Application of phytophthora infestans response gene StRTP5 in regulation and control of potato disease resistance

Abstract

The invention belongs to the technical field of gene engineering, and relates to application of a potato late blight bacterium response gene StRTP5 in regulation and control of potato disease resistance. The invention provides application of a potato late blight response gene StRTP5 in regulation and control of potato disease resistance, the potato late blight response gene StRTP5 comprises a gene StRTP5a and a gene StRTP5b, the gene StRTP5a and the gene StRTP5b negatively regulate and control resistance of potatoes to phytophthora infestans and alternaria solani, and the gene StRTP5a and the gene StRTP5b have potential to be used for creating potato broad-spectrum disease-resistant materials.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and relates to application of a potato late blight pathogen responsive gene StRTP5 in regulation of potato disease resistance. BACKGROUND

[0002] Potato is an important food crop next to wheat, rice and corn, but potato is susceptible to various biological and non-biological stress during its production process. Among them, potato late blight caused by Phytophthora infestans is an important factor restricting potato production, and using disease-resistant genes to improve and cultivate disease-resistant potato varieties is a more economical and effective measure to control diseases. At present, although the dominant disease-resistant gene (R gene) plays a role in potato disease-resistant breeding, the resistance mediated by the R gene has physiological race specificity, and the resistance is easily overcome by new pathogens appearing in the field, and there is a prominent problem of loss of resistance of the variety in production. The plant disease resistance pathway mediated by the susceptible gene (S gene) has potential for molecular design of crop broad-spectrum disease resistance, and has important theoretical and practical significance for crop disease resistance and biological breeding.

[0003] StRTP5a and StRTP5b are two homologous genes that are significantly induced by P. infestans and have similar expression patterns. The two genes are highly homologous to Arabidopsis RTP5 encoding WD40 protein. Studies have shown that Arabidopsis RTP5 negatively regulates the resistance of Arabidopsis to parasitic P. infestans, and StRTP5a, StRTP5b and RTP5 and their homologous genes are widely distributed in plants, suggesting that StRTP5a, StRTP5b and their homologous genes may have conserved biological functions in plants, but the function of StRTP5a and StRTP5b in response to P. infestans infection is still unknown.

[0004] Therefore, using Agrobacterium-mediated potato genetic transformation technology, transgenic lines overexpressing and silencing the genes StRTP5a and StRTP5b are obtained, and then the obtained transgenic lines are tested for resistance to P. infestans and other pathogens (early blight pathogens) of potato, which provides materials and reference information for further analyzing the mechanism of the genes StRTP5a and StRTP5b in potato disease resistance. SUMMARY

[0005] Therefore, the application provides application of a potato late blight pathogen responsive gene StRTP5 in regulation of potato disease resistance. The genes StRTP5a and StRTP5b negatively regulate the resistance of potato to P. infestans and Alternaria solani, indicating that the genes StRTP5a and StRTP5b have potential for creating potato broad-spectrum disease-resistant materials.

[0006] To achieve the technical purpose, the application adopts the following technical solutions:

[0007] In one aspect, the application provides application of a potato late blight pathogen response gene StRTP5 in regulating potato disease resistance, wherein the potato late blight pathogen response gene StRTP5 comprises a gene StRTP5a and a gene StRTP5b,

[0008] The nucleotide sequence of the gene StRTP5a is shown as SEQ ID NO: 1.

[0009] The nucleotide sequence of the gene StRTP5b is shown as SEQ ID NO: 2.

[0010] Preferably, the regulation of potato disease resistance comprises:

[0011] a) overexpression of the gene StRTP5a and the gene StRTP5b to enhance the susceptibility of potato to late blight and early blight; and / or

[0012] b) silencing of the gene StRTP5a and the gene StRTP5b to enhance the resistance of potato to late blight and early blight.

[0013] Preferably, the amino acid sequence of the protein encoded by the gene StRTP5a is shown as SEQ ID NO: 3.

[0014] Preferably, the amino acid sequence of the protein encoded by the gene StRTP5b is shown as SEQ ID NO: 4.

[0015] Preferably, the pathogenic bacteria of the late blight is Phytophthora infestans.

[0016] Preferably, the pathogenic bacteria of the early blight is Alternaria solani.

[0017] In another aspect, the application provides a potato gene silencing strain,

[0018] The potato gene silencing strain is obtained by silencing a potato late blight pathogen response gene StRTP5.

[0019] The potato late blight pathogen response gene StRTP5 comprises a gene StRTP5a and a gene StRTP5b.

[0020] The nucleotide sequence of the gene StRTP5a is shown as SEQ ID NO: 1.

[0021] The nucleotide sequence of the gene StRTP5b is shown as SEQ ID NO: 2.

[0022] Silencing the gene StRTP5a and the gene StRTP5b enhances the resistance of potato to late blight and early blight.

[0023] In another aspect, the present application provides an expression vector,

[0024] The expression vector contains a potato late blight response gene StRTP5;

[0025] The potato late blight response gene StRTP5 includes a gene StRTP5a and a gene StRTP5b;

[0026] The nucleotide sequence of the gene StRTP5a is shown as SEQ ID NO: 1;

[0027] The nucleotide sequence of the gene StRTP5b is shown as SEQ ID NO: 2.

[0028] In another aspect, the present application provides a method for breeding potato varieties resistant to late blight and early blight, which comprises the step of silencing the gene StRTP5a and the gene StRTP5b.

