Panax notoginseng PnMYB7 gene and application thereof

Abstract

The application discloses a Panax notoginseng PnMYB7 gene, wherein the nucleotide sequence is shown as SEQ ID NO:1, the gene has transcription activation activity, the gene expression is induced by exogenous salicylic acid and fusarium oxysporum infection, the gene transient expression in tobacco leaves increases the resistance of tobacco to fusarium oxysporum infection, the gene is genetically transformed into tobacco to obtain PnMYB7 transgenic tobacco plants, the expression level of PAL4, WIPK, PR1c and Definsin defense genes can be improved, and the gene has application potential in resisting fusarium oxysporum infection in plants.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a Panax notoginseng PnMYB7 gene and its application in improving the antibacterial ability of plants. Background Technology

[0002] Sanqi ( Panax notoginseng Panax notoginseng (Burk.) FH Chen is a traditional Chinese medicinal herb belonging to the Araliaceae family and the Panax genus. It is known as the "King of Ginseng" and its main chemical components include saponins, polysaccharides, amino acids, flavonoids, alkaloids, and other substances. It possesses beneficial pharmacological effects such as hemostasis, blood tonification, lowering blood sugar and lipids, immune regulation, and anti-inflammation. In recent years, with the rapid development of the domestic Chinese medicine industry, the market sales volume of Panax notoginseng has increased significantly, thus contributing to the economic growth of Yunnan Province.

[0003] MYB transcription factors participate in numerous physiological responses in plants, such as environmental responses and stress responses, and play irreplaceable roles in many aspects. As important regulators related to plant resistance, MYB transcription factors have attracted much attention and become a research hotspot for improving plant resistance, thanks to advancements in whole-genome sequencing and experimental techniques. MYB transcription factors contain a highly conserved MYB domain at their N-terminus. A single MYB domain consists of 50 to 52 amino acids, typically including three conserved tryptophan residues, encoding three α-helices to form a helical-turn-helical structure. Based on the number of conserved domains, they are classified into four types: 1R-MYB (MYB-related), R2R3-MYB, 4R-MYB, and 3R-MYB, with R2R3-MYB transcription factors being the most numerous found in plants. R2R3-MYB transcription factors contain two conserved MYB domains at their N-terminus and play a crucial role in plant physiological responses to biotic stress, abiotic stress, and anabolic metabolism.

[0004] Biological stress is a collective term for the harm caused to plants by organisms such as microorganisms, pests, and animals. Many genes and proteins are affected by biological stress, among which the R2R3-MYB transcription factor family is a crucial member, playing an important role in the response to biological stress. Jiang et al. cloned and identified a new disease-resistant R2R3-MYB gene, VqMYB154, from the disease-resistant grape variety "Danfeng-2". Their study found that pathogens such as *Pseudomonas syringae* directly activated VqMYB154 and its promoter. Further identification revealed that this gene participates in the biosynthesis of plant antitoxins, promotes the expression of multiple downstream genes, and enhances their resistance to pathogens, thus playing a positive role in the plant's defense response. Previous research by Xie et al. showed that the OsMYB63 transcription factor is the transcriptional activator of the cellulose synthase CESA gene. Phenotypic identification indicated that the OsMYB63 transcription factor positively regulates rice resistance to *Xanthomonas xanthomonas* and promotes sclerenchyma cell wall development, thereby conferring resistance to bacterial blight in rice.

[0005] Abiotic stresses, including drought, salinity, extreme temperatures, and sugar stress, can also adversely affect plant growth and development. To complete the entire growth cycle, plants regulate relevant metabolic levels under stress to maintain cellular homeostasis. Studies have found that overexpression of the MbMYBC1 gene in Arabidopsis thaliana can activate the expression of downstream drought-related AtRD29a promoters, AtSOD1, and AtP5CS1, indicating that MbMYBC1 has a positive response to drought stress. In tartary buckwheat, treatment with mannitol and NaCl induced upregulation of the FtMYB41 gene expression in seedlings, suggesting that the FtMYB41 gene can actively respond to drought stress.

