Application of ginseng transcription factor pgmyb14 in improving plant drought tolerance

Abstract

The present application belongs to the field of biological genetic engineering, and relates to application of ginseng transcription factor PgMYB14 in improving drought tolerance of plants, wherein a sequence of the PgMYB14 protein is shown as SEQ ID NO. 1, and a sequence of the PgMYB14 gene is shown as SEQ ID NO. 2. The results of the present application show that heterologous expression of the PgMYB14 gene in Arabidopsis thaliana can activate a response pathway to drought and osmotic stress, and up-regulate biosynthesis metabolism of phenylpropanoids, suberin and wax. In addition, PgMYB14 can enhance the drought stress tolerance of Arabidopsis thaliana by removing active oxygen and up-regulating expression of drought stress related genes.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological genetic engineering, and relates to application of ginseng transcription factor PgMYB14 in improving plant drought tolerance. BACKGROUND

[0002] Ginsenosides accumulate in the roots of ginseng, which may endow ginseng with resistance to various potential biological stresses, such as antibacterial activity against pathogens and anti-herbivory activity against insects and other herbivorous animals.

[0003] Drought is considered as one of the most serious environmental stress factors affecting plant growth and crop yield. MYB transcription factors are involved in regulating the response and tolerance mechanisms of plants to drought stress due to their diversified functions (WANG X P, NIU YL, ZHENG Y. Multiple functions of MYB transcription factors in abiotic stress responses [J]. Int J Mol Sci, 2021, 22(11): 6125-6139). In Arabidopsis thaliana, overexpression of gene AtMYB12 not only significantly increases the accumulation of antioxidant active substances flavonoids to improve the tolerance to drought and saline-alkali, but also up-regulates the expression of genes involved in flavonoid biosynthesis, abscisic acid (ABA) biosynthesis, proline biosynthesis, stress response and reactive oxygen species (ROS) scavenging in transgenic plants, resulting in significant increase in ABA, proline content, superoxide dismutase (SOD) and peroxidase (POD) activities, and significant decrease in H2O2 and malondialdehyde (MDA) contents (WANG F B, KONG W L, WONG G, et al. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana [J]. Mol Genet Genomics, 2016, 291(4): 1545-1559).

[0004] The MYB gene family constitutes a large and diverse family of transcription factors in the plant kingdom, which plays a crucial role in regulating plant growth and development, stress response, and metabolic processes. MYB proteins generally contain one or more highly conserved DNA-binding domains, known as MYB domains, which confer their ability to bind to specific DNA sequences. Despite the conservation of the DNA-binding domain among MYB family members, the overall protein structure and function can exhibit significant diversity due to differences in species, genes, and environmental conditions (Yiling, Jiang Xiuming, Xu Xiangyang. Research Progress of Plant Transcription Factor MYB Gene Family [J]. Molecular Plant Breeding, 2016(8): 10. DOI: 10.13271 / j.mpb.014.002050.).

[0005] Even in the same species, the structure and function of MYB family members show significant diversity. For example, in rice, overexpression of the OsMYB6 gene has been shown to improve the drought and salt tolerance of transgenic plants, revealing the positive regulatory function of the gene in plant response to abiotic stress (Tang Y H, Bao X X, Zhi YL, et al. Overexpression of a MYB family gene, OsMYB6, increases drought and salinity stress tolerance in transgenic rice [J]. Frontiers in Plant Science, 2019, 10(18): 168-185.). In addition, the OsMYB103L gene is involved in regulating the synthesis of secondary cell walls in rice (Ye, Y., Liu, B., Zhao, M., Wu, K., Cheng, W., Chen, X., Liu, Q., Liu, Z., Fu, X., & Wu, Y. (2015). CEF1 / OsMYB103L is involved in GA-mediated regulation of secondary wall biosynthesis in rice. Plant molecular biology, 89(4-5), 385-401.), and the OsMYB112 gene is involved in regulating anthocyanin biosynthesis in rice (Xiang Z. Functional study of rice MYB family transcription factor OsMYB112. Diss. Chongqing University.). The OsMYBS2 gene regulates the light protection mechanism of rice by inhibiting the expression of the OsPsbS1 gene (Fu, X., Liu, C., Li, Y., Liao, S., Cheng, H., Tu, Y., Zhu, X., Chen, K., He, Y., & Wang, G. (2021). The coordination of OsbZIP72 and OsMYBS2 with reverse roles regulates the transcription of OsPsbS1 in rice. The New phytologist, 229(1), 370-387.).Meanwhile, the expression of OsMYB4 gene is closely related to the disease resistance of rice, and may play a role in regulating the defense response of plants to pathogens (Pooja, S., Sweta, K., Mohanapriya, A., Sudandiradoss, C., Siva, R., Gothandam, K. M., & Babu, S. (2015). Homotypic clustering of OsMYB4 binding site motifs in promoters of the rice genome and cellular-level implications on sheath blight disease resistance. Gene, 561(2), 209-218.).

[0006] The same situation also exists in the MYB family members of ginseng. In ginseng, the transcription factor PgMYB2 has been shown to specifically bind to specific sequences in the promoter region of the PgDDS gene, thereby positively regulating the transcription of PgDDS, and thus promoting the biosynthesis and accumulation of ginsenosides (LIU T, LUO T, GUO X Q, et al. PgMYB2, a MeJA-responsive transcription factor, positively regulates the dammarenediol synthase gene expression in Panax ginseng [J]. Int J Mol Sci, 2019, 20(9): 15.). When PgMYB38 gene is overexpressed, it is found that the expression level of key enzyme genes regulating 2,3-oxidosqualene synthesis is significantly reduced, indicating that PgMYB38 may play a negative regulatory role in the biosynthesis of ginsenosides (Chi. Screening and functional analysis of PgMYB38 in Panax ginseng [D]. Jilin Agricultural University.).