[0029] In another aspect, the present application provides a method for breeding potato varieties resistant to pathogenic Phytophthora and Alternaria solani,

[0030] The method includes the step of silencing the gene StRTP5a and the gene StRTP5b;

[0031] The nucleotide sequence of the gene StRTP5a is shown as SEQ ID NO: 1;

[0032] The nucleotide sequence of the gene StRTP5b is shown as SEQ ID NO: 2.

[0033] SEQ ID NO: 1 is shown as follows:

[0034]

[0035] SEQ ID NO: 2 is specified as follows:

[0036]

[0037] SEQ ID NO: 3 is specified as follows:

[0038] “MRSSMSPDYSSSAPSTSRSSPPSAASQNLNCKHSNVFQLLTRREVAPRTKRTSRKF WGENTKCTLDSYGLKRGVGSDARQGLISWVEAESLQHLSAKYCSLLPPPRSTIAAAFSPDGRTLASTHGDHTVKIIDCQTGKCLKVMSGHRRTPWVVRFHPLYPEILASGSLDHEVRLWDAKTAECIGSRDFYRPIASIAFHAQGEVLAVASGHKLYIWHYNRRGEASSPAIILKTRRSLRAVHFHPHGAPFLLTAEVNDLDSSDSSITRATSPGNLQYPPPTVYLTDAHSTYQSASANELPIMSQPFLIWPSIARGDPRMPMLQSDTDVGSDSIQQRADTSSSVRLLTYSTPSGQYELLLSPIEQSASPTQEAHTSSSVRENETGTQPLVDPMETDGQPEERNNQFFPFSDPAYWELPFLQGWLIGRSQATRSELSGATINPSTYGELENPSAVPLVISSNSHPRSGRSGSRHRSSRSRVIPVDGAGDGAAPVNVMHDESDSQISIGRIQSEIATSLAAAAAAELPCTVKLRIWPYDIKVPCAALDAERCCLIIPHAVLCSEMGAHFSPCGRFLAACVACILPNLDSDPGFHGHLHHDTMAAATSPTRHPVAAHQVMYELRIYSLEEETFGSVLAARAIRAAHCLTSIQFSPTSEHLLLAYGRRHSSLLKSVVIDGDTTIPIYTILEVYRVSDMELVRVLPSTEDEVNVACFHPSVGGGLVYGTKEGKLRILQYDNSNSLGRTISCSPVENMLEVPTYALEG”.

[0039] SEQ ID NO: 4 is specified as follows:

[0040] "MRGSLWPENSSSDSSTSNPIHVPLPPPPPGWNSPSLPIEQNPNCKNRCGNVFQLLTR REVSPRDKRSSKKLWDENSKYCAHSYEKLKSQVARDPRRDLISWVEAESLRHFSAKYCPL LPPPRSTIAAAFSPDGKTLASTHGDHTVKIIDCQSGKCLKVLSGHRRTPWVVRFHPLHS EILASGSLDHEVRLWDAKTAECIGSRDFYRPIASIAFHAQGEVLAVASGHKLYMWHYN RRGEASSPAIILKTRRSLRAVHFHPHGAPYLLTAEVNDLDSSDPLMTFATSLGNLRYPP PTVYLTDAHSTYRSASANELPIMSLPFMIWPSIARGDPRMPLQQSNADMGSDSTQNRAD TSASVRLLTYSTPSGQYELLLSPVEPTLSPAQEAQTSSSVRDTENASNPVVDPMETDVP TEERNNQFFPFSDPAYWDLPFLQGWLIGQSQAGRRAIHSEHSGATNIVSAYGEVEHP PAVPSIISNSNHPRSGRSGSRHRSSHSRAIPVAGSGDSAVPINIAHNESDSQAFMSRFQ SEIATSLTAAAASELPCTVKLRVWPYDIKVPCAPLDAEKCRLIIPHAVLCSEMGAHFSP CGRFLAACVACISPSMEADPGFHGQFRHDAATSPTRHPIAAHPVMYELRIYSLEEANF GRVLASRLIRAAHCLTSIQFSPTSEHLLLAYGRRHGSLLKSIVIDGDTTLPVYTILEVY RVSDMELVRVLPSAEDEVNVACFHPLVGGGLVYGTKEGKLRILQFDKSNGLDCTISCS PDEDMLEVPTYALEG".

[0041] The present application has the following advantages over the prior art:

[0042] (1) The genes StRTP5a and StRTP5b are up-regulated in response to infection by P. infestans and have similar expression patterns.

[0043] (2) The genes StRTP5a and StRTP5b both encode a protein containing four WD40 repeat domains, and the genes encoding homologous proteins are widely distributed in plants.

[0044] (3) By means of Agrobacterium-mediated genetic transformation technology, potato transgenic lines with simultaneous silencing and separate overexpression of genes StRTP5a and StRTP5b are obtained.

[0045] (4) It is clear that genes StRTP5a and StRTP5b act as negative regulation of disease resistance of potato to P. infestans and A. solani, and it is confirmed that genes StRTP5a and StRTP5b have potential for creating potato broad-spectrum disease-resistant materials. Specifically, overexpression of genes StRTP5a and StRTP5b enhances the susceptibility of potato to late blight and early blight; silencing of genes StRTP5a and StRTP5b enhances the resistance of potato to late blight and early blight. BRIEF DESCRIPTION OF DRAWINGS

[0046] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0047] Figure 1 Figure is an expression pattern analysis diagram of genes StRTP5a and StRTP5b in response to P. infestans infection.