[0006] Panax notoginseng is a traditional Chinese medicine, commonly used to treat cardiovascular and cerebrovascular diseases with significant effects. Yunnan Province is a major cultivation area for Panax notoginseng and a pillar industry of Yunnan's medicinal herb industry. However, Panax notoginseng's preference for warm, humid environments and its large-scale monoculture over many years creates favorable conditions for the occurrence and spread of diseases. Root rot can occur throughout the entire growth and development process of Panax notoginseng, accounting for 75% to 80% of economic losses caused by various diseases, making it a major factor restricting the development of the Panax notoginseng industry. Currently, the most common method for controlling root rot is still the use of chemical fungicides, but this cannot completely eliminate the disease and also leads to problems such as pesticide residues and environmental pollution. Therefore, seeking green, safe, and effective control measures is crucial for the development of the Panax notoginseng industry. Furthermore, identifying genes in Panax notoginseng related to plant disease resistance is of great significance for revealing the disease resistance mechanism and cultivating disease-resistant varieties. Summary of the Invention

[0007] This invention provides a Panax notoginseng PnMYB7 gene, the nucleotide sequence of which is shown in SEQ ID NO:1, encoding a protein with the amino acid sequence shown in SEQ ID NO:2; it is derived from Panax notoginseng ( Panax notoginseng The gene sequence was cloned from the (Burk.) FHChen plant. The full-length gene sequence is 894 bp. The encoded PnMYB7 protein consists of 297 amino acids and has a molecular weight of approximately 32 kDa.

[0008] Another objective of this invention is to apply the aforementioned Panax notoginseng PnMYB7 gene to improve the plant's resistance to Fusarium oxysporum (Fusarium oxysporum). Fusarium oxysporum In the resistance of ).

[0009] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution: 1. Total RNA was extracted from Panax notoginseng plants grown in a greenhouse at the College of Life Sciences and Technology, Kunming University of Science and Technology, Yunnan Province. Primers were designed based on the gene sequence. Using Panax notoginseng cDNA as a template, the full-length fragment and each deleted fragment of the MYB7 gene were amplified using the designed primers. The amplified fragment was ligated into the pMD-19T vector and transformed into Escherichia coli DH5α. Then, the fragments were plated on ampicillin-containing plates, and positive colonies were picked for sequencing. The gene sequence encoding the MYB7 protein was identified, and its sequence is shown in SEQ ID NO:1. The protein is encoded by 297 amino acids, and the amino acid sequence is shown in SEQ ID NO:2. 2. The target fragments were recovered from the correctly sequenced pMD-19T-MYB7F, pMD-19T-MYB7N, and pMD-19T-MYB7C, and ligated into the yeast expression vector pGBKT7 plasmid digested with restriction endonucleases to obtain recombinant plasmids pGBKT7-PnMYB7F, pGBKT7-PnMYB7N, and pGBKT7-PnMYB7C. Each recombinant plasmid was transformed into Saccharomyces cerevisiae Y2HGold strain using the LiAC method and plated on SD / -Trp medium for culture to obtain recombinant yeast strains Y2HGold-pGBKT7, Y2HGold-pGBKT7-PnMYB7F, Y2H Gold-pGBKT7-PnMYB7N, and Y2HGold-pGBKT7-PnMYB7C. The above yeast strains were cultured on SD / -Trp-His-Ade medium, which confirmed that the PnMYB transcription factor has a transcriptional activation function and determined that the transcriptional activation domain is located at the C-terminus.

[0010] 3. The transcriptional level of the PnMYB7 gene in Panax notoginseng leaves was analyzed using qRT-PCR under salicylic acid (SA) treatment, Fusarium oxysporum infection, and co-treatment with salicylic acid and Fusarium oxysporum. The results showed that salicylic acid treatment, Fusarium oxysporum infection, and co-treatment with both could induce PnMYB7 gene expression.

[0011] 4. Using Panax notoginseng cDNA as a template, the PnMYB7 fragment was amplified with specific primers. Then, it was ligated with the pRI101-GFP vector digested by restriction endonuclease using homologous recombination and transformed into Escherichia coli DH5α to obtain the recombinant plasmid pRI101-GFP-PnMYB7 and the recombinant Escherichia coli strain DH5α-pRI101-GFP-PnMYB7 containing the recombinant expression plasmid.

[0012] 5. The correctly sequenced pRI101-GFP-PnMYB7 recombinant plasmid was transformed into Agrobacterium LBA4404 using a repeated freeze-thaw method to obtain the recombinant Agrobacterium strain LBA4404-pRI101-GFP-PnMYB7 containing the recombinant expression plasmid. First, transient expression was used to preliminarily explore the role of PnMYB7 in plant resistance to biotic stress. Then, the recombinant Agrobacterium strain LBA4404-pRI101-GFP-PnMYB7 was genetically transformed into tobacco leaves using the leaf disc method. After screening and culture, transgenic tobacco containing the Panax notoginseng PnMYB7 gene was successfully obtained. The expression levels of four related defense genes, PAL4 (phenylalanine ammonia-lyase 4), WIPK (wound-inducing protein kinase), Definsin, and PR1c (PR gene), in PnMYB7 transgenic tobacco and wild-type tobacco leaves were detected.

