysis was performed from 65 °C to 95 °C for 20 min; and three biological replicates were performed. Roots and untreated samples were used as controls. -ΔΔCt The relative expression level of the SbMYB113-like gene was calculated using a method.

[0050] 2.8 Measurement of physiological and biochemical indicators

[0051] Tobacco leaves from wild-type and transgenic tobacco were collected under normal conditions and after low-temperature treatment at 4°C. The contents of malondialdehyde (MDA, BC0025), superoxide dismutase (SOD, BC5165), peroxidase (POD, BC0095), and catalase (CAT, BC0205) were determined using commercial kits (Soluble Biotech, Beijing).

[0052] 2.9 Data Statistics and Analysis

[0053] Data were organized using Excel 2019 and statistical analysis and graphing were performed using GraphPad Prism v10.1.2. Data are expressed as mean ± standard deviation (Mean ± SD, n=3 biological replicates). Two-way ANOVA was used to compare differences between groups. <0.05 indicates a significant difference. <0.01 indicates a highly significant difference.

[0054] III. Results

[0055] 3.1 Cloning and sequence analysis of the SbMYB113-like gene from Scutellaria baicalensis

[0056] This study used Scutellaria baicalensis cDNA as a template to clone SbMYB113-like, obtaining a 252 bp specific band. Sequencing comparison confirmed that it was indeed SbMYB113-like from Scutellaria baicalensis, indicating that the gene cloning was successful. Figure 1(Part A of the gene). SbMYB113-like gene structure analysis showed that the total length of the gene's CDS sequence was 252 bp, encoding 88 amino acids. The gene contains 2 exons and 1 intron. The first exon starts at 1 bp and terminates at 118 bp (length 118 bp); the second exon starts at 201 bp and terminates at 334 bp (length 134 bp); the intron is 82 bp in length (119-200 bp). Figure 1 Part B). Conserved domain analysis showed that the SbMYB113-like protein contains a highly conserved MYB domain, consisting of a single MYB sequence. Figure 1 Part C). Figure 1 In the image, A: Electrophoresis diagram of PCR product of SbMYB113-like gene from Scutellaria baicalensis; B: Structure of SbMYB113-like gene; C: Analysis of SbMYB113-like protein domains.

[0057] 3.2 Homology analysis of SbMYB113-like protein from Scutellaria baicalensis

[0058] Using the BLAST tool in the NCBI database, seven amino acid sequences with high homology to SbMYB113-like sequences were searched and downloaded. The results showed that the SbMYB113-like amino acid sequence of *Scutellaria baicalensis* had a similarity of 82.50% to *Physalis alkekengi* EtMYB113 (KAL7101630.1), followed by *Lysimachia christinae* TfMYB113 (ARW29630.1), *Gynostemma pentaphyllum* SaMYB113 (GER30734.1), *Castor bean* RcMYB113 (XP_048229653.1), *Phyllanthus urinaria* PcMYB113 (KAK7374553.1), *Abrus precatorius* ApMYB113 (XP_027351419.1), and *Plantago asiatica* TaMYB113 (AIT76576.1), with similarities of 82.05%, 80.95%, and 80.95%, respectively. %, 79.52%, 77.11%, 75.90%, 74.70%. Phylogenetic analysis using MEGA 11.0 showed that Scutellaria baicalensis SbMYB113-like is closely related to Corydalis yanhusuo TfMYB113, Physalis alkekengi EtMYB113, and Astragalus membranaceus SaMYB113, and relatively distantly related to Castor bean RcMYB113, Trifolium repens TaMYB113, Leonurus japonicus PcMYB113, and Abrus precatorius ApMYB113. Figure 2 ).