[0048] Figure 2 Figure is a sequence alignment diagram of StRTP5a and StRTP5b with Arabidopsis RTP5 (AtRTP5) protein.

[0049] Figure 3 Figure is a phylogenetic tree of StRTP5a and StRTP5b and their homologous proteins.

[0050] Figure 4 Figure is a schematic diagram of T-DNA region of RNA gene silencing (RNAi) and overexpression vectors; A and B are schematic diagrams of T-DNA region of vectors expressing genes StRTP5a and StRTP5b, respectively; C is a schematic diagram of T-DNA region of RNAi vector pHells12-StRTP5a / bi.

[0051] Figure 5 Figure is a flow chart of Agrobacterium-mediated potato genetic transformation; A is co-cultivation of potato leaves with Agrobacterium; B is formation of potato leaf callus; C is formation of callus and adventitious buds; D is induction of adventitious bud rooting; E is root differentiation of adventitious buds; F is regenerated potato seedlings.

[0052] Figure 6Molecular identification of potato genetic transformation lines; A is RNAi gene silencing line Kan r of StRTP5a / b; B is the gene silencing level of StRTP5a and StRTP5b in RNAi gene silencing lines detected by RT-qPCR; C is the amplification of overexpression line Kan r ; D is the accumulation of target protein in overexpression lines detected by Western blotting.

[0053] Figure 7 Growth phenotype of RNAi and overexpression lines of StRTP5a / b.

[0054] Figure 8 StRTP5a and StRTP5b negatively regulate potato resistance to Phytophthora infestans; A is the disease incidence of potato lines with simultaneous silencing and overexpression of StRTP5a and StRTP5b after inoculation with P. infestans strain 88069 for 4 days; B and C are the statistical analysis of lesion area and P. infestans colonization biomass of potato lines with simultaneous silencing and overexpression of StRTP5a and StRTP5b, respectively, after inoculation with P. infestans;

[0055] Désirée is the wild type control; * is P < 0.05.

[0056] Figure 9 StRTP5a and StRTP5b negatively regulate potato resistance to Alternaria solani; A is the disease incidence of potato lines with simultaneous silencing and overexpression of StRTP5a and StRTP5b after inoculation with A. solani strain TA-0410 for 5 days; B is the statistical analysis of lesion area of potato lines with simultaneous silencing and overexpression of StRTP5a and StRTP5b, respectively, after inoculation with A. solani; Désirée is the wild type; * is P < 0.05. DETAILED DESCRIPTION

[0057] Hereinafter, the technical solutions of the present application will be described in conjunction with examples, but the present application is not limited to the following examples. The experimental methods and detection methods described in each example are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0058] Figure 6Molecular identification of potato genetic transformation lines; A is the RNAi gene silencing line Kan r of amplification, where the internal reference gene is StEF1α; M is DNA Marker; + is the positive control vector pHells12-StRTP5a / b i; - is ddH2O. B is the gene silencing level of StRTP5a and StRTP5b in the RNAi gene silencing line detected by RT-qPCR; where the internal reference gene is StEF1α. The expression level of StRTP5a and StRTP5b in wild-type potato is standardized to 1, and the results are the average of 3 biological replicates ± standard error; * is P < 0.05. C is the amplification of overexpression line Kan r of amplification, where the internal reference gene is StEF1α; + is the positive control vector pART27-GFP-StRTP5a; - is ddH2O. D is the accumulation of target protein in overexpression lines detected by Western blotting; Désirée is wild type; Ponceau S is the amount of RuBisCO protein in total protein detected by staining; α-GFP is the amount of GFP-StRTP5a and GFP-StRTP5b protein in StRTP5a and StRTP5b overexpression lines.

[0059] Materials and methods of example 1

[0060] 1.1 Experimental materials

[0061] 1.1.1 Plant material

[0062] Test-tube seedlings of potato (Solanum tuberosum L.) variety Désirée were preserved by the Potato Research Group of the College of Agriculture, Northwest A&F University.

[0063] 1.1.2 Strain, vector material

[0064] Bacterial strains: Agrobacterium tumefaciens strain GV3101 (pMP90) was purchased from Shanghai Uridian Biotechnology Co., Ltd.; Escherichia coli strain Mach T1 was purchased from ThermoFisher Scientific Company.

[0065] Pestalotiopsis strains: Phytophthora infestans (88069) was a gift from Professor Francine Govers of Wageningen University in the Netherlands. Alternaria solani (TA-0410) was a gift from Dr. Tang Wei of the School of Life Sciences, Yunnan Normal University.

[0066] Vector material: Gateway compatible RNAi vector pHELLSGATE 12 (Helliwell Chris and Waterhouse Peter. 2003. Constructs and methods for high-throughput gene silencing in plants. Methods, 30(4): 289-295). Plant expression vector pART27-GFP carrying GFP tag (Fan Guangjin, Yang Yang, Li Tingting, Lu Wenqin, Du Yu, Qiang Xiaoyu, Wen Qujiang, Shan Weixing. 2018. A Phytophthora capsici RXLR Effector Targets and Inhibits a Plant PPIase to Suppress Endoplasmic Reticulum-Mediated Immunity. Molecular Plant, 11(8): 1067-1083.).