[0007] The prior art discloses cloning of ginseng Pg MYB4 gene by RT-PCR technology, constructing an expression vector, transforming it into Arabidopsis thaliana by Agrobacterium-mediated inflorescence dipping method, obtaining transgenic Arabidopsis thaliana, taking wild-type Arabidopsis thaliana as a control, and detecting physiological indexes related to drought resistance of the plants. The full length of the ginseng Pg MYB4 gene obtained is 735 bp, encoding 245 amino acids, and the relative molecular weight thereof is predicted to be 27 914; under the condition of drought stress, the growth state of the transgenic Arabidopsis thaliana is obviously better than that of the wild-type Arabidopsis thaliana, the transgenic Arabidopsis thaliana has a smaller reduction range of relative chlorophyll content in leaves, a significantly increased proline content and a lower water loss rate. It is shown that the ginseng Pg MYB4 gene has the ability of resisting drought stress (Meng X Y, Xie H M, Cai K X, et al. Construction of ginseng Pg MYB4 gene expression vector and analysis of drought resistance of Arabidopsis thaliana [J]. Chinese Traditional and Herbal Drugs, 2016, 47(17): 4. DOI:10.7501 / j.issn.0253-2670.2016.17.022.).

[0008] The sequence of the MYB14 gene in ginseng is quite different from that of the Pg MYB4 gene, and the research on the function thereof is still insufficient. SUMMARY

[0009] The application aims to provide application of ginseng transcription factor PgMYB14 in improving drought resistance of plants.

[0010] In order to achieve the above-mentioned purpose, the technical scheme adopted by the application is:

[0011] The application of ginseng transcription factor PgMYB14 in improving drought resistance of plants, wherein the sequence of the PgMYB14 gene is shown as SEQ ID NO. 2.

[0012] The sequence of the PgMYB14 protein is shown as SEQ ID NO. 1.

[0013] SEQ ID NO. 1 is:

[0014] MVRAPCCEKMGLKKGPWTRDEDQILINHVTLYGHGNWRALPKQAGLLRCGKSCRLRWTNYLRPDIKRGNFSEEEEETIISLHEVLGNRWSAIAARLPGRTDNEIKNVWHTHLKKKLKKNQLETTSPDQVLSKSEPKPMKHSTTQSTSHDDLPNSPQSITSTSSEDNNMSIHTAEAFDDTFWSELFSADENSGNTSDNSGDTTDFNRCAQLQFPEFGCENNLNMNDGMDFFWYDILTKSAGELLEF.

[0015] The sequence of the PgMYB14 gene is shown as SEQ ID NO. 2.

[0016] SEQ ID NO. 2 is:

[0017] ATGGTGAGAGCTCCTTGCTGTGAAAAGATGGGGTTGAAGAAAGGTCCATGGACTCGCGACGAAGATCAGATTCTCATCAACCATGTCACTCTTTATGGCCATGGCAACTGGCGGGCTCTCCCTAAACAAGCCGGTTTGTTGAGGTGTGGAAAGAGTTGTAGACTTCGGTGGACGAATTATTTGAGACCGGACATTAAACGAGGAAACTTTAGTGAAGAAGAGGAGGAAACCATCATCAGTTTACACGAAGTATTAGGAAATAGATGGTCAGCAATTGCAGCAAGATTACCAGGACGTACCGACAATGAAATCAAAAATGTGTGGCACACCCACTTAAAAAAGAAACTCAAGAAAAACCAATTAGAAACTACTAGCCCTGATCAAGTATTATCCAAGTCGGAACCAAAACCAATGAAGCACTCTACCACTCAATCAACATCCCACGATGACCTCCCAAATTCACCGCAATCAATCACCAGCACCAGCAGCGAAGATAATAACATGTCCATCCACACCGCCGAGGCTTTTGATGACACTTTTTGGTCAGAATTATTCTCCGCCGATGAAAACTCTGGTAATACAAGCGACAATTCCGGTGACACAACTGATTTTAACCGATGTGCTCAGCTTCAATTTCCGGAATTTGGATGTGAAAATAATTTAAACATGAATGATGGTATGGACTTTTTTTGGTACGACATTCTCACAAAATCAGCTGGGGAGTTGCTTGAATTCTAA.

[0018] The application of the ginseng transcription factor PgMYB14 in improving the expression level of genes related to plant drought tolerance, wherein the sequence of the PgMYB14 gene is shown as SEQ ID NO. 2.

[0019] In one preferred embodiment, the plant drought tolerance related gene comprises one or more of AtCBF1, AtDREB2A, AtCOR47 and AtKIN1.

[0020] The application of ginseng transcription factor PgMYB14 in improving the activity of O-acyltransferase of plants, characterized in that the sequence of the PgMYB14 gene is shown as SEQ ID NO. 2.

[0021] The application of ginseng transcription factor PgMYB14 in improving the activity of POD, SOD or CAT enzyme of plants, characterized in that the sequence of the PgMYB14 gene is shown as SEQ ID NO. 2.

[0022] The application of ginseng transcription factor PgMYB14 in reducing the activity of MDA enzyme of plants, characterized in that the sequence of the PgMYB14 gene is shown as SEQ ID NO. 2.

