Application of DaD27 gene in regulating the resistance of ginseng potato to anthracnose

Abstract

The present invention belongs to the field of genetic engineering technology and specifically relates to <h2 style=";text-align:left;direction:ltr">DaD27 The application of the gene in regulating the resistance of ginseng and potato to anthracnose. <h2 style=";text-align:left;direction:ltr"> DaD27 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein expressed is shown in SEQ ID NO.2. The present invention utilizes virus-induced gene silencing technology to construct <h2 style=";text-align:left;direction:ltr"> DaD27 Gene-silencing of ginseng potato plants, and determination of ABA content and anthracnose resistance index in the silenced ginseng potato, proved <h2 style=";text-align:left;direction:ltr"> DaD27 The function of genes in the resistance of ginseng potato to anthracnose.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology and specifically relates to DaD27 Application of genes in regulating ginseng potato resistance to anthracnose. Background Art

[0002] Ginseng potatoes are herbaceous vines of the Dioscorea family and the Dioscorea genus. Anthracnose is the main disease in ginseng potato production, which easily infects the leaves of ginseng potatoes and causes the death of the entire plant in severe cases, often resulting in a large-scale reduction in ginseng potato production or even a total crop failure, causing serious economic losses. Dioscorea alata .

[0003] At present, the prevention and control measures of anthracnose are mainly based on chemical control, which is easy to cause environmental pollution. Long-term use leads to drug resistance of pathogens. Severe residues of chemical reagents can cause poisoning of humans and livestock. Therefore, the prevention and control of anthracnose should accelerate the cultivation of high-quality anthracnose-resistant new germplasm. Breeding disease-resistant varieties is the most fundamental way to prevent the large-scale occurrence of yam anthracnose. However, the disease resistance mechanism of ginseng potato anthracnose is unknown. Exploring the molecular mechanism of ginseng potato resistance to anthracnose is of great significance to improving the resistance of ginseng potato to anthracnose and cultivating new disease-resistant varieties.

[0004] It is reported that DWARF27, a key enzyme encoding strigolactone synthesis in Arabidopsis, negatively regulates 9-cis-violetaxanthin, a precursor of abscisic acid synthesis, thereby negatively regulating abscisic acid synthesis. Recent studies have shown that abscisic acid can regulate the closure of stomata, thereby improving the drought resistance of plants. At present, it is necessary to develop key enzyme genes for strigolactone synthesis that negatively regulate abscisic acid to participate in new applications in other areas. Summary of the invention

[0005] In order to develop a new application of a key enzyme gene for the synthesis of strigolactones that negatively regulates abscisic acid in other aspects, the present invention has discovered a ginseng potato gene that regulates the resistance to anthracnose of ginseng potato. DaD27 Genes provide ginseng DaD27 Application of genes in improving plant stress resistance. To achieve the above object, the present invention adopts the following technical solution.

[0006] The present invention provides DaD27 Application of gene in regulating ginseng potato resistance to anthracnose, the DaD27 The nucleotide sequence of the gene is shown in SEQ ID NO.1. DaD27 The gene is a key enzyme gene for the synthesis of strigolactones that negatively regulates abscisic acid, and the gene can be used to regulate the anthracnose resistance of ginseng and potato. The present invention provides the application of the gene in regulating the anthracnose resistance of ginseng and potato, and develops a new application of a key enzyme gene for the synthesis of strigolactones that negatively regulates abscisic acid in other aspects.

[0007] Preferably, the DaD27The amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO.2.

[0008] Wherein, the ginseng potato DaD27 The method for obtaining the gene is as follows: using the transcriptome sequencing sequence of ginseng potato, nucleotide sequence comparison and retrieval were performed in the ginseng potato whole genome and transcriptome database, and the obtained ginseng potato DaD27 The cDNA sequence of the gene was obtained by designing specific primers D27_F and D27_R using the software Primer Premire 5.0 and amplifying the cDNA of Ginseng potato as a template. DaD27 The nucleotide sequence of a gene.

[0009] Among them, the URL of the whole genome and transcriptome database is: https: / / yambase.org / .

[0010] Ginseng DaD27 cDNA sequence of gene: Dioal.11G002500.1.

[0011] The nucleotide sequence of D27_F is shown in SEQ ID NO.3:

[0012] 5′-ATGGAGCTCATCTCCTTCCC-3′.