[0067] 1.1.3 Reagents and instruments

[0068] Main reagents: high-fidelity DNA polymerase Fast Pfu (full form gold), restriction endonuclease EcoR I and Xba I (New England Biolabs), T4 DNA ligase (Thermo Scientific), ordinary agarose gel DNA recovery kit (TIANGEN), plasmid small extraction kit (TIANGEN), PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TakaRa), UltraSYBR Miture (Kangwei Century), ClonExpress II One Step Cloning Kit (C112-01, Novozyme), Gateway TM BP Clonase TM II Enzyme Mix and Gateway TM LR Clonase TM II Enzyme Mix (Thermo Fisher Scientific).

[0069] Main instruments: constant temperature biochemical incubator (SPX-380, Heilongjiang Dongtuo), shaker (MAXQ 8000, Thermo), clean bench (SW-CJ-2FD, Suning Antai), light incubator, plant culture room (23℃), 4℃ refrigerator (HYC-940, Haier), -20℃ refrigerator (DW-25L300, AUCMA), -80℃ refrigerator (ULTS1651, Thermo Scientific), ultraviolet-visible spectrophotometer (UV1600A, UNIC), metal bath (OSE-100C, TIANGEN), ultrapure water instrument (UPY-111-103, Youpu), electronic analytical balance (CP313, OHAUS), high-pressure steam sterilization pot (SX-500, TOMY), ice maker (SIM-F140AOL), electrophoresis instrument (DYY-7C, Beijing Liuyi), PCR instrument (S1000, Bio-Rad), pH meter (Seven campest S210, METTLER TOLEDO).

[0070] 1.1.4 Main culture medium and reagent formula

[0071] (1) Rye medium (RSA, Table 1)

[0072] Table 1 Rye medium

[0073] Reagent Amount Sucrose 15g Agar 4g Rye add to 500 mL

[0074] 121℃, high pressure sterilization for 20min.

[0075] (2) Potato dextrose agar medium (PDA, Table 2)

[0076] Table 2 Potato dextrose medium

[0077] Reagent Amount Potato juice 1000 mL Glucose 20g Agar 14g

[0078] 121℃, high pressure sterilization for 20min.

[0079] (3) LB medium (PDA, Table 3-Table 4)

[0080] Table 3 LB liquid medium

[0081] Reagent Amount NaCl 10g Yeast extract 5g Tryptone 10g [dH2O] 1L

[0082] 121℃, high pressure sterilization for 20min.

[0083] Table 4 LB solid medium

[0084] Reagent Amount NaCl 10g Yeast extract 5g Tryptone 10g Agar 7.5g [dH2O] 1L

[0085] 121°C, autoclaved for 20 min.

[0086] (4) Potato tissue culture medium (Tables 5-7)

[0087] Table 5 MS liquid medium (1 L)

[0088]

[0089]

[0090] 121°C, autoclaved for 20 min.

[0091] Table 6 Callus induction medium (CIM)

[0092] Reagent Amount MS 4.43g Glucose 16.0g MES 0.5g Agar 8.0g NAA 5 mg 6-BA 0.1 mg [dH2O] up to 1 L

[0093] 121°C, autoclaved for 20 min.

[0094] Table 7 Shoot induction medium (SIM)

[0095] Reagent Amount MS 4.43g Glucose 16.0g MES 0.5g Agar 8.0g NAA 0.02 mg GA3 0.1 mg ZT 2.0 mg [dH2O] up to 1 L

[0096] 121°C, autoclaved for 20 min.

[0097] (5) Modified CTAB method: DNA extraction solution = DNA extraction buffer: Nuclei lysis buffer: N-lauroylsarcosine sodium = 40:50:5 volume ratio (Tables 8-10).

[0098] Table 8 DNA extraction buffer (1 L)

[0099]

[0100]

[0101] Table 9 Nuclei lysis buffer (1 L)

[0102] Reagent Amount Tris-HCl (pH=7.5) 200 mM NaCl 2M CTAB 20g EDTA (pH=8.0) 50 mM

[0103] Table 10 N-lauroylsarcosine sodium solution (1 L)

[0104] Reagent Amount N-lauroylsarcosine 100g

[0105] (6) MES solution (Table 11)

[0106] Table 11 MES solution formulation

[0107] Reagent Amount MES 1.066g MgCl2.6H2O 1.0165g ddH2O 500 mL

[0108] pH adjusted to 5.6, 121°C, autoclaved for 20 min.

[0109] 1.2 Experimental methods

[0110] 1.2.1 Plant culture

[0111] The potato plantlets were cultured in a plant incubator at 23℃, 16h light / 8h dark, and light intensity of 3000lx. When acclimatizing, the potato seedlings with roots were cultured in vermiculite for about one week, and then transferred to the culture soil.

[0112] 1.2.2 Extraction of plant RNA

[0113] The extraction of potato total RNA was performed according to the instructions of the plant total RNA extraction kit (DP149, TIANGEN).

[0114] 1.2.3 Synthesis of cDNA

[0115] The synthesis of cDNA was performed according to the instructions of the PrimeScript TM RTregent Kit with gDNAEraser kit (TaKaRa), and the main steps included:

[0116] ① Removal of genomic DNA (Table 12)

[0117] Table 12 Reaction system for removing genomic DNA (10 μL system)

[0118]

[0119]

[0120] Reaction conditions: 42℃, 2min; 4℃ forever.