[0059] 3.3 Bioinformatics Analysis of SbMYB113-like Proteins

[0060] The physicochemical property prediction shows that the molecular formula of SbMYB113-like protein is C 428 H 691 N 137 O 119 S7, the relative molecular mass is 9.88 kD, the theoretical isoelectric point (pi) is 9.59, the instability coefficient is 30.78 (the stability protein threshold is less than 40), and the overall hydrophilic coefficient is -0.964 (less than 0 is hydrophilic, and vice versa is hydrophobic), so the SbMYB113-like protein is a stable hydrophilic protein Figure 3 Part A). Six phosphorylation sites (threshold > 0.5) are predicted in the sequence of SbMYB113-like protein, including 2 serine (Ser) sites, 3 threonine (Thr) sites, and 1 tyrosine (Tyr) site, indicating that the phosphorylation modification process mainly with serine, threonine and tyrosine may affect the activity regulation of the protein Figure 3 Part B). The signal peptide prediction result shows that there is no signal peptide structure in the sequence of SbMYB113-like protein Figure 3 Part C), and SbMYB113-like protein has no transmembrane domain Figure 3 Part D), which meets the basic characteristics of transcription factor and belongs to non-secretion protein. The protein secondary structure analysis shows that the random coil accounts for the largest proportion of 56.63%, followed by alpha helix of 38.55%, and extended chain accounts for 4.82% Figure 3 Part E). The tertiary structure of SbMYB113-like protein is shown in the following figure Figure 3 Part F). In Figure 3 Part A: the prediction result of the hydrophilicity of SbMYB113-like protein; Part B: the prediction of the phosphorylation site of SbMYB113-like protein; Part C: the prediction analysis of the signal peptide of SbMYB113-like protein; Part D: the prediction display of the transmembrane domain of SbMYB113-like protein; Part E: the prediction of protein secondary structure (blue represents alpha helix, red represents extended chain, green represents beta turn, and purple represents random coil); and Part F: the prediction of protein tertiary structure.

[0061] 3.4, SbMYB113-like transcription factor binding site and cis-acting element analysis

[0062] SbMYB113-like contains 42 transcription factor binding sites of 14 transcription factor families, of which ERF accounts for the highest proportion of 24%; C2H2 type zinc finger protein (C2H2), five half-cysteine transcription factor binding sites (BBR-BPC), basic leucine zipper domain protein (bZIP) account for the second highest proportion, a total of 34%; the rest of the transcription factor binding sites are B3, Dof, GATA, AP2, ARF, LBD, MYB, NAC, Trihelix, G2-like, accounting for less than 42% (Table 1) Figure 4 The transcription factors involved in plant stress regulation indicate that SbMYB113-like gene may participate in Scutellaria baicalensis Georgi stress regulation by regulating these transcription factors. In cis-acting element analysis, in addition to the promoter characteristic elements, 67 cis-acting elements were detected in the 2000 bp upstream promoter sequence of SbMYB113-like gene in Scutellaria baicalensis Georgi, which were mainly related to stress response, light response, hormone response and cell development; of which, 15 stress response elements (ABRE, STRE, MBS, W box, ABRE4, ABRE3a), 11 light response elements (G-Box, GT1-motif, chs-CMA1a), 9 hormone response elements (GARE-motif, ERE, TATC-box, TGACG-motif, CGTCA-motif), and 10 cell development elements (as-1, circadian) Figure 4 This indicates that SbMYB113-like gene may be involved in Scutellaria baicalensis Georgi stress response. Figure 4 A: SbMYB113-like gene transcription factor binding sites; B: cis-acting elements of SbMYB113-like 2000 bp upstream sequence.

[0063] 3.5, Expression pattern of Scutellaria baicalensis Georgi SbMYB113-like

[0064] In order to reveal the tissue-specific expression and expression pattern of Scutellaria baicalensis Georgi SbMYB113-like gene under low temperature stress, qRT-PCR was used to analyze the expression of SbMYB113-like gene in the roots, flowers and leaves of Scutellaria baicalensis Georgi. The results showed that SbMYB113-like was expressed in the roots, flowers and leaves of Scutellaria baicalensis Georgi, but the expression in flowers was the highest, which was 8.86 times and 2.24 times of that in roots and leaves, respectively Figure 5 A part of the results). Under 4℃ low temperature treatment, the relative expression of SbMYB113-like in the roots and leaves of Scutellaria baicalensis Georgi significantly increased at the beginning of treatment (0-6 h); with the increase of treatment time, the relative expression of SbMYB113-like in the roots and leaves of Scutellaria baicalensis Georgi significantly decreased at 12-24 h treatmentFigure 5 SbMYB113-like has obvious tissue specificity and low temperature stress response function. In Figure 5 A: Relative expression level of SbMYB113-like in different tissues; B: Relative expression level of SbMYB113-like in root and leaf under 4 ℃ treatment.