[0013] The nucleotide sequence of D27_R is shown in SEQ ID NO.4:

[0014] 5'-TCATGCTTGTAATTTAGGACAAATTGG-3'.

[0015] The present invention utilizes virus-induced gene silencing technology to construct DaD27 Gene silencing of ginseng potato plants, and determination of abscisic acid content and anthracnose resistance index in the silenced ginseng potato, proved DaD27 The function of genes in the resistance of ginseng and potato to anthracnose. Among them, the English name of virus-induced gene silencing is Virus-induced gene silencing, VIGS. The English name of abscisic acid is Abscisic acid, ABA. The further scheme is as follows:

[0016] The present invention uses virus-induced gene silencing technology to make the above-mentioned DaD27 Gene silencing is used to obtain silenced plants with enhanced anthracnose resistance. Wherein, the plant is ginseng potato.

[0017] Preferably, the virus-induced gene silencing technique is to silence DaD27 The recombinant vector of the gene is transferred into the ginseng potato, so that the DaD27 Gene silencing can improve the anthracnose resistance of ginseng potato.

[0018] Preferably, the silence DaD27 The recombinant vector of the gene is constructed using the VIGS virus vector DaD27 Gene silencing vector.

[0019] Preferably, the silence DaD27 The recombinant vector of the gene includes a VIGS virus vector and the DaD27 Gene.

[0020] Preferably, the VIGS viral vector comprises a tobacco rattle virus pTRV1 vector or a tobacco rattle virus pTRV2 vector.

[0021] Preferably, the silence DaD27 The recombinant vector of gene was used to construct DaD27 Gene VIGS silencing system. DaD27 Gene VIGS silencing system is also called DaD27 Gene-enhanced VIGS system in ginseng potato leaves.

[0022] Preferably, the ginseng potato DaD27 The gene VIGS silencing system includes an Agrobacterium liquid containing an auxiliary vector and a silencing DaD27 Agrobacterium culture containing the recombinant vector of the gene.

[0023] The auxiliary vector is a tobacco rattle virus pTRV1 vector.

[0024] Preferably, the silence DaD27 The recombinant vector of the gene is a tobacco rattle virus pTRV2 vector containing a target gene fragment, the target gene fragment is derived from ginseng potato leaves, the target gene fragment is a DaD27 gene specific fragment, and its nucleotide sequence is shown in SEQ ID NO.7. DaD27 The recombinant vector of a gene is referred to as a recombinant vector.

[0025] The present invention constructs a method based on the auxiliary vector DaD27 The specific steps of the VIGS system for ginseng and potato leaves are as follows:

[0026] (1) Construction of recombinant vector

[0027] The expression of the present invention DaD27 The recombinant vector of the gene was amplified by primers TRV_D27_F and TRV_D27_R to obtain the nucleotide sequence shown in SEQ ID NO.7 and then inserted into the tobacco rattle virus pTRV2 vector. Xba I and BamHI restriction site; that is, the 5' end of the first position of SEQ ID NO.7 is adjacent to Xba I restriction site, the last 1 digit of SEQ ID NO.7 is adjacent to the 3' end BamH I restriction site, and the recombinant vector pTRV2-DaD27 was obtained. The recombinant vector pTRV2-DaD27 is also referred to as the recombinant vector.

[0028] The nucleotide sequence of TRV_D27_F is shown in SEQ ID NO.5:

[0029] 5'-AAGGTTACCGAATTCTCTAGAGGATTGGGACTATGAGAGCTTTGT-3'.

[0030] The nucleotide sequence of TRV_D27_R is shown in SEQ ID NO.6:

[0031] 5'-CGTGAGCTCGGTACCGGATCCCCACTGTTCTCCAAATACCTGCA-3'.

[0032] The nucleotide sequence of SEQ ID NO.7 contains an XbaI restriction site at the 5' end and a BamHI restriction site at the 3' end. The nucleotide sequence is as follows:

[0033] tctagaGGATTGGGACTATGAGAGCTTTGTTGATGTCTCAAAGAGAGTTATGATGGGTAGGAACAGGACCCAGCAACAGGTTGTTGTTAGGGAGGTCCTTCTTTCTATGCTTCCCCCTGGTGCTCCTGCTCAGTTTAGGAAATTGTTTCCACCAAC AAAATGGGCAGCTGAATTCAATGCTGCATTGACAGTGCCTTTCTTCCATTGGCTTGTTGGTCCCTCTGAGGTTGTGGAAGTTGAGGTGAATGGTGTAAAGCAAAAGAGTGGAGTGCATATAAAAAAATGCAGGTATTTGGAGAACAGTGGggatcc.

