Application of potato gene StPYL6

Abstract

The invention relates to the technical field of gene engineering and plant biology, and discloses application of a potato gene StPYL6. The functional verification and application research of the potato soft rot disease-resistant gene StPYL6 are provided, an excess expression vector of the StPYL6 gene is subjected to genetic transformation of an Hubei potato No.3 plant by using a transgenic technology driven by a 35S promoter, and it is found that the soft rot resistance of transgenic potatoes of which the expression quantity of the StPYL6 gene is remarkably improved is obviously enhanced, and the transgenic potatoes of which the expression quantity of the StPYL6 gene is remarkably improved are used for screening the soft rot disease-resistant gene StPYL6. The activity of ROS scavenging enzymes SOD, POD and CAT is enhanced, which proves that the StPYL6 gene plays an important role in soft rot resistance.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and plant biotechnology, specifically to a potato gene. StPYL6 Applications. Background Technology

[0002] Potatoes are highly susceptible to various pathogens during growth, development, and post-harvest storage, severely impacting yield and quality. Among these, Enterobacter soft rot (Enterobacter) is particularly vulnerable. Enterobacteriaceae Bacterial soft rot caused by ) Bacterial soft rot Soft rot is a major bacterial disease affecting potatoes. Currently, there are no effective control measures. Therefore, identifying resistance genes and cultivating resistant varieties are the most effective means of controlling potato soft rot, addressing the urgent need to combat this disease.

[0003] Research on PYL in potato abiotic stress response has mainly focused on the effects of PYL on abiotic stresses such as drought. Studies on PYL in biotic stress response are relatively lacking, and existing techniques have not reported on the regulation of biotic stresses such as soft rot in potatoes. Summary of the Invention

[0004] To address the above problems, this invention provides a potato gene. StPYL6 In its application, this invention screens a [subject] in potatoes. StPYL6 This gene, located in the cytoplasm and nucleus, enhances the disease resistance of potato plants.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a potato gene StPYL6 The potato gene StPYL6 The nucleotide sequence of the CDS region is shown in SEQ ID No. 1.

[0006] This invention also provides a method using potato genes. StPYL6 The encoded protein, the amino acid sequence of which is shown in SEQ ID No. 2.

[0007] The present invention provides an overexpression vector containing a nucleotide sequence as shown in SEQ ID No. 1.

[0008] This invention provides a potato gene StPYL6 Application in improving potato resistance to soft rot.

[0009] Compared with existing technologies, this invention provides a potato gene. StPYL6 Its application has the following beneficial effects: (1) Enhancing the resistance of potato to soft rot The present application provides a potato soft rot disease resistance gene StPYL6 The function verification and application research of the present application, using 35S promoter driven transgenic technology, the overexpression vector of the gene is genetically transformed into potato No. 3 plant, it is found that StPYL6 The transgenic potato with significantly improved gene expression has significantly enhanced resistance to soft rot, and the activities of ROS scavenging enzymes SOD, POD and CAT are enhanced, which proves that StPYL6 The gene plays an important role in resisting soft rot. StPYL6

[0010] (2) Improve the economic and ecological benefits of potato crop production The present application effectively enhances the resistance of potato to soft rot bacteria by analyzing the key resistance gene of potato soft rot. The application of this transgenic technology system not only alleviates the threat of disease to potato yield and quality, but also greatly reduces the amount of chemical pesticides through the establishment of endogenous disease resistance mechanism, reduces the risk of pesticide residue pollution, and promotes the sustainable operation of farmland ecosystem. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 The phylogenetic tree of PYL family members in Arabidopsis, tomato and potato.

[0012] Figure 2 The expression heat map of the gene under different stress conditions. StPYLs

[0013] Figure 3 The interaction of StPYLs and StPP2C1-4 in yeast.

[0014] Figure 4 The subcellular localization of StPYL6.

[0015] Figure 5 The in vitro protease activity of StPYL6, which verifies the inhibitory effect of StPYL6 on StPP2C4 through in vitro protease activity test.

[0016] Figure 6 The disease phenotype record of WT and StPYL6 -OE transgenic line seedlings inoculated with soft rot bacteria for 0 d to 8 d.

[0017] Figure 7 The determination result graph of antioxidant enzyme (CAT, POD, SOD) activity and MDA, PRO content in the leaves of WT and StPYL6 -OE transgenic line 12 h after inoculation with soft rot bacteria.