[0023] A plasmid, the overexpression plasmid PgMYB14 gene.

[0024] In one preferred embodiment, the PgMYB14 gene is obtained by connecting the PgMYB14 gene with the digested vector after the vector is digested.

[0025] In one preferred embodiment, the vector is a pCAMBIA1301s plasmid.

[0026] An agrobacterium competent cell, the agrobacterium competent cell overexpresses the PgMYB14 gene.

[0027] In one preferred embodiment, the agrobacterium competent cell is obtained by transforming the plasmid into a DH5a competent cell.

[0028] The application also protects the application of the plasmid or the agrobacterium competent cell in enhancing the drought tolerance of plants.

[0029] In one preferred embodiment, the plant is Arabidopsis thaliana, rice or cotton.

[0030] The application clones the PgMYB14 gene and constructs a corresponding plant expression vector. The gene is successfully transferred into wild-type Arabidopsis thaliana by agrobacterium-mediated floral dip transformation. PCR technology is used to identify the genomic DNA and transcription mRNA level to screen out positive Arabidopsis thaliana plants stably overexpressing the PgMYB14 gene.

[0031] The present application relates to transcriptomic analysis of transgenic lines. The analysis results show that in the PgMYB14 overexpressing Arabidopsis lines, the "response to water" category in biological process is the most significant by gene ontology (GO) enrichment analysis, followed by "response to osmotic stress" and "response to water deficiency" categories. Further, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis reveals that the "cutin, suberin and wax biosynthesis" pathway is the most significant, and enrichment of the "phenylpropanoid metabolism" pathway is also observed.

[0032] Compared with the prior art, the present application has the following beneficial effects:

[0033] Experiments prove that the heterologous expression of PgMYB14 gene in Arabidopsis activates the response pathway to drought and osmotic stress, and up-regulates the biosynthetic metabolism of phenylpropanoids, suberin and wax. In addition, PgMYB14 can enhance the drought stress tolerance of Arabidopsis by removing reactive oxygen species and up-regulating the expression of drought stress related genes. In addition, the ginseng PgMYB14 transcription factor, its coding gene, and the overexpression recombinant vector containing the gene involved in the present application can be applied to the genetic transformation of economic crops such as rice and cotton, and provide reliable gene resources and theoretical basis for plant stress resistance molecular breeding. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Figure 1 is the expression profile of PgMYB14 in ginseng callus under salt stress and drought stress treatment; Figure A and Figure B respectively show the changes of PgMYB14 gene expression after treating ginseng callus under 150 mM NaCl and 15% PEG conditions; PgActin is the internal reference gene, and the data shown represent the mean ± standard deviation (n = 3, t test, *P < 0.05 and **P < 0.01);

[0035] Figure 2 Figure 2 is the identification of PgMYB14 transgenic Arabidopsis plants; Figure A: PCR identification of hygromycin resistance gene (550 bp) in transgenic Arabidopsis leaf tissue; Lane 1: DNA Marker 2000; Lane 2: negative control: WT genomic DNA; Lanes 3, 4, 5: genomic DNA of transgenic lines (OE-2, OE-4, OE-6); Lane 6: positive control: empty vector plasmid; Figure B: RT-qPCR verification of PgMYB14 expression level in transgenic Arabidopsis; AtEF1a is the internal reference gene, and the data shown represent the mean ± standard deviation (n = 3, t test, *P < 0.05 and **P < 0.01);

[0036] Figure 3GO enrichment analysis of differentially expressed genes in OE-2 and WT samples; the horizontal axis represents RichFactor, and the vertical axis represents GO entries; different colors and shapes represent biological processes, cell composition, and molecular functions, respectively; the color gradient represents the size of the pvalue value, while the size of the shape corresponds to the number of target genes enriched in the corresponding pathway; Figure A: up-regulated differential genes. Figure B: down-regulated differential genes;

[0037] Figure 4 KEGG enrichment analysis of differentially expressed genes in OE-2 and WT samples; the horizontal axis represents RichFactor, and the vertical axis represents Pathway; the color gradient represents the size of the pvalue value, while the size of the bubble corresponds to the number of target genes enriched in the corresponding pathway; Figure A: up-regulated differential genes; Figure B: down-regulated differential genes;

[0038] Figure 5 PgMYB14 overexpression enhances drought tolerance of Arabidopsis; Figure A and Figure B show the phenotypic characteristics of PgMYB14 transgenic plants and WT plants under normal growth conditions and drought stress conditions, respectively, and the scale bar in the figure is 1 cm; Figure C-F: determination results of POD, SOD, and CAT activities and MDA content of WT and transgenic plants under normal growth conditions and drought stress conditions; the experiment was repeated three times independently, and 12 plants of each strain were used in the experiment; the data shown represent the mean ± standard deviation (n≥3, t test, *P<0.05 and **P<0.01); Figure G-J: expression levels of drought stress-related genes in PgMYB14 transgenic plants and WT plants before and after stress treatment; AtEF1a is the internal reference gene, and the data shown represent the mean ± standard deviation (n=3, t test, *P<0.05 and **P<0.01). DETAILED DESCRIPTION

[0039] Example 1 PgMYB14 expression pattern analysis

[0040] 1.1 Induction of ginseng callus

[0041] (1) A certain amount of ginseng callus was evenly spread on a 6,7-V plate containing 20 μM ABA and cultured at 25°C in the dark. The sample was collected after 0, 4, 8, 12, 24, and 48 h and quickly frozen using liquid nitrogen, followed by storage at -80°C for preservation;

[0042] (2) Drought induction: 15% PEG (15% PEG is a commonly used drought inducer) was added to the 6,7-V medium, and the callus was evenly spread on the plate. The rest of the operation was the same as the ABA induction treatment;

[0043] (3) NaCl induction: 150 mM NaCl was added to the 6,7-V medium, and the callus was evenly spread on the culture plate. The rest of the operation was the same as the ABA induction treatment.