[0034] (2) DaD27 Construction of gene VIGS silenced plants

[0035] The recombinant vector pTRV2-DaD27, tobacco rattle virus pTRV2 vector and tobacco rattle virus pTRV1 vector in step (1) were transformed into Agrobacterium GV3101 competent cells respectively, and PCR identification was positive to obtain bacterial liquid containing the recombinant vector, tobacco rattle virus pTRV2 vector and tobacco rattle virus pTRV1 vector respectively.

[0036] (3) The bacterial solution containing the recombinant vector, tobacco rattle virus pTRV2 vector, and tobacco rattle virus pTRV1 vector in step (2) was placed in LB liquid medium containing 50 mg / L Kan and 25 mg / L Dif, and cultured at 28°C in a shaker at 200 rpm with an OD of 600 to 1.0, centrifuge at room temperature and discard the supernatant, collect the bacteria, resuspend the bacteria with an equal volume of MS infection solution, mix by pipetting, and then mix the recombinant vector pTRV2-DaD27, tobacco rattle virus pTRV2 vector and tobacco rattle virus pTRV1 vector in equal volumes, and let stand in the dark for 4 hours; inject the MS infection solution into the back of ginseng and potato leaves to achieve the target gene DaD27 Gene silencing and control plant construction, in which the recombinant vector pTRV2-DaD27 and the tobacco rattle virus pTRV1 vector were mixed with MS infection fluid and injected into the plant as the target gene DaD27 The silenced plants and the plants injected with the mixed infection solution of tobacco rattle virus pTRV2 vector and tobacco rattle virus pTRV1 vector were used as control plants. The real-time quantitative PCR method was used to detect the expression of primers TRV_D27_F and TRV_D27_R in each VIGS plant. DaD27 The expression level of the gene.

[0037] The formula of MS infection solution is: MS medium + 10mM / L MES + 200μM / L AS + 10mM / LMgCl2, pH = 5.6. The Chinese name of MES is 2-morpholineethanesulfonic acid; the Chinese name of AS is acetosyringone.

[0038] Preferably, the plant is Ginseng tuber.

[0039] The present invention provides DaD27 Application of gene-silencing ginseng and potato materials in regulating ginseng and potato resistance to anthracnose. Under normal growth conditions, the target plant DaD27 The expression of the gene can increase the abscisic acid content in the leaves of ginseng and potato, close the stoma of the leaves, and significantly enhance the anthracnose resistance of ginseng and potato plants. DaD27 The study of the molecular mechanism of genes and anthracnose resistance has guiding significance.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention provides DaD27 The invention provides a gene for negatively regulating ABA synthesis. DaD27 The acquisition of the gene and the functional verification of its related materials in anthracnose resistance show that the present invention has potential application prospects in improving plant resistance to anthracnose. The present invention provides the application of the gene in regulating the anthracnose resistance of ginseng and potato, and develops a new application of a key enzyme gene for the synthesis of strigolactones that negatively regulates abscisic acid to participate in other aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The ginseng potato in the present invention DaD27 Cloning of genes.

[0043] Figure 2 The resistant and susceptible ginseng potato germplasm of the present invention was inoculated / not inoculated with anthracnose pathogens on the 1st day, the 3rd day, and the 5th day in the leaves DaD27 Changes in gene expression.

[0044] Figure 3 For the present invention DaD27 The leaves of gene silenced plants VIGS-DaD27 and control VIGS-CK DaD27 Gene expression status.

[0045] Figure 4 For the present invention DaD27 The phenotype of the gene silenced plant VIGS-DaD27 ginseng potato leaves inoculated with anthracnose pathogen; Figure 4 Figure A in the figure shows the control VIGS-CK ginseng potato leaves; Figure 4 Figure B in the figure is DaD27 Leaves of gene silenced VIGS-DaD27 ginseng potato.