[0018] ​​Figure 8 The pGHPB vector spectrum. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Unless otherwise specified, all reagents used in this invention are commercially available reagents. The experimental procedures involved in the embodiments, such as the construction of expression vectors, transformation of expression vectors, purification of proteins, and preparation of transgenic plants, are all routine operations familiar to those skilled in the art.

[0021] Example 1

[0022] This example is a phylogenetic analysis of potato StPYLs. Find Arabidopsis thaliana using the NCBI online website ( Arabidopsis thaliana The PYLs protein sequence and gene number reported in L. were BLAST-aligned with those in potato (Potato) using a Solanaceae database. Solanum tuberosum L.) and tomatoes ( Solanum lycopersicum The protein sequences and gene numbers corresponding to the gene were obtained. The protein sequences were downloaded and saved as FASTA files. A phylogenetic tree was constructed using MEGA11 software using the Neighbor-joining Tree (NJ tree) method. ClustalW was used for analysis, with 1000 bootstrap tests and the default Poisson test for calculating genetic distances. The constructed phylogenetic tree was further refined using the Chiplot online website (https: / / www.chiplot.online / ).

[0023] Evolutionary analysis results as follows Figure 1As shown, the PYLs family members can be divided into three subfamilies. Among them, the members of subfamily I are: potato StPYL11, StPYL12, StPYL13, StPYL14, Arabidopsis AtPYL7, AtPYL8, AtPYL9, AtPYL10 and tomato SlPYL1, SlPYL2, SlPYL3, SlPYL10, SlPYL15; the members of subfamily II are: potato StPYL5, StPYL6, StPYL7, StPYL8, StPYL9, StPYL10, Arabidopsis AtPYL4, AtPYL5, AtPYL6, AtPYL11, AtPYL12, AtPYL13 and tomato SlPYL4, SlPYL6, SlPYL7, SlPYL9, SlPYL11; the members of subfamily III are: potato StPYL1, StPYL2, StPYL3, StPYL4, Arabidopsis AtPYR1, AtPYL1, AtPYL2, AtPYL3 and tomato SlPYL5, SlPYL8, SlPYL12, SlPYL13, SlPYL14.

[0024] Example 2

[0025] This example is a heat map of the expression of StPYLs genes under different stress conditions In order to explore the expression pattern of StPYLs family members under exogenous stress, the transcriptome data under different stress treatments in PGSC, the potato genome database, was analyzed. The results showed that under different stress conditions, StPYLs The family members showed different expression patterns. Compared with the control group, the members of subfamily II StPYL5 , StPYL6 , StPYL7 and StPYL8 were significantly induced under Pectobacterium carotovorum Pc3 , Salt, ABA and Mannitol treatments, suggesting that this subfamily member may play an important role in the response of potato to biotic and abiotic stress Figure 2 ).

[0026] Example 3

[0027] This example is the yeast interaction analysis of potato StPYLs and StPP2C1-4 PYLs as ABA receptors need to bind to PP2Cs to function. In order to explore the interaction between StPYLs and StPP2Cs, yeast two-hybrid test was performed.

[0028] The CDS sequence of StPYLs was constructed into pGBKT7 vector by homologous recombination (ClonExpress II One Step Cloning Kit kit, Novagen, Cat No: C112), the CDS sequence of StPP2Cs was amplified and constructed into pGADT7 vector by homologous recombination (the primer sequences are shown in Table 1), and the pGBKT7-StPYLs and pGADT7-StPP2Cs recombinant plasmids were co-transformed into Y190 yeast strain. Single colonies were resuspended with 20 μL of ddH2O, and 3 μL of the resuspension was spotted on solid medium with ABA or DMSO, respectively. After 36 h of culture at 30°C, the glass culture plate was opened in the fume hood and the trichloromethane was poured over the yeast bacteria along the glass edge. After 15 min of standing, the trichloromethane was poured out, 15 mL of staining solution was quickly and uniformly added with an electric pipette, and after the staining solution was solidified, it was inverted in a 37°C incubator for 16 h of staining. The results were observed and photographed.

