Use of potato gene stpyl6

Abstract

This invention relates to the fields of genetic engineering and plant biotechnology, and discloses a potato gene. StPYL6 Applications. This invention provides a potato soft rot resistance gene. StPYL6 Functional verification and application research, utilizing 35S promoter-driven transgenic technology, to StPYL6 Genetic transformation of Emasu 3 potato plants using gene overexpression vectors revealed... StPYL6 Transgenic potatoes with significantly increased gene expression levels showed markedly enhanced resistance to soft rot, and the activities of ROS scavenging enzymes SOD, POD, and CAT were enhanced, demonstrating... StPYL6 StPYL6 StPYL6 StPYL6 StPYL6 StPYL6 StPY Genes play an important role in resistance to soft rot.

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:

[0006] 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.

[0007] 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.

[0008] The present invention provides an overexpression vector containing a nucleotide sequence as shown in SEQ ID No. 1.

[0009] This invention provides a potato gene StPYL6 Application in improving potato resistance to soft rot.

[0010] Compared with existing technologies, this invention provides a potato gene. StPYL6Its application has the following beneficial effects:

[0011] (1) Enhance the resistance of potatoes to soft rot

[0012] This invention provides a potato soft rot resistance gene. StPYL6 Functional verification and application research, utilizing 35S promoter-driven transgenic technology, to StPYL6 Genetic transformation of Emasu 3 potato plants using gene overexpression vectors revealed... StPYL6 Transgenic potatoes with significantly increased gene expression levels showed markedly enhanced resistance to soft rot, and the activities of ROS scavenging enzymes SOD, POD, and CAT were enhanced, demonstrating... StPYL6 Genes play an important role in resistance to soft rot.

[0013] (2) Improve the economic and ecological benefits of potato crop production

[0014] This invention effectively enhances the resistance of potatoes to the soft rot pathogen by analyzing key resistance genes for potato soft rot. The application of this transgenic technology system not only alleviates the threat of the disease to potato yield and quality, but also significantly reduces the amount of chemical pesticides used and lowers the risk of pesticide residue pollution by establishing an endogenous disease resistance mechanism, thus promoting the sustainable operation of farmland ecosystems. Attached Figure Description

[0015] Figure 1 Phylogenetic trees of PYL family members in Arabidopsis thaliana, tomato, and potato.

[0016] Figure 2 For different stress conditions StPYLs Gene expression heatmap.

[0017] Figure 3 The interaction between StPYLs and StPP2C1-4 in yeast.

[0018] Figure 4 Subcellular localization of StPYL6.

[0019] Figure 5 The image shows the in vitro protease activity of StPYL6. The inhibitory effect of StPYL6 on StPP2C4 was verified by in vitro protease activity assay.

[0020] Figure 6 WT inoculated with soft rot fungi from 0 to 8 days and StPYL6 -OE transgenic line seedling disease phenotype record.

[0021] Figure 7 WT and 12 h after inoculation with soft rot fungus StPYL6Figure 1 shows the results of the determination of antioxidant enzyme (CAT, POD, SOD) activities and MDA and PRO contents in the leaves of -OE transgenic lines.

[0022] Figure 8 The pGHPB vector spectrum. Detailed Implementation

[0023] 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.

[0024] Unless otherwise specified, all reagents used in this invention are commercially available reagents.

[0025] 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.

[0026] Example 1

[0027] This example is a phylogenetic analysis of potato StPYLs.

[0028] 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 / ).

[0029] Evolutionary analysis results as follows Figure 1As shown, the PYL family members can be divided into three subfamilies. Subfamilies I consist of: potato (StPYL11, StPYL12, StPYL13, StPYL14), Arabidopsis (AtPYL7, AtPYL8, AtPYL9, AtPYL10), and tomato (SlPYL1, SlPYL2, SlPYL3, SlPYL10, SlPYL15); Subfamilies II consist of: potato (StPYL5, StPYL6, StPYL7, StPYL8, StPYL9, StPYL10), Arabidopsis (AtPYL4, At...). PYL5, AtPYL6, AtPYL11, AtPYL12, AtPYL13 and tomato SlPYL4, SlPYL6, SlPYL7, SlPYL9, SlPYL11; members of subfamily III are: potato StPYL1, StPYL2, StPYL3, StPYL4, Arabidopsis AtPYR1, AtPYL1, AtPYL2, AtPYL3 and tomato SlPYL5, SlPYL8, SlPYL12, SlPYL13, SlPYL14.