[0046] Figure 5 For the present invention DaD27 ABA content in leaves of gene silenced plant VIGS-DaD27.

[0047] Figure 6 For the present invention DaD27 Stomatal status of the leaves of the gene silenced plant VIGS-DaD27 after inoculation with anthracnose pathogen; Figure 6 Figure A in the figure shows the control VIGS-CK ginseng potato leaves; Figure 6 Figure B in the figure is DaD27 Leaves of gene silenced VIGS-DaD27 ginseng potato. DETAILED DESCRIPTION

[0048] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0049] Embodiment 1: DaD27 Cloning of gene fragments

[0050] 1. Use the plant RNA extraction kit provided by TaKaRa to extract RNA from ginseng potato leaves.

[0051] Among them, plant RNA extraction kit: MiniBEST Plant RNA Extraction Kit.

[0052] 2. cDNA synthesis method

[0053] The RNA extracted as above was used as a template and reverse transcription was performed using a reverse transcription kit provided by TaKaRa to obtain the first-strand cDNA.

[0054] Among them, reverse transcription kit: PrimeScript™ RT Reagent Kit.

[0055] 3. Using the transcriptome sequence of ginseng potato, nucleotide sequence comparison and retrieval were performed in the ginseng potato whole genome and transcriptome database to obtain the ginseng potato DaD27 The cDNA sequence of the gene was obtained. Based on this sequence, specific primers D27_F and D27_R were designed. The cDNA of Ginseng potato was used as a template for amplification. The PCR amplification reaction system is shown in Table 1.

[0056] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, for a total of 30 cycles, and finally extension at 72°C for 10 min.

[0057] The PCR products were detected by 2% agarose gel electrophoresis. Figure 1 shown.

[0058] Among them, the URL of the whole genome and transcriptome database is: https: / / yambase.org / .

[0059] Ginseng DaD27 cDNA sequence of gene: Dioal.11G002500.1.

[0060] The nucleotide sequence of D27_F is shown in SEQ ID NO.3:

[0061] 5′-ATGGAGCTCATCTCCTTCCC-3′.

[0062] The nucleotide sequence of D27_R is shown in SEQ ID NO.4:

[0063] 5'-TCATGCTTGTAATTTAGGACAAATTGG-3'.

[0064] Table 1 PCR amplification reaction system

[0065]

[0066] 4. Sequencing of PCR products to obtain ginseng potato DaD27 The nucleotide sequence of the gene is shown in SEQ ID NO.1:

[0067] ATGGAGCTCATCTCCTTCCCCAGCACAATTGCTAGAGTTCCACCAGCAAAAGGAGCAGTGCTAATGAGAAGTAAACAATGGCAAACCATTAATGGTCCTTGTATTATCAGGAATGGTGGAATAGCTGAACCATCAGGCATGCCTGCACCAATGGGGCAGAAGACACAGTACAAAGATGGCTTTTCTGAGAAGGCTTTCATGTCTCTGTTTGCCCGGAAGATGGGGAAGTTTGCTTCAGACACTAAGTCCAACAACAACAAGAGTGAGAGAGGCTTTTGGGATTGGGACTATGAGAGCTTTGTTGATGTCTCAAAGAGAGTTATGATGGGTAGGAACAGGACCCAGCAACAGGTTGTTGTTAGGGAGGTCCTTCTTTCTATGCTTCCCCCTGGTGCTCCTGCTCAGTTTAGGAAATTGTTTCCACCAACAAAATGGGCAGCTGAATTCAATGCTGCATTGACAGTGCCTTTCTTCCATTGGCTTGTTGGTCCCTCTGAGGTTGTGGAAGTTGAGGTGAATGGTGTAAAGCAAAAGAGTGGAGTGCATATAAAAAAATGCAGGTATTTGGAGAACAGTGGTTGTGTTGGGATGTGTGTCAATATGTGCAAGTTTCCAACTCAGGATTTCTTCACCAATGACTTTGGACTTCCTTTAACCATGAATCCAAATTTTGAAGATATGAGTTGTGAAATGATTTATGGCCAAGTCCCACCTCCTTTTGAAGAGGACCCTGTTTCAAAGCAGCCTTGCTATGCTGACTTCTGTTCCCTGGCCACTCCCAGCTCTCCAATTTGTCCTAAATTACAAGCATGA。

[0068] DaD27 The nucleotide sequence of the gene consists of 813 bases.