[0029] The results showed that StPYL1, StPYL2, StPYL4, StPYL7 and StPYL13 did not interact with StPP2Cs without exogenous ABA treatment. The StPYLs subfamily II members StPYL5, StPYL6, StPYL8, StPYL9 and StPYL10 interacted with StPP2Cs without ABA, indicating that the interaction between these family members and StPP2C had a certain basic activity; 20 μmol / L ABA treatment enhanced the interaction between StPYLs and StPP2Cs, among which the interaction between StPYL5, StPYL6, StPYL8, StPYL9, StPYL10 and StPP2C4 was most significantly affected by ABA treatment. The subfamily I members StPYL12, StPYL13 and StPYL14 hardly changed the interaction effect with StPP2Cs with or without ABA. The above results can indicate that the subfamily III members have no basic activity in interacting with StPP2Cs and their interaction is strongly induced by ABA, the subfamily II members have low basic activity in interacting with StPP2Cs and their interaction is strongly induced by ABA, and the subfamily I members are hardly induced by ABA. According to the results of Example 1 and Example 2, the subfamily II members are strongly induced and respond to stress, and StPYL6 in the subfamily II is significantly induced and expressed under pathogenic bacteria and drought stress conditions, so StPYL6 in the subfamily II is selected as a candidate gene for subsequent experiments. Figure 3 ).

[0030] Table 1 Primer sequences

[0031] Example 4 This embodiment is the subcellular localization of potato StPYL6 gene The coding sequence of StPYL6 was inserted into pGHPB vector (vector map as shown in Figure 8 The correct sequencing recombinant plasmid and empty plasmid were transformed into Agrobacterium GV3101 competent cells, respectively. The bacterial cells were resuspended in 100 mL tobacco resuspension solution (10 mL of 0.1 mol / L MES (pH 5.6), 10 mL of 0.1 mol / L MgCl2, 15 μL of 1 mol / L AS, and ddH2O to 100 mL), and the resuspended Agrobacterium was resuspended to OD 600 = 1.0, and was placed in the dark for 4 h. After standing, a needleless syringe was used to inject the 1-month-old tobacco leaves, and the tobacco leaves containing the mCherry nuclear localization marker were observed using a laser confocal microscope after being placed in the dark for 24-36 h. The tobacco injected with empty pGHPB Agrobacterium was used as a control.

[0032] The results show that the GFP fluorescence signal of StPYL6 appears in the cytoplasm and nucleus, and it is speculated that StPYL6 is localized in the cytoplasm and nucleus Figure 4 ).

[0033] Example 5 This embodiment is the effect of potato StPYL6 on StPP2C4 protein phosphatase activity (1) Acquisition of target gene and construction of vector The coding sequences of StPYL6 and StPP2C4 were obtained from the Sol Genomics Network (SGN, https: / / solgenomics.net / ). Specific primers were designed to amplify the CDS sequence of StPYL6 and connect it to the pET-28a vector with His tag to construct the pET28a-His6-StPYL6 expression vector. Specific primers were designed to amplify the CDS sequence of StPP2C4 and connect it to the pMAL-c2x vector with MBP tag to construct the pMAL-c2x-MBP-StPP2C4 expression vector.

[0034] (2) Protein expression and purification The recombinant plasmids pET28a-His6-StPYL6 and pMAL-c2x-MBP-StPP2C4 correctly sequenced were transformed into E. coli competent BL21 (DE3) pLysS, respectively; after culture, single colonies were picked and inoculated into 5 mL of LB liquid medium containing the corresponding antibiotics (kanamycin for pET-28a and ampicillin for pMAL-c2x), and cultured at 37°C, 220 rpm overnight; the overnight culture was transferred into fresh medium at a ratio of 1:100, and the culture was continued until the OD 600 ≈0.9, IPTG was added to a final concentration of 1 mmol / L for induction, wherein StPYL6 was induced at 16°C, 150 rpm for 16 hours, and StPP2C4 was induced at 23°C, 150 rpm for 16 hours; the bacterial cells were collected by centrifugation, and the StPYL6 and StPP2C4 bacterial cells were resuspended with lysis buffer I and lysis buffer II, respectively; then the bacterial solution was frozen in a -80°C refrigerator for 30 min until the bacterial cells were completely frozen, and then the bacterial cells were taken out from the -80°C refrigerator and melted in a 30°C water bath; after the bacterial cells were completely melted, they were again placed in a -80°C refrigerator for 30 min, and then thawed in a water bath; the freeze-thawing was repeated twice, and the bacterial cells were ultrasonically broken on ice (power 300 W, ultrasonic 3 s, intermittent 5 s, total time 20 min); the bacterial cells were centrifuged at 4°C, 12000 g for 30 min, and the supernatant was collected.