[0030] Example 2

[0031] This example shows the expression heatmap of the StPYLs gene under different stress conditions.

[0032] To investigate the expression patterns of StPYLs family members under exogenous stress, transcriptome data from the Potato Genome Database (PGSC) under different stress treatments were analyzed. The results showed that under different stress conditions, StPYLs Family members exhibited different expression patterns. Compared to the control group, subfamily II members... StPYL5 , StPYL6 , StPYL7 and StPYL8 In soft rot fungi Pc3 Expression was significantly induced under treatment with Salt, ABA, and Mannitol, suggesting that members of this subfamily may play an important role in the potato's response to biotic and abiotic stresses. Figure 2 ).

[0033] Example 3

[0034] This example illustrates the yeast interaction analysis between potato StPYLs and StPP2C1-4.

[0035] As ABA receptors, PYLs need to bind to PP2Cs to function. To investigate the interaction between StPYLs and StPP2Cs, a yeast two-hybrid experiment was conducted.

[0036] The CDS sequence of StPYLs was constructed into the pGBKT7 vector via homologous recombination (ClonExpress II One Step Cloning Kit, Novizan, catalog number: C112). The CDS sequence of StPP2Cs was amplified and constructed into the pGADT7 vector via homologous recombination (primer sequences are shown in Table 1). The pGBKT7-StPYLs and pGADT7-StPP2Cs recombinant plasmids were co-transformed into Y190 yeast strain. Single colonies were picked and resuspended in 20 μL of ddH2O. 3 μL of the resuspended liquid was spotted onto solid medium supplemented with ABA or DMSO. After incubation at 30℃ for 36 h, the glass culture plate was opened in a fume hood, and chloroform was gently poured along the edge to cover the yeast cells. After standing for 15 min, the chloroform was poured out, and 15 mL of staining solution was quickly and evenly added using a power pipette. After the staining solution solidified, the plate was inverted and incubated at 37℃ for 16 h for staining. The staining was observed and photographed.

[0037] The results showed that without exogenous ABA treatment, StPYL1, StPYL2, StPYL4, StPYL7, and StPYL13 did not interact with StPP2Cs. Subfamily II members StPYLs (StPYL5, StPYL6, StPYL8, StPYL9, and StPYL10) interacted with StPP2Cs without ABA, indicating that these family members have a certain basic activity in their interaction with StPP2Cs. Treatment with 20 μmol / LABA enhanced the interaction between StPYLs and StPP2Cs, with the interaction between StPYL5, StPYL6, StPYL8, StPYL9, and StPYL10 and StPP2C4 being most significantly enhanced by ABA treatment. Subfamily I members StPYL12, StPYL13, and StPYL14 showed almost no change in their interaction with StPP2Cs with or without ABA. The above results indicate that the interaction between subfamily III members and StPP2Cs has no basal activity and their interaction is strongly induced by ABA; the interaction between subfamily II members and StPP2Cs has low basal activity and is strongly induced by ABA; and the interaction between subfamily I members and StPP2Cs is almost uninducible by ABA. Combining the results of Examples 1 and 2, it is evident that subfamily II members exhibit a strong stress-induced response, and StPYL6 in subfamily II is significantly induced under pathogen and drought stress conditions. Therefore, StPYL6, a representative gene from subfamily II, was selected as a candidate gene for subsequent experiments. Figure 3 ).

[0038] Table 1 Primer Sequences

[0039]

[0040] Example 4

[0041] This example demonstrates the subcellular localization of the potato StPYL6 gene.

[0042] Using the specific primer pair StPYL6-GFP-F / R (primer sequences are shown in Table 1), and the Novizan ClonExpressII One Step Cloning Kit-C112, the coding sequence of StPYL6 was inserted into the pGHPB vector according to the infusion vector construction method (vector map shown in Table 1). Figure 8 As shown in the figure, the pGHPB vector carries a GFP fluorescent tag. The correctly sequenced recombinant plasmid and the empty vector plasmid were transformed into Agrobacterium GV3101 competent cells, respectively. The bacterial cells were resuspended in 100 mL of tobacco resuspension buffer (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 added to 100 mL). The shaken Agrobacterium cells were then resuspended at OD200. 600 =1.0, and allowed to stand in the dark for 4 h. After standing, the leaves of one-month-old *Agrobacterium tumefaciens* were injected with the drug using a needle-free injector. After 24 to 36 h in the dark, the leaves containing mCherry nuclear localization markers were observed using a laser confocal microscope. Tobacco injected with empty pGHPB *Agrobacterium* served as a control.