[0065] 3.6, Subcellular localization analysis of SbMYB113-like protein

[0066] To explore the position of SbMYB113-like protein in the cell, the transgenic tobacco was observed by laser confocal microscopy. The results showed that the protein generated by 35S::GFP Agrobacterium competent cells GV3101 with empty vector plasmid was widely distributed in the cell membrane and nucleus, and there was also a certain distribution in the rest of the cell; the protein generated by 35S::SbMYB113-like-GFP Agrobacterium competent cells GV3101 with recombinant vector plasmid was mainly distributed in the position of cell membrane (Fig. 3.6), which indicated that SbMYB113-like protein was located on the cell membrane. Figure 6

[0067] 3.7, Phenotype and physiological response characteristics of SbMYB113-like overexpression tobacco under low temperature stress

[0068] To detect the biological function of SbMYB113-like gene, the overexpression vector of the gene was constructed, and tobacco was heterologously transformed. Three independent strains (OE2, OE7 and OE12) were selected for subsequent verification. Under the conventional culture conditions at 25 ℃, the growth of each group of tobacco was in good condition; under the low temperature stress treatment at 4 ℃, the wild type (WT) tobacco still maintained relatively good phenotype characteristics, while the growth phenotype of overexpression strains OE2, OE7 and OE12 was significantly inhibited by low temperature stress, and the affected degree was significantly greater than that of WT group (Fig. 3.7A). Figure 7 Further determination of antioxidant enzyme activity physiological indicators showed that the content of malondialdehyde (MDA) in WT increased by 1.35 times after low temperature stress, while the content of MDA in SbMYB113-like overexpression strains increased by about 2.78 times (Fig. 3.7B). Figure 7 ​SbMYB113-like overexpression was unable to effectively maintain the stability of the membrane system of transgenic plants. Compared with non-stress conditions, the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in WT lines were significantly up-regulated by 57.36%, 57.43% and 107.13% (P<0.01) after low temperature stress, while the antioxidant enzyme activities of SbMYB113-like overexpression lines under low temperature stress did not change significantly compared with the control group under normal growth conditions Figure 7 C-E, which indicates that the overexpression of SbMYB113-like plants is destroyed and unable to effectively resist the damage caused by cold stress. Figure 7 A: Phenotype of wild type (WT) and SbMYB113-like overexpression (OE) plants before and after low temperature treatment; B: Malondialdehyde (MDA) content; C: Superoxide dismutase (SOD) activity; D: Catalase (CAT) activity; E: Peroxidase (POD) activity.

[0069] The cis-element analysis of Scutellaria baicalensis SbMYB113-like found that the promoter region contains rich hormone response elements (GARE-motif, ERE, TATC-box, TGACG-motif, CGTCA-motif) and stress response elements (ABRE, STRE, MBS, W box, ABRE4, ABRE3a). Plant hormone abscisic acid (ABA) is an important signal molecule for plant response to abiotic stress, and the ABA signaling pathway mediated by it plays a key role in plant response to abiotic stress. The present application determines the expression level by low temperature treatment of Scutellaria baicalensis and using qRT-PCR method. The results show that with the extension of low temperature treatment time, the relative expression of SbMYB113-like in the roots and leaves of Scutellaria baicalensis significantly increases first and then decreases. This indicates that SbMYB113-like may regulate the cold resistance of plants by responding to temperature changes. SbMYB113-like is a stable, hydrophilic, small molecule transcription factor without transmembrane structure, with typical MYB family structure characteristics, and may regulate its activity through phosphorylation modification. Multiple sequence alignment results show that Scutellaria baicalensis SbMYB113-like has high homology with Physostegia virginiana EtMYB113, Tuberose TfMYB113 and Dandelion SaMYB113.