[0069] DaD27 The amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO.2:

[0070] MELISFPSTIARVPPAKGAVLMRSKQWQTINGPCIIRNGGIAEPSGMPAPMGQKTQYKDGFSEKAFMSLFARKMGKFASDTKSNNNKSERGFWDWDYESFVDVSKRVMMGRNRTQQQVVVREVLLSmLPPGAPAQ FRKLFPPTKWAAEFNAALTVPFFHWLVGPSEVVEVEVNGVKQKSGVHIKKCRYLENSGCVGMCVNMCKFPTQDFFTNDFGLPLTMNPNFEDMSCEMIYGQVPPPFEEDPVSKQPCYADFCSLATPSSPICPKLQA.

[0071] Example 2: After the invasion of the ginseng and potato anthracnose pathogen DaD27 Expression analysis of genes in resistant and susceptible potato germplasms

[0072] 1. Prepare a 40cm×60cm porcelain plate, spray it with alcohol and then rinse it with sterile water, evenly spread the straw paper soaked with sterile water on the bottom of the porcelain plate to keep it moist, and place a hollow plastic grid on it; use 75% alcohol by volume to scrub the surface of the leaves of resistant and susceptible ginseng potatoes, and use a sterile needle to poke four to five holes on the left and right sides of the leaf veins. Inoculate the blank culture medium on the left side of the leaf vein as the control group and the anthracnose on the right side of the leaf vein as the treatment group. Among them, the control group is also called the CK group.

[0073] The resistant and susceptible ginseng potato varieties are D284 and D404, which are from Shangyou City and Anyuan County, Jiangxi Province, respectively.

[0074] The control group used a 5mm-aperture puncher to punch blank culture medium from a blank PDA culture medium, and used a sterilized inoculation needle to place it on the left side of the leaf vein of the resistant and susceptible potato germplasms. The treatment group used a 5mm-aperture puncher to punch mycelium blocks from the edge of the colony, and used a sterilized inoculation needle to place them on the right side of the leaf vein of the same leaf of the resistant and susceptible potato germplasms. Cotton balls soaked in 75% alcohol by volume were placed on the left and right culture media to isolate external bacteria. Each germplasm sample was repeated three times, and finally the inoculated leaves were placed on the grid rack in the porcelain plate. After all inoculations were completed, plastic wrap was applied to the porcelain plate, and the porcelain plate was uniformly placed in an incubator with a relative humidity of 95% and a temperature of 26°C. After 24 hours of dark moisturizing treatment, it was cultured under normal light for 7 days. The cotton was removed on the second day after inoculation, and the disease was investigated every other day. Among them, relative humidity is abbreviated as RH in English.

[0075] 2. Take blank culture medium of resistant and susceptible ginseng potato germplasm and leaves from the inoculated area treated with anthracnose, extract total RNA from leaves, and reverse transcribe to synthesize first-chain cDNA; use first-chain cDNA as template and EF-1α The gene was used as the internal reference gene, and the expression of the gene was detected by fluorescence quantitative PCR.

[0076] The total RNA of leaves was extracted using the plant RNA extraction kit provided by TaKaRa. Plant RNA extraction kit: MiniBEST Plant RNA Extraction Kit.

[0077] The PCR system was performed in 20 μL using the fluorescent quantitative reagent provided by Takara, including: 1 μL forward primer, 0.5 μL reverse primer, 100 ng template, 10 μL SYBR® Premix Ex Taq™ II Mix 2X, and RNase-free water to make up to 20 μL.

[0078] Among them, the fluorescent quantitative reagent is: SYBR® Premix Ex Taq™ II.

[0079] The forward primer itself has a concentration of 10 μM; the reverse primer itself has a concentration of 10 μM in 0.5 μL.

[0080] The template was cDNA from leaves of Ginseng tuber.

[0081] The procedure of the fluorescent quantitative PCR amplification preferably includes: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 20 s, for a total of 40 cycles.

[0082] The fluorescent quantitative PCR detection uses a primer pair consisting of D27_F and D27_R to detect DaD27 The primer pair consisting of EF-1α-F and EF-1α-R was used to detect the internal reference gene EF-1α expression.