[0043] The results showed that the GFP fluorescence signal of StPYL6 appeared in both the cytoplasm and the nucleus, suggesting that StPYL6 is located in the cytoplasm and nucleus. Figure 4 ).

[0044] Example 5

[0045] This example illustrates the effect of potato StPYL6 on the activity of StPP2C4 protein phosphatase.

[0046] (1) Acquisition of target gene and construction of vector

[0047] The coding sequences of StPYL6 and StPP2C4 were obtained from the Solanaceae Genome Database (Sol Genomics Network, SGN, https: / / solgenomics.net / ). Specific primers were designed to amplify the CDS sequence of StPYL6, which was then ligated into a His-tagged pET-28a vector to construct the pET28a-His6-StPYL6 expression vector. Specific primers were designed to amplify the CDS sequence of StPP2C4, which was then ligated into a MBP-tagged pMAL-c2x vector to construct the pMAL-c2x-MBP-StPP2C4 expression vector.

[0048] (2) Protein expression and purification

[0049] The correctly sequenced recombinant plasmids pET28a-His6-StPYL6 and pMAL-c2x-MBP-StPP2C4 were transformed into competent E. coli cells BL21(DE3)pLysS, respectively. After culturing, single colonies were picked and inoculated into 5 mL of LB broth containing the corresponding antibiotics (kanamycin for pET-28a and ampicillin for pMAL-c2x), and cultured overnight at 37°C with shaking at 220 rpm. The overnight culture was then transferred to fresh culture medium at a 1:100 ratio and cultured until OD200. 600 ≈0.9, add IPTG to a final concentration of 1 mmol / L for induction. StPYL6 was induced at 16℃ and 150 rpm for 16 hours, and StPP2C4 was induced at 23℃ and 150 rpm for 16 hours. Centrifuge to collect the bacterial cells, and resuspend the StPYL6 and StPP2C4 cells with lysis buffer I and lysis buffer II, respectively. Then, freeze the bacterial solution at -80℃ for 30 min until the cells are completely frozen. Then, remove the cells from -80℃ and thaw them in a 30℃ water bath. After the cells are completely thawed, freeze them at -80℃ for 30 min, and then thaw them in a water bath. Repeat the freeze-thaw cycle twice. Sonicate on ice (300W power, 3s sonication, 5s interval, total time 20 min), centrifuge at 12000g for 30 min at 4℃, and collect the supernatant.

[0050] Ni-NTA affinity purification of StPYL6: The supernatant was incubated with pretreated Ni-NTA agarose resin at 4°C with gentle shaking for 1 hour. The mixture was loaded into a chromatography column, the flow-through was collected, the resin was washed with wash buffer I to remove non-specifically bound proteins, the target protein was eluted with elution buffer I, and the eluent was collected stepwise.

[0051] Amylose resin affinity purification of StPP2C4: The supernatant was incubated with pretreated Amylose resin at 4°C with gentle shaking for 1 hour. The mixture was loaded into a chromatography column, the flow-through was collected, the resin was washed with wash buffer II to remove non-specifically bound proteins, and the target protein was eluted with elution buffer II containing 10 mmol / L maltose. The eluent was collected stepwise.

[0052] Samples from each stage of the purification process (whole bacteria, supernatant, washing, elution) were subjected to SDS-PAGE, stained with Coomassie Brilliant Blue, and the purification effect and protein purity were analyzed. The protein on the SDS-PAGE gel was transferred to a PVDF membrane, and the specificity of StPYL6 was detected using anti-His antibody, while that of StPP2C4 was detected using anti-MBP antibody. The concentration of the purified protein was determined using the BCA method.

[0053] The following are a series of buffer solutions used in the purification of StPYL6:

[0054] 100mL lysis buffer I: 50mL AX2 buffer, 10mmol / L imidazole, protease inhibitor, ddH2O to bring volume to 100mL

[0055] 100mL Wash Buffer I: 50mL AX2 buffer, 30mmol / L imidazole, protease inhibitor, ddH2O to bring volume to 100mL

[0056] 10 mL Elution Buffer I: 5 mL AX2 buffer, 250 mmol / L imidazole, protease inhibitor, ddH2O to bring volume to 10 mL

[0057] The following are a series of buffers used in the purification process of StPP2C4:

[0058] 100mL Lysis Buffer II: 10mL 10xTBS buffer, 10mmol / L imidazole, 10mmol / L manganese chloride, protease inhibitor, ddH2O to bring volume to 100mL

[0059] 100mL Wash Buffer II: 10mL 10xTBS buffer, 30mmol / L imidazole, 10mmol / L manganese chloride, protease inhibitor, ddH2O to bring volume to 100mL

[0060] 20 mL Elution Buffer II: 2 mL 10x TBS buffer, 250 mmol / L imidazole, 10 mmol / L manganese chloride, protease inhibitor, 1 mmol / L LTECP, ddH2O to bring volume to 20 mL.