[0070] The application expresses SbMYB1130-like gene in tobacco by using Agrobacterium-mediated genetic transformation method, and the wild type tobacco is infected by Agrobacterium, and the results show that the gene significantly reduces the adaptability of tobacco to low temperature stress. When plants respond to low temperature stress, they can accumulate cold stress response related genes to resist cold stress related substances, including proline, soluble sugar and osmotic adjustment substances, thereby improving the low temperature stress resistance of plants. These substances participate in regulating the osmotic potential of cold stressed plants, the defense function of cell membrane, the stability of cell membrane structure and the removal of reactive oxygen species (ROS). In this study, under low temperature stress treatment, transgenic tobacco shows higher membrane lipid peroxidation level (MDA content increases) and antioxidant enzyme activity (SOD, CAT, POD activity decreases). The expression level of OsMYB2 increases under cold stress, salt stress and drought stress treatment, and the overexpression of OsMYB2 enhances the expression of proline synthetase and transporter genes. The plants overexpressing OsMYB2 have less H2O2 and malondialdehyde content, and the activities of antioxidant enzymes such as peroxidase, superoxide dismutase and catalase are enhanced, therefore, OsMYB2 participates in the low temperature stress response of rice and plays a regulatory role in low temperature stress resistance. OsMYBS3 responds to low temperature stress and inhibits the expression of cold signal-induced OsDREB1B, thereby negatively regulating the cold stress resistance of rice. Strawberry R2R3-MYB FvMYB82 participates in the regulation of plant cold stress resistance. Compared with wild type and transformed empty vector Arabidopsis, Arabidopsis ectopic expression FvMYB82 strain has higher proline and chlorophyll content under cold stress treatment, and the activities of SOD, POD and CAT are also stronger; but the MDA content is reduced. The overexpression of SbMYB1130-like may reduce the antioxidant defense system of plants, and fail to effectively protect the stability of cell structure and function, thereby reducing the stress resistance of plants.

Claims

1. The application of an SbMYB113-like gene in improving the low-temperature tolerance of Scutellaria baicalensis, characterized in that, The nucleotide sequence of the SbMYB113-like gene is shown in SEQ ID NO:

1. By reducing the overexpression of the SbMYB113-like gene in Scutellaria baicalensis, the antioxidant defense system of Scutellaria baicalensis is enhanced, thereby protecting the stability of cell structure and function and improving the tolerance of Scutellaria baicalensis to low temperature stress.

2. The application of the SbMYB113-like gene according to claim 1 in improving the low-temperature tolerance of Scutellaria baicalensis, characterized in that, cDNA sequences were genetically transformed into plants using Agrobacterium GV3101.

3. The application of the SbMYB113-like gene according to claim 2 in improving the low-temperature tolerance of Scutellaria baicalensis, characterized in that, The plant in question is Nicotiana benthamiana.

4. The application of the SbMYB113-like gene according to claim 2 in improving the low-temperature tolerance of Scutellaria baicalensis, characterized in that, The process of genetically transforming cDNA sequences into plants using Agrobacterium GV3101 is as follows: The process of constructing the SbMYB113-like gene into a plant expression vector and introducing it into plant cells is as follows: Subcellular localization homologous recombination primers were designed based on the SbMYB113-like gene sequence, the sequence was amplified, and the target sequence was constructed into the pYBA-1132 vector. Using the Agrobacterium infection method, Agrobacterium GV3101 containing the recombinant vector plasmid 35S::SbMYB113-like-GFP was resuspended in the infiltration buffer and then injected into the lower epidermis of the plant. The plant was then placed in a plant culture chamber with a temperature of 22±2 ℃ and a photoperiod of 16 h light / 8 h dark for 48 h.

5. A cDNA sequence of the SbMYB113-like gene from Scutellaria baicalensis, characterized in that, It is one of the following nucleotide sequences: 1) having the sequence shown in SEQ ID NO:1; 2) a DNA sequence that hybridizes to the cDNA sequence of SEQ ID NO:1 and has SbMYB113-like transcription factor active protein.

6. Primers for amplifying the cDNA sequence of the Scutellaria baicalensis SbMYB113-like gene as described in claim 5, characterized in that, The sequences of the primers are shown in SEQ ID NO:2 to SEQ ID NO:

9.

7. A recombinant expression vector, characterized in that, The vector used is pYBA-1132 and contains the SbMYB113-like gene as described in claim 1.

8. An SbMYB113-like protein, characterized in that, The molecular formula of SbMYB113-like protein is C 428 H 691 N 137 O 119 S7 has a relative molecular mass of 9.88 kD, a theoretical isoelectric point of 9.59, an instability coefficient of 30.78, and an overall hydrophilicity coefficient of -0.

964.

9. The application of the SbMYB113-like gene according to claim 8 in improving the low-temperature tolerance of Scutellaria baicalensis, characterized in that, The SbMYB113-like protein is located on the cell membrane.

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