[0083] Wherein, the nucleotide sequence of D27_F is shown in SEQ ID NO.3:

[0084] 5′-ATGGAGCTCATCTCCTTCCC-3′.

[0085] The nucleotide sequence of D27_R is shown in SEQ ID NO.4:

[0086] 5'-TCATGCTTGTAATTTAGGACAAATTGG-3'.

[0087] The nucleotide sequence of EF-1α-F is shown in SEQ ID NO.8:

[0088] 5'-TCAGGCTGACTGTGCTGTCCT-3'.

[0089] The nucleotide sequence of EF-1α-R is shown in SEQ ID NO.9:

[0090] 5′-GTGGTGGCGTCCATCTTGTT-3′.

[0091] Test results such as Figure 2 As shown in the figure, after the invasion of the ginseng and potato anthracnose pathogen, the leaves of the DaD27 The gene was significantly down-regulated in disease-resistant germplasm, P<0.05.

[0092] Example 3: Ginseng and potato DaD27 Construction of VIGS-DaD27 gene silenced plant

[0093] 1. Construction of recombinant vector pTRV2-DaD27

[0094] Pick DaD27 A nucleotide sequence as shown in SEQ ID NO.7 in the gene sequence is obtained by amplification using primers TRV_D27_F and TRV_D27_R. The obtained nucleotide sequence is inserted into the tobacco rattle virus pTRV2 vector Xba I and BamH I restriction site; that is, the 5' end of the first position of SEQ ID NO.7 is adjacent to Xba I restriction site, the last 1 digit of SEQ ID NO.7 is adjacent to the 3' end BamH I restriction site, and the recombinant vector pTRV2-DaD27 was obtained. The recombinant vector pTRV2-DaD27 is also called plant expression vector pTRV2-DaD27 and recombinant vector.

[0095] Among them, the tobacco rattle virus pTRV2 vector was purchased from Newpro Biotechnology Cat.#V012780.

[0096] The nucleotide sequence of TRV_D27_F is shown in SEQ ID NO.5:

[0097] 5'-AAGGTTACCGAATTCTCTAGAGGATTGGGACTATGAGAGCTTTGT-3'.

[0098] The nucleotide sequence of TRV_D27_R is shown in SEQ ID NO.6:

[0099] 5'-CGTGAGCTCGGTACCGGATCCCCACTGTTCTCCAAATACCTGCA-3'.

[0100] The nucleotide sequence of SEQ ID NO.7 contains Xba I restriction site, 3' end contains BamH I restriction site, the nucleotide sequence of which is as follows:

[0101] tctagaGGATTGGGACTATGAGAGCTTTGTTGATGTCTCAAAGAGAGTTATGATGGGTAGGAACAGGACCCAGCAACAGGTTGTTGTTAGGGAGGTCCTTCTTTCTATGCTTCCCCCTGGTGCTCCTGCTCAGTTTAGGAAATTGTTTCCACCAAC AAAATGGGCAGCTGAATTCAATGCTGCATTGACAGTGCCTTTCTTCCATTGGCTTGTTGGTCCCTCTGAGGTTGTGGAAGTTGAGGTGAATGGTGTAAAGCAAAAGAGTGGAGTGCATATAAAAAAATGCAGGTATTTGGAGAACAGTGGggatcc.

[0102] 2. Ginseng and potato DaD27 Construction of gene silenced plant VIGS-DaD27;

[0103] The tobacco rattle virus pTRV1 vector, tobacco rattle virus pTRV2 vector and recombinant vector pTRV2-DaD27 were transferred into Agrobacterium GV3101 by freeze-thaw method. The specific steps are as follows:

[0104] Two 50 μL Agrobacterium GV3101 competent EP tubes were pre-thawed on ice, and 3 μL of 200 ng / μL tobacco rattle virus pTRV1 vector, tobacco rattle virus pTRV2 vector and recombinant vector pTRV2-DaD27 were taken out and added to three tubes of Agrobacterium competent respectively. The EP tubes were quickly plunged into liquid nitrogen and frozen for 5 minutes. After being taken out, they were transferred to a 37°C water bath to thaw for 5 minutes, and then 500 μL of LB liquid was added to a 28°C shaking incubator. After shaking the bacteria at 200 rpm for 2 hours, the plates were spread on LB solid culture medium containing 25 μg / mL rifamycin and 50 μg / mL kanamycin for growth, positive single clones were screened, and the above-mentioned primers TRV_D27_F and TRV_D27_R were used for colony PCR amplification to identify positive clones, and Agrobacterium GV3101 containing tobacco rattle virus pTRV1 vector, tobacco rattle virus pTRV2 vector and plant expression vector pTRV2-DaD27 was obtained.