[0035] Ni-NTA affinity purification of StPYL6: the supernatant was slowly incubated with pretreated Ni-NTA Agarose resin at 4°C for 1 hour, the mixture was loaded into a chromatography column, the flow-through was collected, the resin was washed with washing buffer I to remove non-specifically bound impurities, and the target protein was eluted with elution buffer I, and the eluate was collected step by step.

[0036] Amylose resin affinity purification of StPP2C4: the supernatant was slowly incubated with pretreated Amylose resin at 4°C for 1 hour, the mixture was loaded into a chromatography column, the flow-through was collected, the resin was washed with washing buffer II to remove non-specifically bound impurities, and the target protein was eluted with elution buffer II containing 10 mmol / L maltose, and the eluate was collected step by step.

[0037] Samples at each stage of the purification process (whole bacteria, supernatant, washing, elution) were taken for SDS-PAGE, and the purified proteins were analyzed for purity and effect by Coomassie blue staining. The proteins on the SDS-PAGE gel were transferred to a PVDF membrane, and the StPYL6 was detected with an anti-His antibody, and the StPP2C4 protein specificity was detected with an anti-MBP antibody; the concentration of the purified protein was determined by BCA method.

[0038] A series of buffers used in the purification process of StPYL6 are as follows: 100 mL Lysis Buffer I: 50 mL AX2 buffer, 10 mmol / L imidazole, protease inhibitors, ddH2O to 100 mL) 100 mL Wash Buffer I: 50 mL AX2 buffer, 30 mmol / L imidazole, protease inhibitors, ddH2O to 100 mL) 10 mL Elution Buffer I: 5 mL AX2 buffer, 250 mmol / L imidazole, protease inhibitors, ddH2O to 10 mL) A series of buffers used in the purification of StPP2C4 are as follows: 100 mL Lysis Buffer II: 10 mL 10xTBS buffer, 10 mmol / L imidazole, 10 mmol / L manganese chloride, protease inhibitors, ddH2O to 100 mL) 100 mL Wash Buffer II: 10 mL 10xTBS buffer, 30 mmol / L imidazole, 10 mmol / L manganese chloride, protease inhibitors, ddH2O to 100 mL) 20 mL Elution Buffer II: 2 mL 10xTBS buffer, 250 mmol / L imidazole, 10 mmol / L manganese chloride, protease inhibitors, 1 mmol / L TECP, ddH2O to 20 mL) 1 L TBS Buffer II: 500 mmol / L Tris, 1.5 mol / L sodium chloride, pH 7.5 1 L AX2 buffer: 100 mmol / L NaH2PO4, 600 mmol / L NaCl, pH 8.

[0039] (3) Protease activity reaction The total reaction system is 100 μL, and PnPP is added before sample loading in a flat-bottom 96-well plate. The reaction system is shown in the following table.

[0040] Table 2 PP2C protease activity reaction system

[0041] The buffer formula in Table 2 is: 165 mmol / L TAE buffer (pH=7.9) + 300 mmol / L potassium acetate + 0.5% BSA (bovine serum albumin).

[0042] The results showed that without the addition of ABA, StPYL6 had no significant effect on the activity of StPP2C4. However, the addition of 50 μmol / L ABA inhibited the activity of StPP2C4, and this inhibition intensified with increasing proportion of StPYL6 in the reaction system. When the ratio of StPP2C4 to StPYL6 was 1:4, the activity of StPP2C4 was inhibited by more than 67%. In conclusion, StPYL6 inhibits the activity of StPP2C protein phosphatase in an ABA-dependent manner.

[0043] Example 6 This embodiment is StPYL6 -OE transgenic potato disease resistance phenotype analysis (1) Obtaining transgenic materials: Specific primers StPYL6-pHGHPB-F / R were designed to amplify the CDS sequence of StPYL6 using potato cDNA as a template (primer sequences are shown in Table 1). The CDS sequence of StPYL6 was then ligated into the pHGHPB vector via homologous recombination (ClonExpress II One Step Cloning Kit, Novizan, catalog number: C112). The correctly sequenced positive plasmid was then transformed into Agrobacterium strain GV3101. Using the E'masu 3 potato material as background material, genetic transformation was performed to obtain three independent positive transgenic lines. StPYL6 -OE 1 / 2 / 3.