[0013] The advantages and technical effects of this invention are as follows: The Panax notoginseng PnMYB7 gene of this invention can increase the disease resistance of tobacco. The Panax notoginseng PnMYB7 gene is a transcription factor. Compared to the limitations of overexpressing a single resistance gene, overexpression of a disease resistance-related transcription factor can regulate the expression of a series of downstream functional genes, which is equivalent to simultaneously overexpressing multiple disease resistance-related genes in the plant, thereby improving the plant's disease resistance. This transgenic technology can improve the disease resistance of plants themselves, is green and efficient, and has important application value for breeding disease-resistant plant varieties. Attached Figure Description

[0014] Figure 1Figure 1 shows the construction and detection of the transgenic yeast of this invention. Figure A shows the enzyme digestion detection of the pGBKT7-MYB7F plasmid, Figure B shows the enzyme digestion detection of the pGBKT7-MYB7N plasmid, and Figure C shows the enzyme digestion detection of the pGBKT7-MYB7C plasmid. In the figures, M represents the DNA molecular weight standard; 1, 3, and 6 are the pGBKT7-MYB7F, pGBKT7-MYB7N, and pGBKT7-MYB7C plasmids, respectively; 2, 4, and 5 are the enzyme digestion detection of the pGBKT7-MYB7F, pGBKT7-MYB7N, and pGBKT7-MYB7C plasmids, respectively. Eco RⅠ and Pst I. Enzyme-digested plasmids; Figure 2 The above diagram shows the transcriptional activation activity analysis of the transgenic yeast of this invention. The upper diagram is a schematic diagram of the structure of the PnMYB7 gene deletion fragment, the lower left diagram shows the growth on tryptophan-deficient medium (SD / -T), and the lower right diagram shows the growth on tryptophan / histidine / adenine (SD / -ATH)-deficient medium. Figure 3 This invention describes the transcriptional level of the PnMYB7 gene in the leaves of Panax notoginseng under various treatments; wherein: Figure A shows the transcriptional level of the PnMYB7 gene in the leaves of Panax notoginseng under various treatments. 6 Figure B shows the transcription level of the PnMYB7 gene after treatment with Fusarium oxysporum spore suspension at 1 / mL; Figure C shows the transcription level of the PnMYB7 gene after treatment with 200 μmol / L SA; Figure C shows the transcription level of the PnMYB7 gene after 30 min of sterile water soaking (pretreatment) followed by 10 μmol / L SA treatment. 6 The transcription level of the PnMYB7 gene under treatment with Fusarium spore suspension per mL; Figure D shows the root soaking in 200 μmol / L SA for 30 min (pretreatment), followed by treatment with 10 μmol / L SA for 24 h. 6 Transcriptional level of PnMYB7 gene under treatment with Fusarium spore suspension (spores / mL); Figure 4 This invention illustrates the changes in leaf lesions after transient expression of the PnMYB7 gene in tobacco leaves and inoculation with Fusarium oxysporum. Figure A shows the electrophoretic detection of pRI101-GFP-PnMYB7 transformed into LBA4404 Agrobacterium; Figure B shows the leaf phenotype of tobacco leaves infected with Fusarium oxysporum 5 days after transient expression of the PnMYB7 gene; Figure C shows the leaf lesion area of ​​tobacco leaves infected with Fusarium oxysporum 5 days after transient expression of the PnMYB7 gene. *** indicates a highly significant difference compared to the control (P<0.001). Figure 5 This invention relates to the PCR detection of transgenic tobacco plants; where M is the DNA molecular weight standard; positive control 1: PCR product with PnMYB7-19T as template; 2, 3: PCR products with PnMYB7 recovered product as template; WT is PCR product with wild-type tobacco DNA as template; Figure 6Figure 1 shows the expression levels of relevant defense genes in the leaves of PnMYB7 transgenic tobacco of this invention; Figure A shows the transcription level of PAL4 gene in M1, M2, and M3 transgenic tobacco lines; Figure B shows the transcription level of WIPK gene in M1, M2, and M3 transgenic tobacco lines; Figure C shows the transcription level of Definsin gene in M1, M2, and M3 transgenic tobacco lines; Figure D shows the transcription level of PR1c gene in M1, M2, and M3 transgenic tobacco lines. Detailed Implementation

[0015] The present invention will be further described in detail below through embodiments, but the content of the present invention is not limited thereto. Unless otherwise specified, the methods in this embodiment shall be operated in accordance with conventional methods, and the reagents used shall be conventional reagents or reagents prepared in accordance with conventional methods unless otherwise specified.