[0105] Among them, the tobacco rattle virus pTRV1 vector is an auxiliary vector. The tobacco rattle virus pTRV1 vector is also called pTRV1 vector.

[0106] The tobacco rattle virus pTRV2 vector was used as a control group.

[0107] The plant expression vector pTRV2-DaD27 is the recombinant vector pTRV2-DaD27.

[0108] 4 μL of Agrobacterium liquid containing tobacco rattle virus pTRV1 vector, tobacco rattle virus pTRV2 vector and recombinant vector pTRV2-DaD27 were inoculated into 10 mL of LB medium containing 25 μg / mL rifamycin and 50 μg / mL kanamycin, and cultured at 28°C and 250 r / min shaking. After about 20 hours, the OD value was measured using a spectrophotometer and cultured until OD 600 When the value was 1.0, centrifuge at 3500r / min for 10min and discard the supernatant. Add 10mL MS infection solution, break up the resuspended bacteria with a pipette, mix them evenly, mix the Agrobacterium containing the tobacco rattle virus pTRV1 vector with the Agrobacterium containing the recombinant vector pTRV2-DaD27 and the tobacco rattle virus pTRV2 vector in equal proportions, then let it stand for 4h, and inject it into the back of the ginseng potato leaves with a syringe to construct DaD27 Gene silenced plant VIGS-DaD27 and control plant VIGS-CK.

[0109] Among them, the formula of MS infection solution is: MS + 10mM / L MES + 200μM / L AS + 10mM / L MgCl2, pH = 5.6.

[0110] The Chinese name of MES is 2-morpholineethanesulfonic acid; the Chinese name of AS is acetosyringone.

[0111] 3. Detection of gene silencing efficiency in VIGS silenced plants

[0112] DaD27 Gene expression detection: DaD27 RNA was extracted from leaves of gene silenced plants VIGS-DaD27 and control plants VIGS-CK and reverse transcribed into cDNA. DaD27 The gene expression level was detected. The method of extracting RNA and reverse transcribing it into cDNA was the same as in Example 1. The detection results are shown in Figure 3 shown.

[0113] Results DaD27 Gene silenced plant VIGS-DaD27DaD27 The expression of the gene was significantly lower than that of the control VIGS-CK, indicating that the silenced plants were successfully constructed.

[0114] Example 4: Ginseng and potato DaD27 Gene silencing plant VIGS -DaD27 Functional validation in anti-anthrax

[0115] 1. Ginseng and potato DaD27 Gene silencing plant VIGS -DaD27 Phenotype of anthrax inoculation

[0116] The anthracnose bacteria were inoculated into ginseng potatoes using the bacterial cake method. DaD27 Gene silencing plant VIGS -DaD27 For the leaves of VIGS-CK and control plants, four to five holes were pierced on the left and right sides of the leaf veins with a sterile needle. The left side of each leaf was inoculated with a blank PDA medium. The right side was inoculated with anthrax bacteria, and a cotton ball soaked with 75% alcohol was used to cover the bacterial cake. After the inoculation, it was placed in an incubator with a relative humidity of 95% and a temperature of 26°C. After 24 hours of dark moisturizing treatment, it was cultured under normal light. The cotton ball was removed 48 hours after inoculation. The ginseng potato could be observed on the 5th day of inoculation. DaD27 The leaf lesions of the gene silenced plant VIGS-DaD27 expanded more slowly than those of the control plant VIGS-CK, indicating enhanced disease resistance. Figure 4 shown.

[0117] Depend on Figure 4 It can be seen that ginseng DaD27 The leaf lesions of the gene-silenced plant VIGS-DaD27 expanded more slowly than those of the control plant VIGS-CK, indicating enhanced disease resistance.