[0061] 1L TBS buffer II: 500mmol / L Tris, 1.5mol / L sodium chloride, pH 7.5

[0062] 1L AX2 buffer: 100mmol / L NaH2PO4, 600mmol / L NaCl, pH 8.

[0063] (3) Protease activity reaction

[0064] The total reaction volume was 100 μL, which was added to a flat-bottomed 96-well plate. PnPP was added before analysis. The reaction system is shown in the table below.

[0065] Table 2. PP2C protease activity reaction system

[0066]

[0067] The buffer formulation in Table 2 is: 165 mmol / L TAE buffer (pH=7.9) + 300 mmol / L potassium acetate + 0.5% BSA (bovine serum albumin).

[0068] 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.

[0069] Example 6

[0070] This embodiment is StPYL6 -OE transgenic potato disease resistance phenotype analysis

[0071] (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.

[0072] (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.

[0073] (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.

[0074] (4) Soft rot bacteriaPc3 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.

[0075] (5) Soft rot fungi Pc3 Preparation of working bacteria: Pick a single colony and incubate overnight at 28°C and 150 rpm. Centrifuge at 25°C and 12000 rpm for 3 min. Remove the supernatant and resuspend the bacteria in 1 mL of fresh LB broth (this concentration is C1). Take 0.9 mL of ddH2O into a new centrifuge tube and add 0.1 mL of [unspecified ingredient]. Pc3 OD was measured using a UV spectrophotometer. 600 (This concentration is C2). Based on 1.0 OD 600 =10 9 The formula for calculating CFU concentration is C1 = C2 * 10 * 10. 9 CFU / mL, calculate using the following formula: C1*V1=C2*V2. Dilute the stock solution with ddH2O to adjust the bacterial concentration to 10. 7 CFU / mL can be used as a working solution for later use.

[0076] (6) Pathogen inoculation treatment: Select 3-week-old WT and healthy WTs with uniform growth. StPYL6 -OE transgenic seedlings were injected with 100 μL of the solution at the base of the stem using a 1 mL syringe. Pc3 The working solution was cultured at 28℃ for 16h / 8h. The disease phenotype was observed and photographed. Ten seedlings from each line were used as 10 independent biological replicates. Leaf samples were taken at 0h and 12h of treatment and frozen at -80℃.

[0077] Observation of the disease phenotype of the plants revealed that WT plants showed interfoliar chlorosis two days after inoculation. StPYL6 -OE1 showed main stem bending 2 days after inoculation, but recovered to normal on the 4th day and continued to grow normally until the 9th day. StPYL6 -OE2 and StPYL6 -OE3 transgenic plants did not show symptoms of infection. On the 6th day after inoculation, the leaves of WT plants began to fall off, the symptoms worsened, and the entire plant died. StPYL6 -OE transgenic lines grew normally and showed no obvious symptoms of disease. In summary, overexpression... StPYL6 Enhanced plant resistance to soft rot fungi Pc3 resistance ( Figure 6 ).

[0078] Example 7

[0079] This embodiment is WT,StPYL6 -OE1 and StPYL6 Determination of antioxidant enzyme (CAT, POD, SOD) activity and analysis of MDA and PRO content in OE2 transgenic potato leaves

[0080] Inoculation in Example 6 Pc3 The antioxidant enzyme activity and MDA and PRO content were measured in leaf samples at 0h and 12h.

[0081] (1) CAT content determination (catalase (CAT) kit, Suzhou Gres Biotech: G0105W):

[0082] a. Sample preparation: Take 0.1g of sample into a 2mL centrifuge tube, add 1mL of extraction solution, and centrifuge at 4℃, 12000rpm for 10min.

[0083] b. Collect the supernatant and place it on ice for testing. Preheat the microplate reader for 30 minutes and adjust it to 510 nm.

[0084] c. Blank tube test: Mix 80 μL of reagent I, 20 μL of reagent II, and 100 μL of reagent III thoroughly. Take 10 μL of this mixture and immediately mix it with the sample to initiate the colorimetric reaction. Record the absorbance value at this point as blank A.