[0044] 1.2 Total RNA extraction from ginseng callus

[0045] (1) Add 1 ml Trizol to an RNase-Free centrifuge tube in advance and place it on ice. Use a mortar and pestle to grind the sample into a powder, and add liquid nitrogen to the mortar as needed during the process. Take 50 mg of powder and add it to the centrifuge tube, then mix it with a vortex mixer; let it stand at room temperature for 5 min;

[0046] (2) 12,000 x g 4°C centrifugation for 5 min, carefully pipette 950 μL supernatant into a new RNase free centrifuge tube, add chloroform in an amount of 1 / 5 of the volume of Trizol, mix thoroughly; let it stand at room temperature for 5 min;

[0047] (3) 12,000 x g 4°C centrifugation for 10 min, the homogenate will be divided into three layers: the supernatant containing RNA, the middle protein layer, and the lower organic phase;

[0048] (4) Pipette 450 μl of supernatant into another new RNase free centrifuge tube (do not pipette out the middle protein layer); add isopropanol in an amount equal to the volume of Trizol, mix thoroughly, and let it stand at room temperature for 10 min;

[0049] (5) Transfer the above mixture to the adsorption column RA, centrifuge at 13,000 rpm for 2 min, discard the filtrate;

[0050] (6) Add 500 μL of deproteinization solution RW1, let it stand at room temperature for 3 min, centrifuge at 13,000 rpm for 30 s, discard the filtrate;

[0051] (7) Add 500 μL of rinse solution RW pre-added with anhydrous ethanol, centrifuge at 13,000 rpm for 30 s, discard the filtrate; repeat this operation once;

[0052] (8) Place the adsorption column back into the empty collection tube and centrifuge at 13,000 rpm for 2 min;

[0053] (9) Place the adsorption column in a new RNase free centrifuge tube, open the lid and let it stand at room temperature for 2 min to evaporate the residual ethanol; add 30 μL of RNase free water preheated to 70-90°C to the adsorption membrane, let it stand at room temperature for 2 min, and centrifuge at 12,000 rpm for 1 min.

[0054] After the RNA was taken, 2 μL was taken for agarose gel electrophoresis to evaluate the integrity of the RNA. The unused RNA sample should be stored in the refrigerator at -80℃ for subsequent reverse transcription or other purposes.

[0055] 1.3 Reverse transcription to synthesize the first strand of cDNA

[0056] (1) Removal of genomic DNA

[0057] In a 0.2 mL PCR tube without RNase contamination, the reagents of the reverse transcription reaction system were added in sequence. The reverse transcription reaction system is as follows:

[0058] Table 1 Reverse transcription reaction system

[0059]

[0060] After mixing the above reagents, the sample was incubated at 42℃ for 2 minutes in the PCR instrument, then taken out and immediately placed on ice for subsequent use.

[0061] (2) Synthesis of the first strand of cDNA

[0062] 4 μL of 5×HiScriptⅡEnzyme Mix was added to the PCR tube containing the above reaction solution, and the following reaction program was performed in the PCR instrument: 50℃, 15 min; 85℃, 5 s. After the reaction was completed, the synthesized cDNA sample was diluted by 10 times and stored in the refrigerator at -20℃.

[0063] 1.4 RT-qPCR analysis

[0064] Novozyme ChamQ Universal SYBR qPCR Master Mix (Q711-02) system is as follows:

[0065] Table 2 RT-qPCR reaction system

[0066]

[0067] The amplification program is as follows:

[0068] Table 3 RT-qPCR reaction program

[0069]

[0070] PgACT was used as the internal reference gene, and each sample was repeated three times. 2 -ΔΔCt Method was used to measure the relative difference of gene transcription level, and the expression level of each gene was analyzed and compared.

[0071] The primer sequences used are as follows:

[0072] Primer Sequence (5′→3′)

[0073] qRT-PgMYB14-F:ATTCACCGCAATCAATCACC(SEQ ID NO.3)

[0074] qRT-Pg MYB14-R:TCGCTTGTATTACCGGAGTT(SEQ ID NO.4)

[0075] qRT-PgACT-F:TGCCCCAGAAGAGCACCCTGT(SEQ ID NO.5)

[0076] qRT-PgACT-R: AGCATACAGGGAAAGATCGGCTTGA (SEQ ID NO.6)

[0077] The results are as follows Figure 1 As shown. Figure 1 In Figure A, under sodium chloride (NaCl) salt stress treatment, the expression level of PgMYB14 showed a gradual upward trend, reaching a peak after 8 hours, and then began to gradually decrease. After PEG simulated drought treatment, the expression level of PgMYB14 increased, reached a peak after 4 hours, and then gradually decreased, as shown in Figure 4. Figure 1 As shown in B. The experimental results showed that PgMYB14 could respond to salt stress and drought stress treatment.