[0016] Example 1: Panax notoginseng ( P. notoginseng Cloning and identification of the PnMYB7 gene in plants Total RNA was extracted from Panax notoginseng using the Trizol method. Following the instructions of the reverse transcription kit provided by TaKaRa, cDNA, the reverse transcription product of the total RNA from Panax notoginseng, was obtained. Based on the multiple cloning site of the pGBKT7 vector and the CDS sequence of the target gene PnMYB7, primers with different restriction enzyme sites were designed to amplify the full-length, N-terminal, and C-terminal fragments of the PnMYB7 gene. The forward primer PnMYB7F for amplifying the full-length PnMYB7 gene was 5'- GAATCC ATGGCCACCACGACGAAAGAT-3' (underscore is...) Eco RⅠ restriction site), reverse primer PnMYB7R is 5'- GTCGAC CTATTCGATCCTGCTCATCCC-3' (underscore is...) Pst I. Restriction site). The forward primer PnMYB7-NF for amplifying the N-terminal fragment of the gene is 5'- GAATCC ATGGCCACCACGACGAA-3' (underscore is...) Eco RⅠ restriction site), reverse primer PnMYB7-NR is 5'- GTCGAC AGACGAGCTTTTGCGTTTAAG-3' (underscore is...) Pst I. Restriction site). The forward primer PnMYB7-CF for amplifying the C-terminal fragment of the gene is 5'- GAATCC ATGTCCGATGATGGCGC-3' (underscore is...) Eco RⅠ restriction site), reverse primer PnMYB7-CR is 5'- GTCGACCTATTCGATCCTGCTCATCCCA-3' (underscore is...) Pst I. Enzyme cleavage site).

[0017] Using Panax notoginseng cDNA as a template, the full-length PnMYB7 gene, N-terminal and C-terminal fragments were amplified using designed primers. The PCR reaction conditions were: 94℃ pre-denaturation for 3 min, followed by 30 cycles of denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s, and extension at 72℃ for 1 min, with a final reaction time of 10 min at 72℃. The PnMYB7 gene, N-terminal and C-terminal fragments were cloned into the pMD19-T vector, resulting in TA cloning vectors named pMD19-T-PnMYB7F, pMD19-T-PnMYB7N, and pMD19-T-PnMYB7C. The obtained TA cloning vectors were then verified by enzyme digestion. The plasmids pMD19-T-PnMYB7F, pMD19-T-PnMYB7N, and pMD19-T-PnMYB7C that passed the enzyme digestion test were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The PnMYB7 nucleotide sequence is shown in SEQ ID NO:1 of the sequence listing.

[0018] Example 2: Verifying the transcriptional activation activity of the Panax notoginseng PnMYB7 gene and identifying the transcriptional activation domain. 1. The pGBKT7 plasmid was extracted from *E. coli* using a bioplasmid extraction kit, and then... EcoR I. Pst I. The pGBKT7 vector and plasmids pMD19T-MYB7F, pMD19T-MYB7N, and pMD19T-MYB7C were digested with enzymes, and the target fragments PnMYB7F, PnMYB7N, PnMYB7C and the linearized vector fragment pGBKT7 were recovered and ligated.

[0019] 2. Enzyme digestion and ligation reaction: The enzyme digestion reaction conditions are as follows: 37℃ metal bath for 4 h, recover the linearized pGBKT7 fragment after enzyme digestion, and use it as the vector backbone for ligating the target gene; 37℃ water bath for 2-3 h, recover the target fragment product after enzyme digestion; prepare the ligation system according to the following: Solution I 5.0 μL, target fragment 4.0 μL, vector fragment 1.0 μL; react in a 16℃ metal bath for 12 h.

[0020] 3. Transform the ligation products into DH5α competent cells. Once single colonies grow on the plate, confirm the correct positive clones and detect them by digesting the plasmids with enzymes. Results are shown in [Figure number missing]. Figure 1 The results showed that the recombinant plasmid was successfully constructed.