[0121] ② Reverse transcription (Table 13-Table 14)

[0122] Table 13 Reverse transcription reaction system (20 μL)

[0123] Reagent Volume Reaction solution of step 1 10 μL 5x PrimeScript Buffer II 4 μL RT Primer Mix*4 1 μL PrimeScript RT Enzyme Mix I 1 μL RNase free dH2O add to 20 μL

[0124] Table 14 Reverse transcription PCR program (One cycle)

[0125] Temperature Time 37℃ 15 min 85℃ 5s 4℃ Forever

[0126] 1.2.4 Real-time fluorescent quantitative PCR (Table 15-Table 16)

[0127] The Primer-BLAST tool of NCBI was used to input the sequence, set the length of the amplicon (100-300 bp) and the intron crossing option, design the quantitative primer of the target gene, and use the Primer-BLAST tool of NCBI to detect the specificity of the primer.

[0128] Potato reference gene StEF1a, pathogenic oomycete reference gene PiUBC9.

[0129] Table 15 RT-qPCR reaction system

[0130] Component Volume 2x Ultra SYBR Mixture 7.5 μL Forward primer (10 μM) 0.7 μL Reverse primer (10 μM) 0.7 μL cDNA 2.1 μL ddH2O add to 15 μL

[0131] Table 16 RT-qPCR reaction program

[0132]

[0133]

[0134] Using 2 -ΔΔCt The expression level of the target gene relative to the reference gene was calculated.

[0135] 1.2.5 Preparation of competent cells

[0136] The preparation method of E. coli and Agrobacterium competent cells refers to the reported method (Yang Yang. 2023. Mechanism of plant resistance to Phytophthora infestans mediated by mitochondrial RNA processing factors RTP7 and MORF8. [PhD thesis]. Yangling: Northwest A&F University).

[0137] 1.2.6 Identification and phylogenetic analysis of StRTP5a / b homologous proteins

[0138] In the plant genome database Phytozome v13 (https: / / phytozome-next.jgi.doe.gov / ), the sequences of StRTP5a and StRTP5a homologous proteins were obtained by BLASTP, and integrated into a document. Using MEGA software, ClustalW was used for sequence alignment, using the neighbor-joining method (NJ) to construct a phylogenetic tree, and finally using the iTOL website (https: / / itol.embl.de / ) to beautify the phylogenetic tree.

[0139] 1.2.7 Expression pattern analysis of potato genes StRTP5a and StRTP5b (abbreviated as StRTP5a / b) in response to P. infestans infection

[0140] The potato leaves of appropriate leaf age were taken, the petiole was wrapped with wet cotton to keep moist, and zoospores of P. infestans were inoculated. The leaves were cultured at 16℃ in the dark, and the leaves inoculated for 0, 3, 6, 12, 24, 36, 48, 72 and 96 hours were collected, frozen in liquid nitrogen and stored in a refrigerator at -80℃. Total RNA was extracted and reversely transcribed into cDNA, and then fluorescent real-time quantitative PCR was performed to quantify the expression of StRTP5a / b in potato.

[0141] 1.2.8 Cloning of StRTP5a and StRTP5b and construction of corresponding gene overexpression vectors

[0142] Primer design: Vazyme online website (https: / / crm.vazyme.com / cetool / singlefragment.html) was used to design specific primers StRTP5a-F and StRTP5a-R, StRTP5b-F and StRTP5b-R for amplifying the coding regions of StRTP5a and StRTP5b, respectively. The primers were synthesized by Shaanxi Zhongke Yudu Biological Technology Co., Ltd. The primer sequences used in the experiment are as follows in Table 17:

[0143] Table 17 Primer sequences

[0144]

[0145]

[0146] (1) PCR amplification of target genes

[0147] Using cDNA of potato Desiree as a template, the coding region sequences of StRTP5a and StRTP5b were amplified by StRTP5a-F and StRTP5a-R, StRTP5b-F and StRTP5b-R primers, respectively. The PCR amplification system and procedure of the target genes are as shown in Table 18 and Table 19.

[0148] Table 18 PCR amplification reaction system (30 μL reaction system)

[0149]

[0150]

[0151] Table 19 PCR reaction amplification procedure

[0152]

[0153] 125V, 30min, 1% agarose gel, electrophoresis, and according to the DNAMarker, the fragments of the desired size were cut and gel-recovered and purified for standby use.

[0154] (2) Vector linearization

[0155] The plant expression vector pART27-GFP was treated with restriction enzymes EcoR I and Xba I, and the enzyme reaction system was as shown in Table 20:

[0156] Table 20 Enzyme reaction system (40 μL system)

[0157] Component Amount 10x Buffer 4 μL BSA 0.4 μL EcoRI 1.0 μL XbaI 1.0 μL Vector pART27-GFP 1.0 μg ddH2O add to 40 μL

[0158] After incubation at 37°C for 30-60 min, 125 V, 30 min, 1% agarose gel, electrophoresis separation, and gel recovery and purification of the linearized vector pART27-GFP after enzyme digestion.

[0159] (3) Connection of linearized vector and target gene

[0160] The linearized vector obtained in step (2) was connected to the target gene in step (1) using a homologous recombination enzyme, and the connection reaction system was as shown in Table 21:

[0161] Table 21 Homologous recombination reaction system (10 μL system)

[0162]

[0163]

[0164] Incubation at 37°C for 30 min; storage at 4°C.