[0078] Example 2 Construction of gene PgMYB14 overexpression vector

[0079] 2.1 PCR amplification of target fragments

[0080] PCR amplification was performed using PrimeSTAR Max DNA Polymerase from Takara. Primers were designed using Primer Premier 5.0 software and synthesized by Beijing Qingke Biotechnology Co., Ltd. The primer sequences are as follows: Primer Sequence (5′→3′)

[0081] PC1301s-PgMYB14-F: CGGGGTACCATGGTGAGAGCTCCTTGCTGTGAA (SEQ ID NO.7)

[0082] PC1301s-PgMYB14-R: CGCGGATCCTTAGAATTCAGGTAAGTCCC (SEQ ID NO.8)

[0083] The reaction system for PCR amplification is as follows:

[0084] Table 4 PCR amplification system

[0085]

[0086] The PCR reaction program is set as follows:

[0087] Table 5 PCR reaction program

[0088]

[0089] After the reaction is terminated, the liquid on the wall of the test tube is collected by instantaneous centrifugation, and then it is placed on ice for the next agarose gel purification step, or temporarily stored in a -20°C refrigerator.

[0090] 2.2 PCR product purification and expression vector linearization

[0091] The PCR product is subjected to gel electrophoresis to remove non-specific amplification bands, and the Vazyme gel recovery kit is used to purify the target DNA fragment. The PCR product is mixed with 6 times the loading buffer, and agarose gel electrophoresis is performed for 30 minutes. Under the irradiation of ultraviolet light source, the agarose gel region containing the target size DNA fragment is accurately cut and placed in a clean 1.5 ml centrifuge tube. Then weigh the weight of the gel, and the subsequent steps are operated according to the instructions of the kit. Finally, the DNA solution is obtained, which is detected by ultramicro spectrophotometer for concentration and quality, and then stored in a -20°C refrigerator.

[0092] The expression vector pCAMBIA1301s plasmid (the source of the plasmid is referred to the prior art JIANG T, ZHANG Y, ZUOG G, et al. Transcription factor PgNAC72 activates DAMMARENEDIOL SYNTHASE expression to promote ginseng saponin biosynthesis [J]. Plant Physiology, 2024, 195(4): 2952-2969) is cut by BamH I and Kpn I restriction endonuclease, and the same gel recovery experiment is also performed.

[0093] The enzyme cutting system is as follows:

[0094] Table 6 Endonuclease cutting reaction system

[0095]

[0096] 2.3 Vector ligation and E. coli transformation

[0097] The homologous recombination reaction is as follows:

[0098] Table 7 Homologous recombination reaction system

[0099]

[0100] After mixing with a micropipette, the sample was placed in a PCR instrument and incubated at 37°C for 30 minutes. After the reaction, the sample was immediately removed and placed on ice to obtain the recombined vector. Subsequently, an E. coli transformation experiment was performed, and the specific operation steps are as follows:

[0101] (1) One of the DH5a competent cells stored in a -80°C refrigerator was quickly transferred to ice and subjected to thawing treatment in ice for 10 minutes. Subsequently, 10 μL of the ligation product was added to the competent cells, mixed uniformly, and then placed on ice for 15 minutes.

[0102] (2) The centrifuge tube in step (1) was placed in a 42°C water bath for 1 minute of heat shock treatment, and then quickly moved to ice for 2 minutes of ice bath.

[0103] (3) 700 μL of antibiotic-free LB liquid medium was added to the centrifuge tube, gently shaken to mix, and then subjected to 60 minutes of shaking culture at 37°C and 200 rpm.

[0104] (4) Centrifugation was performed at 1,0000 x g for 1 minute to collect the bacteria, and 700 μL of supernatant was removed. The bacteria were resuspended using the remaining approximately 100 μL of medium. After thorough mixing, the bacterial solution was inoculated into LB solid medium containing 100 mg / L kanamycin (Kan), uniformly coated using a spreader, and incubated in a 37°C constant temperature incubator for 12 hours.

[0105] The formula of the antibiotic-free LB liquid medium is as follows (volume is 1 L):

[0106] Table 8 Formula of LB medium

[0107]

[0108] To prepare the LB solid medium, 15 g / L of agar powder was added to the liquid medium, and then subjected to 121°C high-pressure sterilization for 15 minutes. After sterilization, an appropriate amount of antibiotic was added when the high-pressure sterilized medium cooled to about 60°C. The medium can be stored for one week at room temperature.

[0109] 2.4 Identification of positive clones

[0110] Select four transformed colonies of appropriate size and inoculate them into 200 μL of LB liquid medium containing 100 mg / L Kan. Incubate at 37°C and 200 rpm for 4 hours, shaking. Then, take an appropriate amount of the culture medium for PCR identification. The reaction system is as follows:

[0111] Table 9 Bacterial liquid PCR reaction system

[0112]

[0113] The PCR reaction procedure is as follows:

[0114] Table 10 Bacterial liquid PCR reaction program

[0115]

[0116]

[0117] PCR products were analyzed by agarose gel electrophoresis to verify that the resulting bands were single and of the expected size. If the PCR product was identified correctly, a sample of the corresponding bacterial culture was sent to Beijing Qingke Biotechnology Co., Ltd. for DNA sequencing. After sequencing results were confirmed, the remaining bacterial culture was expanded. Subsequently, 300 μL of the fresh bacterial culture was mixed with 700 μL of sterile glycerol, snap-frozen in liquid nitrogen, and stored at -80°C.