[0021] 4. To verify the transcriptional activation activity of the Panax notoginseng PnMYB7 gene, plasmids pGBKT7, pGBKT7-PnMYB7F, pGBKT7-PnMYB7N, and pGBKT7-PnMYB7C were transformed into Y2H Gold, respectively. The transgenic yeast was able to grow on tryptophan-deficient medium, indicating that the recombinant plasmids had been successfully transferred into the yeast strain. The results are shown in [Figure number missing]. Figure 2 Using yeast strains carrying the pGBKT7 plasmid as a negative control, the growth of yeast strains on the auxotrophic medium SD / -Trp-His-Ade indicated that PnMYB7 exhibits strong transcriptional activation activity in yeast, with the transcriptional activation domain concentrated at the C-terminus. The results are shown in [Figure number missing]. Figure 2 .

[0022] Example 3: Expression characteristics of the PnMYB7 gene 1. Preparation of spore suspension: Take 200 μL of the *Fusarium oxysporum* spore solution stored at -80℃ and add it to 150 mL of LDA liquid medium. Incubate at 28℃ and 200 rpm for 3-5 days. Then filter through sterile 400-mesh nylon cloth. The filtrate is the spore suspension. Count the spores using a hemocytometer and adjust the spore suspension concentration to 2 × 10⁻⁶. 8 Cells / mL, store at 4℃ for later use; 2. Select healthy, uniformly growing one-year-old Panax notoginseng plants, wash away the soil, and treat the roots by removing any damage. Divide the plants into 5 groups (5 plants / group) and treat them as follows: Group 1 and Group 2 were pretreated by soaking the roots in 200 μmol / L SA and sterile water for 30 minutes, respectively, and then placed in Hoagland's nutrient solution. After 24 hours, they were treated with 10... 6 The roots were soaked in a suspension of Fusarium oxysporum spores per mL for 30 min, and then the Panax notoginseng plants were placed in Hoagland's nutrient solution for further culture; groups 3-5 were treated with 200 μmol / L SA and 10 6 The roots of Panax notoginseng were soaked for 30 min in a suspension of Fusarium oxysporum spores per mL and sterile water (control group), and then cultured in Hoagland's nutrient solution. All treatments were performed in parallel with 5 plants. Leaves were collected at 4, 12, 24, 48, and 72 h after treatment. All collected plant samples were rapidly treated with liquid nitrogen and then stored at -80℃ for later use.

[0023] 3. Extraction and reverse transcription of RNA from Panax notoginseng leaves in each treatment group: Total RNA was extracted from Panax notoginseng samples using the Trizol method, and cDNA was obtained by following the instructions of the reverse transcription kit provided by TaKaRa.

[0024] 4. Quantitative Real-time PCR (qRT-PCR): The PnACT2 actin gene (KF815706.1) of Panax notoginseng was used as an internal control. Primers for qRT-PCR were designed based on the PnMYB7 gene sequence. The forward primer RT-MYB7-F was 5'-CGGAGGAGGACGACACCA-3', and the reverse primer RT-MYB7-R was 5'-GCCATCATCGGACATAGACG-3'. The forward primer for PnACT2 was RT-PnACT2-F, 5'-TCCAAGGGTGAATATGATGAATCG-3', and the reverse primer RT-PnACT2-R was 5'-AACCTCTCCAAAGAGAATTTCTGAGT-3'. Each qRT-PCR reaction was performed in triplicate. The expression level of the PnMYB7 gene in Panax notoginseng leaves treated with sterile water was used as a control. -ΔΔCt The expression level of the PnMYB7 gene under each treatment was calculated.

[0025] See results Figure 3 This indicates that exogenous application of SA and Fusarium oxysporum infection can induce an increase in PnMYB7 expression. Compared with the sterile water pretreatment group, the expression level of PnMYB7 in Panax notoginseng leaves was significantly increased within 72 hours after SA pretreatment and subsequent inoculation with Fusarium oxysporum, indicating that SA treatment can enhance the transcriptional response of the PnMYB7 gene to Fusarium oxysporum infection.

[0026] Example 4: Analysis of PnMYB7 gene-enhanced tobacco disease resistance PnMYB7-specific primers with restriction enzyme sites were designed. PCR amplification was performed using pMD19T-PnMYB7F as a template. The ORF product containing the corresponding PnMYB7 restriction enzyme sites was cloned in one step to obtain the recombinant plasmid pRI101-GFP-PnMYB7, which was then sequenced for verification. The correctly sequenced pRI101-GFP-PnMYB7 recombinant vector was transformed into strain LBA4404. PCR results are shown below. Figure 4 A. Then, tobacco leaves were inoculated using the injection method. A suspension of *Fusarium oxysporum* spores was injected into the wounds of *Tobacco Benedictinea* leaves that had been transformed for 48 hours and were in good condition, ensuring the wounds were evenly covered with the bacterial suspension. The inoculated leaves were then cultured in a light incubator for 5 days, during which leaf phenotypic changes and lesion changes were observed.