[0118] Example 5: Ginseng and Potato DaD27 Abscisic acid content in gene silenced plants VIGS-DaD27

[0119] The plant hormone abscisic acid enzyme-linked immunosorbent assay kit provided by Kelu (Wuhan) Biotechnology Co., Ltd. was used to detect DaD27 The abscisic acid content in the leaves of the gene-silenced plant VIGS-DaD27 was analyzed.

[0120] Among them, the plant hormone abscisic acid enzyme-linked immunosorbent assay kit: the product number is ELK8563

[0121] The results are as follows Figure 5 shown.

[0122] The results showed DaD27 The ABA content in the leaves of the gene silenced plant VIGS-DaD27 was significantly higher than that in the leaves of the control plant VIGS-CK.

[0123] Example 6: Ginseng and PotatoDaD27 Stomatal detection in leaves of gene silenced plants VIGS-DaD27

[0124] Will DaD27 Place the leaves of the gene silenced plant VIGS-DaD27 and the control plant VIGS-CK on the index finger, fix the leaf with the thumb, gently scratch the leaf with a scalpel to expose the mesophyll cells, and completely tear off the epidermis with tweezers. Drop 2 drops of distilled water in the center of the slide, immerse the torn epidermis in the center of the slide, cover it with a coverslip, observe the state of the stomata under a microscope and take photos to record the results. Figure 6 shown.

[0125] The results showed DaD27 The stomata of the leaves of the gene silenced plant VIGS-DaD27 were less open than those of the control plant VIGS-CK and were in a closed state.

[0126] The present invention provides DaD27 Application of gene in regulating ginseng potato resistance to anthracnose, the DaD27 The nucleotide sequence of the gene is shown in SEQ ID NO.1. DaD27 The gene is a key enzyme gene for the synthesis of strigolactones that negatively regulates abscisic acid, and the gene can be used to regulate the anthracnose resistance of ginseng and potato. The present invention provides the application of the gene in regulating the anthracnose resistance of ginseng and potato, and develops a new application of a key enzyme gene for the synthesis of strigolactones that negatively regulates abscisic acid in other aspects.

[0127] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0128] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once the basic inventive concepts are known, and all such changes and modifications fall within the scope of the present invention.

Claims

1. DaD27 The application of the gene in regulating the resistance of ginseng and potato to anthracnose is characterized in that: Said DaD27 The nucleotide sequence of the gene is shown in SEQ ID NO 1; Virus-induced gene silencing technology was used to DaD27 Gene silencing to obtain silent ginseng potatoes with enhanced anthracnose resistance.

2. according to claim 1 DaD27 The application of the gene in regulating the resistance of ginseng and potato to anthracnose is characterized in that: Said DaD27 The amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO 2.

3. According to claim 1 DaD27 The application of the gene in regulating the resistance of ginseng and potato to anthracnose is characterized in that: The virus-induced gene silencing technique is to silence DaD27 The recombinant vector of the gene is transferred into the ginseng potato, so that the DaD27 Gene silencing can improve the anthracnose resistance of ginseng potato.

4. according to claim 3 DaD27 The application of the gene in regulating the resistance of ginseng and potato to anthracnose is characterized in that: The Silence DaD27 The recombinant vector of the gene is constructed using the VIGS virus vector DaD27 Gene silencing vector.

5. According to claim 4 DaD27 The application of the gene in regulating the resistance of ginseng and potato to anthracnose is characterized in that: The Silence DaD27 The recombinant vector of the gene includes a VIGS virus vector and the DaD27 Gene.

6. According to claim 5 DaD27 The application of the gene in regulating the resistance of ginseng and potato to anthracnose is characterized in that: The VIGS virus vector includes a tobacco rattle virus pTRV1 vector or a tobacco rattle virus pTRV2 vector.

7. According to claim 3 DaD27 The application of the gene in regulating the resistance of ginseng and potato to anthracnose is characterized in that: The Silence DaD27 The recombinant vector of gene was used to construct DaD27 Gene VIGS silencing system.

8. According to claim 7 DaD27 The application of the gene in regulating the resistance of ginseng and potato to anthracnose is characterized in that: The ginseng potato DaD27 The gene VIGS silencing system includes an Agrobacterium liquid containing an auxiliary vector and a silencing DaD27 Agrobacterium solution of the recombinant vector of the gene; The auxiliary vector is a tobacco rattle virus pTRV2 vector.

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