[0118] 2.5 Plasmid extraction

[0119] Adopt Beijing Qingke Biotechnology Co., Ltd. Plasmid Mini Kit is used for small-scale plasmid DNA extraction. All steps are performed at room temperature:

[0120] (1) Take 4 mL of overnight culture and centrifuge at 12,000 × g for 1 min to collect the cells and discard the supernatant as much as possible;

[0121] (2) Add 250 μL of Buffer PA containing RNase A to the bacteria and mix thoroughly by pipetting until no obvious bacterial clumps remain;

[0122] (3) Add 250 μL of Buffer PB and gently invert the tube 6-8 times to fully lyse the cells.

[0123] (4) Add 350 μL of Buffer PC and gently invert 6-8 times to mix thoroughly. Centrifuge at 12,000 rpm for 10 min.

[0124] (5) Aspirate the supernatant into the column, taking care not to aspirate the pellet, centrifuge at 12,000 rpm for 1 min, discard the supernatant, and place the column back into the collection tube;

[0125] (6) Add 600 μΐ^of Buffer PW containing absolute ethanol along the side of the column, centrifuge at 12,000 rpm for 1 min, discard the waste, and repeat this step once;

[0126] (7) Place the column back into the collection tube, centrifuge the empty column at 12,000 rpm for 2 min;

[0127] (8) Place the column into a new clean 1.5 mL microcentrifuge tube, open the lid, and let the residual ethanol evaporate at room temperature for 2 min;

[0128] (9) Add 35-50 μΐ^of Elution Buffer preheated to 60°C to the center of the membrane, let stand at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min. The final plasmid solution can be stored at -20°C.

[0129] Example 3 Construction of transgenic Arabidopsis overexpressing PgMYB14

[0130] 3.1 Preparation of Agrobacterium chemical competent cells

[0131] (1) Take the GV3101 strain (purchased from Shanghai Yingai Biotechnology Co., Ltd.) out of the -80°C freezer and quickly freeze protect in liquid nitrogen, then use a loop to take an appropriate amount of bacterial solution and spread it on YEB solid medium containing Rif (50 μg / mL) for strain recovery. Then, invert culture in a constant temperature incubator at 28°C for 36 hours. The following is the formula of YEB medium:

[0132] Table 11 Formula of YEB medium

[0133]

[0134]

[0135] When preparing YEB solid medium, add 15 g / L agar powder according to the formula, and autoclave at 121°C for 15 minutes. After the medium is cooled to about 50°C, add antibiotics, which can be stored at room temperature for one week.

[0136] (2) Select a single colony of appropriate size and inoculate it into 5 mL of YEB liquid medium containing Rif (50 μg / mL) and shake culture at 28°C, 200 rpm overnight.

[0137] (3) Inoculate 50 μL of the overnight culture into 50 mL of fresh YEB liquid medium and incubate at 28°C and 200 rpm until the OD value of the culture reaches about 0.6 to 0.8. Then, transfer the culture into a 50 mL sterile centrifuge tube and place it in an ice bath for 30 minutes. 600

[0138] (4) Place the centrifuge tube in a 4°C pre-cooled centrifuge and centrifuge at 4,000 rpm for 10 minutes.

[0139] (5) Remove the supernatant and add 10 mL of pre-cooled 0.15 M NaCl solution to the precipitate. Gently pipette the culture on ice to resuspend it, and then centrifuge at 4,000 rpm for 10 minutes at 4°C.

[0140] (6) After removing the supernatant, add 1 mL of pre-cooled 20 mM CaCl2solution to the precipitate and gently pipette the culture on ice to resuspend it. Then, aliquot the culture into pre-cooled 1.5 mL sterile centrifuge tubes, quickly freeze using liquid nitrogen, and store at -80°C for later use.

[0141] 3.2 Transformation of Agrobacterium competent cells

[0142] (1) Take one vial of GV3101 competent cells from the -80°C freezer and thaw them on ice for 10 minutes. In a clean bench, add 2 μL of the plasmid extracted in step 2.5 to the bacterial solution and gently pipette to mix. Then, place the solution on ice for 30 minutes.

[0143] (2) Quickly freeze in liquid nitrogen for 2-5 minutes, and then heat shock in a 37°C water bath for 5 minutes. In a clean bench, add 900 μL of YEB liquid medium, and then incubate at 28°C and 100 rpm for 4-6 hours to recover the bacteria.

[0144] (3) Centrifuge at 8,000 rpm for 1 minute, remove 800 μL of the supernatant, and resuspend the bacteria with the remaining medium. After resuspending the bacterial solution, evenly spread it on the surface of YRK (YEB containing 50 mg / L Rif and 50 mg / L Kan) solid medium, and then incubate it in an inverted manner at 28°C for 2 days.

[0145] (4) Pick single colonies for bacterial liquid PCR identification, following the method in Example 2. Select positive colonies for further culture, genetic transformation, or strain preservation.

[0146] 3.3 Obtaining of transgenic Arabidopsis thaliana

[0147] ​Pick a single positive colony and inoculate it into 3 ml of YEB liquid medium containing the selected antibiotic. Incubate at 28°C and 200 rpm for 12 hours. Then, transfer 300 μL of the bacterial solution into 100 ml of YRK liquid medium and continue to expand the culture until the OD600 value reaches 0.8. At this point, the bacterial solution can be used for genetic transformation experiments in Arabidopsis thaliana.