[0027] Figure 4B Figure 4Results showed that 5 days after inoculation with *Fusarium oxysporum*, mild rot and browning were observed near the inoculation site in transient transgenic tobacco leaves. In contrast, the control group leaves showed more severe browning near the inoculation site, with some mycelial growth, and the lesion area was significantly larger than that of the transgenic tobacco leaves. Tobacco plants transiently expressing the PnMYB7 gene only showed browning without obvious mycelial growth, and the lesion area was smaller than that of the control group, only 22 mm². 2 Clearly, the expression of PnMYB7 in tobacco enhances the resistance of tobacco plants to Fusarium oxysporum.

[0028] Example 5: Construction and Identification of PnMYB7 Transgenic Tobacco Tobacco leaf discs were infected using the leaf disc transformation method with LBA4404 strain transformed with the pRI101-GFP-PnMYB7 recombinant vector and cultured in selection medium. When regenerated tobacco plants emerged, genomic DNA was extracted from the regenerated plants using the CTAB method, and PCR detection was performed using primers specific to amplify PnMYB7. Figure 5 As shown, some regenerated tobacco plants amplified specific bands of the expected size, while the WT control failed to amplify the target band. This indicates that PnMYB7 has been successfully transferred into tobacco, resulting in a total of 9 PCR-positive transgenic tobacco plants.

[0029] Example 6: Analysis of defense gene expression levels in PnMYB7 transgenic tobacco Using the tobacco actin gene NtACT (accession number AB158612.1) as an internal control, the expression levels of PAL4, WIPK, PR1c, and Definsin defense genes in PnMYB7 transgenic tobacco were detected by qRT-PCR. The qRT-PCR primers are as follows: the forward primer Actin-QF for the internal control genes is 5'-TCCCATTGAGCATGGAATAGTAAGC-3', and the reverse primer Actin-QR is 5'-TACATGGCAGGTACATTGAAAGTCT-3'. The forward primer PAL4-QF for detecting PAL4 expression is 5'-CTATTACAACAACGGTTTGCCATCT-3', and the reverse primer PAL4-QR is 5'-CTTTTGGACATGGTTTGTCACTGGA-3'. The forward primer for detecting WIPK expression, WIPK-QF, is 5'-TCAGGCAACTCCCACAACATCC-3', and the reverse primer, WIPK-QR, is 5'-GTTCGTCACCTGCATCGTGGA-3'. The forward primer for detecting Definsin expression, Definsin-QF, is 5'-GCTACCGAGATGGGACCAATGAC-3', and the reverse primer, Definsin-QR, is 5'-CAAGGGCTGGTACAGAAACAACG-3'. The forward primer for detecting PR1c expression, PR1c-QF, is 5'-AGAACCTTTGACCTGGGACGAC-3', and the reverse primer, PR1c-QR, is 5'-ATCCAACACGAACCGAGTTACG-3'.

[0030] qRT-PCR results are shown below Figure 6 Compared with the wild type, the transcription levels of defense genes PAL4, WIPK, Definsin, and PR1c were significantly increased. Among them, PAL4 and WIPK ( Figure 6 A, Figure 6 The transcription level of Definsin and PR1c was highest in the M2 transgenic lines, 32 and 40 times higher than that in wild-type tobacco, respectively. Figure 6 C Figure 6 The transcriptional level of D) was highest in the M3 transgenic line, being 1.6 times and 16 times that of wild-type tobacco, respectively. In the M1 transgenic line, the transcriptional levels of PAL4, WIPK, and PR1c also showed significant changes, reaching 16 times, 32 times, and 11 times that of wild-type tobacco, respectively. These results indicate that the PnMYB7 gene transfer into tobacco can enhance the expression level of defense genes.

Claims

1. A Panax notoginseng PnMYB7 gene enhances the resistance of tobacco to Fusarium oxysporum (… Fusarium oxysporum Application in resistance; the nucleotide sequence of the Panax notoginseng PnMYB7 gene is shown in SEQ ID NO:1.

Images