[0044] (2) Preparation of LB solid culture medium (1L): After the reagents are dissolved, pour them into a 1L blue cap bottle, loosen the cap slightly, and sterilize in an autoclave. After sterilization, pour the culture medium, which has been cooled to 60℃, into a disposable sterile petri dish. After solidification, place it in plastic wrap and store it in a 4℃ refrigerator for later use.

[0045] (3) Soft rot bacteria Pc3 Preservation: Using a sterile pipette tip or a sterilized toothpick, pick single colonies of the soft rot bacteria from the previously prepared solid LB medium after removing them from the refrigerator. Gently transfer the bacteria to antibiotic-free LB liquid medium and incubate at 28°C and 150 rpm for at least 10 hours. Invert the cultured bacteria to mix thoroughly. Transfer 600 μL to a 2 mL preservation tube. Place the tube containing the bacteria on ice for 10 minutes. Add 400 μL of sterilized 50% glycerol (glycerol to water volume 1:1) to a final glycerol concentration of 20%. Mix well, place in liquid nitrogen, and then transfer to a -80°C freezer.

[0046] (4) Soft rot bacteria Pc3 Activation: Take out the pathogen stored at -80℃, use a sterile loop to take an appropriate amount of bacterial solution and streak it on LB solid medium, incubate overnight in a 28℃ incubator, and incubate for no more than 24 hours. Do not shake the culture plate until single colonies grow.

[0047] (5) Pectobacterium carotovorum Pc3 Preparation of working bacteria: pick a single colony in 10 mL of LB liquid medium without antibiotics, 28°C, 150 rpm overnight culture, 25°C, 12000 rpm centrifugation for 3 min, remove the supernatant, and resuspend the bacterial cells with 1 mL of fresh LB liquid medium (this concentration is C1), take 0.9 mL of ddH2O in a new centrifuge tube, add 0.1 mL of working solution to the bacterial cells, and mix well. Pc3 Determine OD 600 with ultraviolet spectrophotometer (this concentration is C2). Based on 1.0 OD 600 =10 9 CFU, the formula is C1=C2*10*10 9 CFU / mL, follow the calculation formula C1*V1=C2*V2, dilute the original solution with ddH2O, adjust the bacterial solution concentration to 10 7 CFU / mL, which can be used as working solution.

[0048] (6) Pathogenic bacteria inoculation treatment: select 3-week-old WT and StPYL6 -OE transgenic lines with consistent growth and healthy growth, use a 1 mL syringe to inject 100 μL of working solution at the base of the plant stem, 28°C, 16h / 8h culture, observe and take pictures to record the disease phenotype, 10 seedlings of each line as 10 independent biological replicates, take 0h and 12h leaf samples and store at -80°C. Pc3

[0049] Observe the plant disease phenotype and find that WT plants show inter-leaf chlorosis after 2 days of inoculation, StPYL6 -OE1 shows stem bending after 2 days of inoculation, but returns to normal on the 4th day, and still grows normally until the 9th day, while StPYL6 -OE2 and StPYL6 -OE3 transgenic plants do not show disease symptoms. On the 6th day of inoculation, WT plant leaves begin to wither, the plant disease worsens, and the whole plant dies, while StPYL6 -OE transgenic lines grow normally and do not show obvious disease symptoms. In summary, overexpression of StPYL6 enhances the resistance of plants to Pectobacterium carotovorum Pc3 . Figure 6 .

[0050] Example 7 This example is to determine the activities of antioxidant enzymes (CAT, POD, SOD) and MDA, PRO content analysis in WT, StPYL6 -OE1 and StPYL6 -OE2 transgenic potato leaves Inoculate Pc3 ​The leaf samples at 0 h and 12 h after treatment were used for antioxidant enzyme activity determination and MDA and PRO content analysis.

[0051] (1) CAT content determination (hydrogen peroxide enzyme (CAT) kit, Suzhou Gaisi Biological: G0105W): a. Sample preparation: 0.1 g of sample was taken in a 2 mL centrifuge tube, 1 mL of extraction solution was added, 4°C, 12000 rpm, centrifugation for 10 min.

[0052] b. The supernatant was collected and placed on ice for testing. The microplate reader was preheated for 30 min and adjusted to 510 nm.

[0053] c. Blank tube detection: 80 μL of reagent one, 20 μL of reagent two and 100 μL of reagent three were mixed, 10 μL was taken and immediately mixed with the sample for color development reaction. The absorbance value at this time was recorded as Ablank.