[0148] Referring to the floral dip transformation method proposed by Zhang et al. (ZHANG X, HENRIQUES R, LIN SS, et al. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method [J]. Nature Protocols, 2006, 1(2): 641-646), the PgERF2 overexpression vector was transformed into the Arabidopsis Col-0 wild-type genome, and the transformed Arabidopsis was placed in an incubator (23°C, 16 hours light / 8 hours dark cycle, light intensity 10,000 lux) until T0 generation transgenic Arabidopsis seeds were harvested. After hygromycin selection and PCR identification, T1 generation transgenic Arabidopsis seedlings were successfully obtained. These transgenic plants will be used for subsequent transcriptomic analysis.

[0149] 2.4 PCR identification of transgenic Arabidopsis

[0150] (1) Extraction of transgenic Arabidopsis genomic DNA

[0151] Arabidopsis seedling gDNA was extracted using the Steady Pure Plant Genomic DNA Extraction Kit from Acryl. For detailed experimental steps, please refer to the kit instructions.

[0152] (2) PCR verification, the primer sequences are as follows:

[0153] Primer Sequence (5′→3′)

[0154] Hyg-F: ACACTACATGGCGTGATTTCAT(SEQ ID NO.9)

[0155] Hyg-R:TCCACTATCGGCGAGTACTTCT(SEQ ID NO.10)

[0156] Table 12 PCR identification reaction system

[0157]

[0158] The reaction conditions are the same as in Example 2.

[0159] The PCR products were collected and subjected to agarose gel electrophoresis, and images were taken using a ChemiDoc XRS gel imager from Bio Rad.

[0160] 2.5 Detection of PgMYB14 gene expression in transgenic lines

[0161] Afterwards, RNA extraction, reverse transcription, RT-qPCR analysis and other experiments were performed on Arabidopsis seedlings, with the steps referring to Example 1.

[0162] The primer sequences are as follows:

[0163] Primer Sequence (5′→3′)

[0164] AtEF1α-F: CACCCTTGGTGTCAAGCAGATGA (SEQ ID NO.11)

[0165] AtEF1α-R:TTGTCTCCCTCGAATCCAGAGATTG (SEQ ID NO.12)

[0166] The RT-qPCR primer sequences of PgMYB14 are shown in Example 1.

[0167] The results are as follows Figure 2 As shown. Figure 2 As shown in Figure A, genomic DNA was amplified by PCR using specific primers containing the selection marker hygromycin resistance gene. The results showed that the expected amplified product of 550 bp was obtained in the transgenic positive plants OE-2, OE-4, and OE-6, while the corresponding fragment was not detected in the wild-type plants. Figure 2 As shown in Figure B, RT-qPCR analysis revealed that PgMYB14 expression levels in transgenic plants were significantly higher than in the wild type. In transgenic plants OE-2, OE-4, and OE-6, PgMYB14 expression levels were increased by 69-fold, 16-fold, and 62-fold, respectively, compared to the wild type. The present invention selected the OE-2 line, which showed the most significant increase in expression, for subsequent analysis. These data confirm that the PgMYB14 gene has been successfully integrated and overexpressed in Arabidopsis.

[0168] Example 4 Transcriptomic Analysis of Transgenic Arabidopsis Overexpressing PgMYB14

[0169] 4.1 Library construction and sequencing

[0170] In this experiment, leaf tissue samples of transgenic line OE-2 and wild-type control (WT) tobacco were collected. These samples were from three independent biological replicates and were used for subsequent RNA extraction, with the steps referring to Example 1. The total RNA of the extracted samples was quality tested and analyzed using a NanoDrop micro-UV spectrophotometer and an Agilent 2100 bioanalyzer. Subsequently, 3 μg of total RNA from each sample was used to construct a cDNA library. These libraries were sequenced using the MGISEQ-T7 sequencing platform using a PE150 read length. In order to obtain clean reads suitable for subsequent analysis, the adapter sequences and low-quality reads were removed. Finally, the clean reads were aligned with the tobacco reference genome sequence using HISAT2 software.

[0171] 4.2 RNA-seq data analysis

[0172] FeatureCounts software was used to calculate the number of fragments per kilobase of transcript per million (FPKM) of each gene in each sample. Differential gene expression between samples was analyzed using DESeq2 software, with the criteria for differentially expressed genes set as an absolute log2FC value >1 and an adjusted P value <0.05. The detected differentially expressed genes (DEGs) were compared with the GO database for each category, and the number of genes in each category was counted. Subsequently, Fisher's exact test was used to assess the significance of protein enrichment in specific GO functional terms, with a threshold q value of ≤0.05 to identify significantly enriched functional annotation GO terms. Furthermore, functional annotation and significant enrichment analysis of DEGs were performed using the KEGG public database, with a threshold q value of ≤0.05.

[0173] The results are as follows Figure 3 shown. Figure 3 In A, GO enrichment analysis of differentially expressed genes showed that upregulated genes were mainly concentrated in O-acyltransferase activity, response to water, response to osmotic stress, response to salt stress, and response to water deficiency in biological processes. These findings suggest that PgMYB14 may play a positive regulatory role in promoting the response of Arabidopsis to drought and osmotic stress. Downregulated genes were mainly enriched in cellular response to hypoxia, cellular response to chemical stimulation, response to chitin, response to nitrogen compounds, and response to ethylene stimulation ( Figure 3 B). Taken together, these results suggest that overexpression of PgMYB14 may enhance the responses of Arabidopsis to drought and osmotic stress, while downregulating its responses to hypoxia, chitin, and other chemicals such as ethylene.