[0085] d. Perform sample analysis in centrifuge tubes according to the following steps:

[0086] Add 10 μL of sample, 70 μL of reagent one, and 20 μL of reagent two sequentially, mix, and take 10 μL of the mixture. Then add 900 μL of reagent one and 290 μL of reagent four, and react at 25℃ for 5 min. Transfer 200 μL of the mixture to an ELISA plate and measure the absorbance value A. ΔA = Ablank - Ameasured.

[0087]

[0088] W: Sample mass (unit: grams); D: Dilution factor, 1 for undiluted.

[0089] (2) SOD assay (Superoxide dismutase (SOD) kit - NBT method, Suzhou Gres Biotech: G0103W):

[0090] a. Sample preparation: Weigh 0.1g of sample into a 2mL centrifuge tube and add 1mL of extraction buffer. Centrifuge at 12000rpm for 10min at 4℃.

[0091] b. Take the supernatant, place it on ice for testing, and preheat the microplate reader to 450nm for measurement.

[0092] c. Before the test, place reagents one, three and four in a 25°C water bath for 10 minutes.

[0093] d. Add reagents to the 96-well microplate according to the instructions, mix well, and let stand at room temperature (25°C) in the dark for 30 minutes. Then measure the absorbance A at 450 nm.

[0094] e. The calculation formula is as follows:

[0095]

[0096]

[0097] W: Sample mass (unit: grams); D: Dilution factor, 1 for undiluted.

[0098] (3) POD assay (peroxidase (POD) kit, Suzhou Greens Biotechnology: G0107W):

[0099] a. Sample preparation: Weigh 0.1g of sample into a 2mL centrifuge tube and add 1mL of extraction buffer. Centrifuge at 12000rpm for 10min at 4℃.

[0100] b. Take the supernatant and place it on ice for testing. After preheating the microplate reader, measure at a wavelength of 470 nm. Before the test, thaw reagents one, two and three to room temperature.

[0101] e. Add the sample, reagents one, two and three to the microplate in sequence according to the instructions, mix immediately, and read the absorbance value A1 at 470nm. After 1 minute, read A2. ΔA = A2 - A1.

[0102]

[0103] W: Sample quality, g

[0104] (4) MDA content determination (malondialdehyde (MDA) kit, Suzhou Greens Biotechnology: G0110W):

[0105] a. Sample preparation: Weigh 0.1g of sample into a 2mL centrifuge tube and add 1mL of extraction buffer. Centrifuge at 12000rpm for 10min at 4℃.

[0106] b. Take the supernatant and place it on ice for testing.

[0107] c. Add the working solution and sample to the centrifuge tube according to the instructions, mix well, incubate in a 95°C water bath for 30 minutes, cool on ice, and centrifuge at 12,000 rpm for 10 minutes.

[0108] d. Transfer 200 μL of supernatant to an ELISA plate and measure the absorbance A at 532 nm and 600 nm, respectively. ΔA = A 532 -A 600 .

[0109]

[0110] W: Sample quality, g

[0111] (5) PRO content determination (proline (PRO) content kit, Suzhou Greens Biotechnology: G0111W):

[0112] a. Sample preparation: Weigh about 0.1g of tissue into a 2mL enzyme-free tube, add 1mL of extraction solution, incubate in a 90℃ water bath for 10min, and centrifuge at 12000rpm for 10min.

[0113] b. Take the supernatant, cool it on ice, and preheat the microplate reader to 520nm.

[0114] c. Add the sample (sample tube) or water (blank tube) to the centrifuge tube as instructed, along with glacial acetic acid and reagent one. Mix well, incubate in a 95°C water bath for 30 minutes, and then cool on ice.

[0115] d. Take 200 μL of supernatant into the microplate and read the absorbance value A at 520 nm. ΔA = Adetermination - Ablank.

[0116]

[0117] W: Sample mass (unit: grams); D: Dilution factor, 1 for undiluted.

[0118] The test results show that StPYL6 The activities of antioxidant enzymes (CAT, POD, SOD) in the -OE transgenic lines were significantly higher than those in the WT lines, as were the contents of the osmotic regulator PRO and the membrane lipid peroxidation product MDA. These results suggest that overexpression... StPYL6 By enhancing the cell's ability to scavenge reactive oxygen species, cell membrane damage is reduced, thus strengthening the plant's disease resistance. Figure 7 ).

Claims

1. Overexpression of potato gene StPYL6 In its application to improving potato resistance to soft rot, the nucleotide sequence of the CDS region of the gene is shown in SEQ ID No. 1.

Images