[0165] (4) Introduction of recombinant plasmid into E. coli

[0166] The homologous recombination product obtained in step (3) was added to the prepared E. coli Mach T1 competent cells, respectively, and mixed gently by pipetting, incubated on ice for 30 min, 42°C water bath for 90 s, iced for 2 min, added with 400 μL of sterilized LB liquid medium, and recovered at 37°C on a shaker at 120 rpm for 2 h. The recovered E. coli liquid was coated on an LB plate containing spectinomycin (Spec) in a clean bench, and incubated at 37°C in an incubator overnight (8-12 h).

[0167] (5) Identification of positive clones

[0168] The single clone colonies of E. coli obtained in step (4) were picked and placed in liquid LB medium containing spectinomycin (Spec), and shaken at 37°C at 200 rpm. After the bacterial solution became turbid, PCR was performed on the bacterial solution using primers StRTP5a-F and StRTP5a-R, and StRTP5b-F and StRTP5b-R, respectively (Table 22).

[0169] Table 22 PCR reaction system of bacterial liquid (20 μL system)

[0170] Component Amount 2x Taq PCR Master Mix 10 μL Forward primer (10 mM) 1 μL Reverse primer (10 mM) 1 μL Bacterial solution 1 μL ddH2O add to 20 μL

[0171] 125V, 30 min, 1% agarose gel, electrophoresis, and the correct band corresponding to the bacterial liquid was sent to Beijing Genki Biotechnology Co., Ltd. Xi'an Branch for DNA sequencing, and the nucleotide sequence of the gene StRTP5a (the amino acid sequence of the encoded protein is shown as SEQ ID NO: 3) and the nucleotide sequence of the gene StRTP5b (the amino acid sequence of the encoded protein is shown as SEQ ID NO: 4) were obtained as shown in SEQ ID NO: 1. Finally, the expression vector plasmid pART27-GFP-StRTP5a and pART27-GFP-StRTP5b containing the target gene were extracted from the correct sequencing bacterial liquid (the schematic diagram of the corresponding vector T-DNA region is shown as Figure 4 A and B).

[0172] (6) Electroporation of Agrobacterium

[0173] 100 ng of the expression vector plasmid containing the target gene obtained in step (5) was added to the competent Agrobacterium tumefaciens GV3101 (pMP90), and electroporation was performed at 1800V. 900 μL of LB liquid medium was added, and the recovery culture was incubated at 28°C with shaking at 120 r / min for 40 min. 100 μL of the recovered Agrobacterium liquid was inoculated on LB plates containing spectinomycin (Spec), gentamicin (Genta) and rifampicin (Rif) in a clean bench, and the plates were incubated at 28°C for 2d. Then the Agrobacterium clones were picked, and the bacterial liquid was used as a template for PCR identification with primers StRTP5a-F and StRTP5a-R, StRTP5b-F and StRTP5b-R. The Agrobacterium liquid containing pART27-GFP-StRTP5a and pART27-GFP-StRTP5b plasmids identified was prepared for subsequent Agrobacterium-mediated potato genetic transformation experiments.

[0174] 1.2.9 Construction of RNAi vector

[0175] ① The VIGS tool (https: / / vigs.solgenomics.net / ) of the Solanaceae Genome Database was used to select specific target silencing sequences of about 170 bp for genes StRTP5a and StRTP5b, respectively.

[0176] (2) Using StRTP5a-F (attBl) and StRTP5a-R (attB2), StRTP5b-F (attBl) and StRTP5b-R (attB2) primers to amplify specific target sequences, respectively, with cDNA of potato Desiree as template.

[0177] (3) Using StRTP5b-F (attBl) and StRTP5a-R (attB2) primers to perform fusion PCR, with the amplification product of step (2) as template.

[0178] (4) Gateway BP recombination reaction was performed with the amplification product of step (3) and entry vector pDONR201, and the recombination product was transferred to E. coli and verified by sequencing.

[0179] (5) The plasmid verified by sequencing was extracted, and Gateway LR reaction was performed with RNAi vector pHELLSGATE12, and the recombination product was transferred to E. coli and sequenced.

[0180] (6) The plasmid verified by sequencing was extracted, and the RNAi gene silencing vector targeting StRTP5a and StRTP5b synchronously was obtained (the schematic diagram of T-DNA region of the vector is shown in Fig. C), which was transferred into Agrobacterium tumefaciens GV3101 by electroporation, and then positive Agrobacterium clones were identified by PCR with primers pHells12-F and pHells12-R (Zhang Jiaxin, Ma Wenzhong, Dang Yaju, Jiao Wenjing, Li Ao, Mai Xuesheng, Qian Guanyu, Shan Weixing, Song Yin. 2025. Construction and genetic transformation of RNA interference vector targeting potato StRTP5a and StRTP5b genes synchronously. Acta Agronomica Sinica, 34(1): 45-53). Figure 4

[0181] 1.2.10 Agrobacterium tumefaciens-mediated genetic transformation of potato

[0182] The method of Agrobacterium tumefaciens-mediated genetic transformation of potato was referred to the method reported by Zhang Jiaxin et al. (Zhang Jiaxin, Ma Wenzhong, Dang Yaju, Jiao Wenjing, Li Ao, Mai Xuesheng, Qian Guanyu, Shan Weixing, Song Yin. 2025. Construction and genetic transformation of RNA interference vector targeting potato StRTP5a and StRTP5b genes synchronously. Acta Agronomica Sinica, 34(1): 45-53), and the main steps were as follows:

[0183] (1) Preparation of bacterial solution. Agrobacterium glycerol bacteria LB plate was streaked, and incubated at 28°C overnight. Single colony was picked and cultured at 28°C, 200 r / min, until OD 600 was about 0.8. The bacterial body was collected at room temperature, 4000 g, resuspended in MS liquid medium, and the concentration was adjusted to OD 600 = 0.6 for use as potato infection solution. ​

[0184] ②Agrobacterium infection. Potato genetic transformation method (An G, Watson B D, Chiang CC. 1986. Transformation of tobacco, tomato, potato, and Arabidopsis thaliana using a binary Ti vector system. Plant Physiology, 81(1): 301-305), cut the leaves into about 0.5 cm 2 sized leaf pieces and place them in a petri dish containing 10 mL MS liquid medium and 80 μL of Agrobacterium resuspension solution with OD 600 = 0.6, 23°C, dark co-culture for 2d.

[0185] ③Potato tissue culture. Absorb the bacterial solution on the surface of the leaf with sterile filter paper, transfer to CIM medium to induce callus differentiation. About 10d, transfer to SIM medium to induce adventitious buds from callus, and change new SIM medium after about 10d. Cut the adventitious buds that grow out and transfer to MS30 solid medium with 50 mg / L Kan for root induction to obtain regenerated lines for screening and identification.

[0186] 1.2.11 Extraction of potato genomic DNA

[0187] Extraction of potato genomic DNA refers to the method reported (Cao Y M. 2025. Mechanism of the effect of the effector protein PpE18 of the parasitic oomycete Phytophthora parasitica on the host ascorbate peroxidase APX3 to inhibit plant immunity. [PhD thesis]. Yangling: Northwest A&F University).

[0188] 1.2.12 Extraction of total protein from plant leaves

[0189] ① Mark and collect samples, freeze in liquid nitrogen.

[0190] ② Grind: Place the sample in a mortar pre-cooled in liquid nitrogen and grind into powder, transfer to a pre-cooled centrifuge tube, weigh the powder, add buffer, vortex to mix, and stand on ice for 30 min.

[0191] ③ 4°C, 12000g centrifugation for 20 min, collect supernatant, store at -80°C for later use.

[0192] 1.2.13 Western blot analysis

[0193] ① Prepare 10% SDS polyacrylamide gel according to the instructions of the rapid gel kit.

[0194] ② Put 10% SDS polyacrylamide gel into 100V electrophoresis tank, sample, electrophoresis for 1 h.

[0195] ③ Transfer membrane: after activating PVDF membrane with methanol for 3 min, put it into wet transfer liquid, put filter paper, PVDF membrane, gel and filter paper in turn, electrophoresis at 250 mA for 110 min.

[0196] ④ Put PVDF membrane into fast blocking liquid and block for about 15 min.

[0197] ⑤ Wash away the excess fast blocking liquid with TBS-T solution.

[0198] ⑥ Put PVDF membrane into Anti-GFP-HRP antibody diluent for incubation at room temperature for about 3 h.

[0199] ⑦ Wash away the excess primary antibody diluent with TBS-T solution, wash for 3 times, each for 15 min.

[0200] ⑧ Develop.

[0201] 1.2.14 Inoculation analysis of P. infestans and A. solani

[0202] Inoculation analysis of P. infestans: Cultivation of P. infestans and production of zoospores refer to the reported method (Yang, Y. 2023. Mechanism of plant resistance to P. infestans mediated by mitochondrial RNA processing factors RTP7 and MORF8. [PhD thesis]. Yangling: Northwest A&F University). Inoculation of potato detached leaves with P. infestans refer to the reported method (Cao, Y. 2025. Mechanism of plant immunity inhibition by P. parasitica effector protein PpE18 targeting host ascorbate peroxidase APX3. [PhD thesis]. Yangling: Northwest A&F University). Take the detached leaves, wrap the petiole with wet cotton, inoculate 13 μL P. infestans spore solution at the symmetrical position of the leaf vein, cover the film and keep it moist, and observe the disease after 4-6 days of culture at 16℃ in the dark. At the same time, stain the diseased area of the leaf with trypan blue, take a photo and calculate the leaf lesion area.

[0203] Inoculation analysis of Alternaria solani: The production of Alternaria solani conidia was performed according to the reported method (Li Q, Feng Y, Li J, Hai Y, Si L, Tan C, Peng J, Hu Z, Li Z, Li C, Hao D, Tang W. 2024. Multi-omics approaches to understand pathogenicity during potato early blight disease caused by Alternaria solani. Frontiers in Microbiology, 15: 1357579). Briefly, a piece of mycelium about 0.5 cm long was scratched with a sterile toothpick and transferred to potato dextrose solid medium (PDA) and cultured in a 25 °C incubator for 7 days or so. Then a small amount of sterile water was poured into the culture dish to wash off the mycelium on the surface of the medium, and a conidial suspension was prepared. The number of conidia was adjusted to about 3.0 x 10 4 The potato leaf pieces were taken, the petiole was wrapped with a wet cotton strip, 13 μL of spore suspension was inoculated symmetrically on both sides of the leaf vein, and the film was covered and moistened. The leaf pieces were cultured in the dark at 23 °C for 3-5 days, photographed and the lesion area was calculated.