[0054] d. Sample detection was carried out in the centrifuge tube according to the following steps: 10 μL of sample, 70 μL of reagent one and 20 μL of reagent two were added in turn, and 10 μL of the mixture was taken after mixing. 900 μL of reagent one and 290 μL of reagent four were added, and the reaction was carried out at 25°C for 5 min. 200 μL of the mixture was transferred to the enzyme-labeled plate, and the absorbance A was measured. ΔA = Ablank-Ameasurement.

[0055]

[0056] W: sample mass (unit: gram); D: dilution multiple, 1 for undiluted.

[0057] (2) SOD determination (superoxide dismutase (SOD) kit-NBT method, Suzhou Gaisi Biological: G0103W): a. Sample preparation: 0.1 g of sample was taken in a 2 mL centrifuge tube, 1 mL of extraction solution was added. 4°C, 12000 rpm, centrifugation for 10 min.

[0058] b. The supernatant was collected and placed on ice for testing. The microplate reader was preheated and adjusted to 450 nm for determination.

[0059] c. Before determination, reagents one, three and four were placed in a 25°C water bath for 10 min.

[0060] d. In the 96-well enzyme-labeled plate, reagents were added in turn according to the instructions, mixed well, and incubated at room temperature for 30 min. The absorbance A was measured at 450 nm.

[0061] e. The calculation formula is as follows:

[0062]

[0063] W: sample mass (unit: g); D: dilution factor, 1 for no dilution.

[0064] (3) POD determination (POD kit, Suzhou Gaisi Biological: G0107W): a. Sample preparation: weigh 0.1 g of sample into a 2 mL centrifuge tube, add 1 mL of extraction solution. 4°C, 12000 rpm, centrifuge for 10 min.

[0065] b. Take the supernatant and place it on ice for testing. After the enzyme reader is preheated, measure the absorbance at a wavelength of 470 nm. Before measurement, thaw reagents 1, 2, and 3 to room temperature.

[0066] e. Add the sample, reagents 1, 2, and 3 to the enzyme plate in order according to the instructions, mix immediately, read the absorbance at 470 nm A1, and read A2 after 1 min. ΔA = A2 - A1.

[0067]

[0068] W: sample mass, g (4) MDA content determination (MDA kit, Suzhou Gaisi Biological: G0110W): a. Sample preparation: weigh 0.1 g of sample into a 2 mL centrifuge tube, add 1 mL of extraction solution. 4°C, 12000 rpm, centrifuge for 10 min.

[0069] b. Take the supernatant and place it on ice for testing.

[0070] c. Add the working solution and sample to the centrifuge tube in order according to the instructions, mix well, then place in a 95°C water bath for 30 min, cool on ice, and centrifuge at 12000 rpm for 10 min.

[0071] d. Take 200 μL of the supernatant to the enzyme plate, and measure the absorbance at 532 nm and 600 nm, respectively. ΔA = A 532 -A 600 .

[0072]

[0073] W: sample mass, g (5) PRO content determination (Proline (PRO) content kit, Suzhou Gaisi Biological: G0111W): a. Sample preparation: weigh about 0.1 g of tissue into a 2 mL enzyme-free tube, add 1 mL of extraction solution, 90°C water bath for 10 min, 12000 rpm, centrifuge for 10 min.

[0074] b. Take the supernatant, cool on ice, and adjust to 520 nm after preheating the enzyme marker.

[0075] c. Add the sample (sample tube plus sample) or water (blank tube plus water) according to the instructions in the centrifuge tube, mix well with glacial acetic acid and reagent one, then cool on ice after 95°C water bath for 30 min.

[0076] d. Take 200 μL of supernatant to the enzyme marker plate, read the absorbance A at 520 nm, and ΔA = A measurement - A blank.

[0077]

[0078] W: sample mass (unit: grams); D: dilution factor, undiluted is 1.

[0079] The results of the determination show that StPYL6 The antioxidant enzyme (CAT, POD, SOD) activity of the -OE transgenic line is significantly higher than that of WT, the osmotic regulator PRO content is also significantly higher than that of WT, and the membrane lipid peroxidation product MDA content is significantly higher than that of WT. The above results show that overexpression of StPYL6 By improving the cell's active oxygen scavenging ability, reducing cell membrane damage and enhancing plant disease resistance ( Figure 7 StPYL6 ).

Claims

1. A potato gene StPYL6 for use in increasing the resistance of potatoes to soft rot, the nucleotide sequence of the CDS region of which is shown as SEQ ID No. 1.

Images