[0174] After in-depth KEGG enrichment analysis of differentially expressed genes, the results are as followsFigure 4 As shown in Table 2, the up-regulated genes were mainly involved in the following biological metabolic pathways: photosynthesis, biosynthesis of cutin, suberin and wax, ribosome, anthocyanin synthesis, RNA polymerase, biosynthesis of various plant secondary metabolites, phenylpropanoid metabolism, phenylalanine, etc. These results suggest that PgMYB14 may promote the biosynthesis of phenylpropanoids, suberin and wax in Arabidopsis. Figure 4 In addition, KEGG enrichment analysis of the down-regulated differential genes revealed that they were mainly enriched in MAPK signaling pathway, plant-pathogen interaction, plant hormone signal transduction pathway, vitamin B6 metabolism, arginine and proline metabolism, etc.

[0175] B), which indicates that overexpression of PgMYB14 in Arabidopsis may have a negative regulatory effect on the resistance to plant pathogens and the metabolism of amino acids and vitamins. Figure 4

[0176] Example 5 Drought stress treatment experiment

[0177] WT and PgMYB14 overexpressing Arabidopsis seeds were sterilized as follows: according to the number of seeds, appropriate centrifuge tubes were selected, 1 ml or 8 ml of sterilization solution containing 20% bleach and 0.1% Triton X-100 prepared with deionized water was added, and after mixing, it was incubated for 8 min. Washed with sterile water for 10 times until the supernatant was clear and colorless. After mixing the seeds with sterile water, they were placed on MS medium and incubated at 4°C for 2 days for vernalization, and then transferred to a light incubator for 7 days. Plants with good growth and a 4-week growth cycle were selected for drought treatment experiment. Before the experiment, the plants were fully irrigated and then stopped watering to keep the soil moisture content consistent. On the 0th, 5th and 10th days of drought treatment, plant leaves were collected for RNA extraction and reverse transcription into cDNA, followed by gene expression analysis (see Reference Example 1). At the same time, the POD, T-SOD and CAT activity assay kits provided by Wuhan Nomad Bioscience Co., Ltd. were used to determine the enzyme activity according to the instructions to evaluate the physiological response of the plants to drought.

[0178] The results are shown in Table 3. Figure 5 After ten days of drought treatment, about 90% of WT plants were observed to show leaf wilting, while the transgenic plants showed more green leaves and milder wilting symptoms. Figure 5 ​A, 5B). Then, we measured the activities of peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) in OE-2 and WT plants under normal growth and stress treatment conditions. The results showed that the activities of POD, SOD and CAT in transgenic Arabidopsis were higher than that of WT under drought stress, which indicated that the transgenic Arabidopsis had stronger antioxidant stress ability. In addition, we found that the content of malondialdehyde (MDA) in WT and OE lines was basically equal under normal growth conditions. Under drought stress, although the accumulation of MDA in WT and OE lines increased, the increase of MDA in OE lines was smaller, and the accumulation of MDA in WT lines increased significantly, which was more than twice that of OE lines (e.g. Figure 5 C-F). These results indicated that overexpression of PgMYB14 could improve the drought tolerance of Arabidopsis.

[0179] To further explore the mechanism of PgMYB14 in regulating plant response to abiotic stress, we detected the expression levels of some abiotic stress-related genes in PgMYB14 and WT plants by real-time quantitative PCR. The results showed that the expression levels of AtCBF1, AtDREB2A, AtCOR47 and AtKIN1 in PgMYB14 transgenic plants were significantly higher than that of WT plants after 5 days and 10 days of drought stress treatment (G-J). These results indicated that PgMYB14 might enhance the drought tolerance of Arabidopsis by promoting the expression of stress response-related genes. Figure 5

[0180] Obviously, the above examples are only examples for clarity, and not limited to the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present invention.​

Claims

1. Application of overexpression of ginseng transcription factor PgMYB14 gene in improving plant drought tolerance, characterized in that: described PgMYB14 The sequence of the gene is shown in SEQ ID NO. 2; the plant is Arabidopsis thaliana.

2. Application of overexpressing the ginseng transcription factor PgMYB14 gene in improving the expression level of genes related to plant drought tolerance, characterized in that: The sequence of the PgMYB14 gene is shown in SEQ ID NO.2; the plant drought tolerance-related genes are selected from one or more of AtCBF1, AtDREB2A, AtCOR47 and AtKIN1; and the plant is Arabidopsis thaliana.

3. Application of overexpressing ginseng transcription factor PgMYB14 gene in increasing plant O-acyltransferase activity, characterized in that: The sequence of the PgMYB14 gene is shown in SEQ ID NO. 2; and the plant is Arabidopsis thaliana.

4. Use of overexpression of ginseng transcription factor PgMYB14 gene in increasing plant POD, SOD or CAT enzyme activity, characterized in that: The sequence of the PgMYB14 gene is shown in SEQ ID NO. 2; and the plant is Arabidopsis thaliana.

5. Application of overexpressing ginseng transcription factor PgMYB14 gene in reducing plant MDA enzyme activity, characterized in that: The sequence of the PgMYB14 gene is shown in SEQ ID NO. 2; and the plant is Arabidopsis thaliana.

6. A plasmid, characterized in that The plasmid overexpresses the PgMYB14 gene, and the sequence of the PgMYB14 gene is shown in SEQ ID NO.

2.

7. An Agrobacterium competent cell, characterized in that The Agrobacterium competent cells overexpress the PgMYB14 gene, and the sequence of the PgMYB14 gene is shown in SEQ ID NO.

2.

8. Use of the plasmid according to claim 6 or the Agrobacterium competent cell according to claim 7 in enhancing drought tolerance in plants; the plant is Arabidopsis thaliana.

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