[0204] Results and analysis of Example 2

[0205] 2.1 Genes StRTP5a (abbreviated as StRTP5a) and StRTP5b (abbreviated as StRTP5b) are up-regulated in response to infection by P. infestans

[0206] To verify the expression of StRTP5a and StRTP5b in response to infection by P. infestans, potato variety Désirée leaves were inoculated with P. infestans zoospores, and P. infestans-infected potato leaf samples were collected at 0, 3, 6, 12, 24, 36, 48, 72 and 96 h, and RT-qPCR analysis was performed. The results of RT-qPCR analysis showed that StRTP5a and StRTP5b could be induced to up-regulate expression in response to infection by P. infestans, and had similar expression trends Figure 1

[0207] Figure 1 ​The expression levels of StRTP5a and StRTP5b at different time points after inoculation of potato leaves with P. infestans were analyzed by RT-qPCR using StEF1a as the internal reference gene. The results were obtained from three biological replicates, and t-test was used for significance detection, *, ** and *** represent P < 0.05, P < 0.01 and P < 0.001, respectively. The results showed that StRTP5a and StRTP5b might play a role in the interaction between potato and P. infestans, and might function in a functional redundancy manner.

[0208] 2.2 Alignment analysis and phylogenetic analysis of StRTP5a and StRTP5b and their homologous proteins

[0209] To explore the consistency degree of protein sequence between StRTP5a and StRTP5b and Arabidopsis RTP5 (AtRTP5), the sequence alignment software ClustralX was used to perform sequence alignment analysis on the three proteins. The results showed that StRTP5a and StRTP5b both contained 4 conserved WD40 domains, and the sequence consistency between StRTP5a (as shown in SEQ ID NO: 3) and StRTP5b (as shown in SEQ ID NO: 4) and AtRTP5 protein was high, and the protein sequence consistency reached 54.5% and 53.9% (as shown in SEQ ID NO: 5), respectively. Figure 2

[0210] ​To further explore the phylogenetic relationship of StRTP5a (Soltu.DM.05G023120) and StRTP5b (Soltu.DM.09G027740) and their homologous proteins, StRTP5a or StRTP5b sequence was used as "query" to perform BlastP alignment in Phytozome v13 (https: / / phytozome-next.jgi.doe.gov / ), and homologous proteins of StRTP5a and StRTP5b were identified from dicotyledonous plants such as tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), pepper (Capsicum annuum), soybean (Glycine max), sunflower (Helianthus annuus), Arabidopsis thaliana and Brassica napus, and monocotyledonous plants such as wheat (Triticum aestivum), rice (Oryza sativa), maize (Zea mays) and sorghum (Sorghum bicolor). Then, the phylogenetic tree of StRTP5a and StRTP5b and their homologous proteins was constructed by using MEGA software with the neighbor-joining method. Phylogenetic tree analysis showed that the coding genes of StRTP5a and StRTP5b and their homologous proteins were not only distributed in dicotyledonous plants, but also in monocotyledonous plants. Figure 3 The results showed that the homologous proteins of StRTP5a / b were from Arabidopsis thaliana, Solanum lycopersicum, Nicotiana tabacum, Capsicum annuum, Glycine max, Helianthus annuus, Brassica napus, Triticum aestivum, Oryza sativa, Zea mays and Sorghum bicolor, respectively. The red bold part is StRTP5a and StRTP5b of potato.

[0211] 2.3 Obtaining and identifying potato gene silencing lines and overexpression lines

[0212] To investigate the function of StRTP5a and StRTP5b, we constructed overexpression vectors pART27-GFP-StRTP5a and pART27-GFP-StRTP5b (Fig. 1 Figure 4 A and B) for overexpression of StRTP5a and StRTP5b, respectively, and RNA silencing (RNAi) vector pHells12-StRTP5a / b i (Fig. 1 Figure 4 C) for RNAi of StRTP5a and StRTP5b, and transformed the vectors into Agrobacterium tumefaciens strain GV3101 for genetic transformation of potato.

[0213] Leaves of potato cultivar Desiree were used as explants and co-cultured with Agrobacterium infection solution carrying the target gene vectors for 2 days (Fig. 1 Figure 5 A), the infected leaves were transferred to sterile filter paper to remove the surface infection solution, and then transferred to callus induction medium for culture to induce callus formation (Fig. 1 Figure 5 B), 10 days later, the leaves with callus formation were transferred to bud induction medium for culture, and new bud induction medium was replaced every 7 to 10 days until the callus differentiated into buds (Fig. 1 Figure 5 C), the buds were cut off and transferred to MS30 solid medium containing 50 mg / L Kan + (Fig. 1 Figure 5 D and E) until potato transgenic plants were obtained (Fig. 1 Figure 5 F).

[0214] Using Agrobacterium tumefaciens-mediated genetic transformation technology, we obtained potato transgenic lines transformed with pHells12-StRTP5a / b i, pART27-GFP-StRTP5a and pART27-GFP-StRTP5b vectors, respectively. The potato lines transformed with pHells12-StRTP5a / b i vector were verified by PCR for the insertion of exogenous DNA fragments, and 8 potato lines that silenced StRTP5a and StRTP5b were obtained, named StRTP5a / b i-1 to 8 (Fig. 1 Figure 6 A). Further verification of the silencing of StRTP5a and StRTP5b by RT-qPCR, we finally obtained 2 potato lines that silenced StRTP5a and StRTP5b simultaneously, StRTP5a / b i-2 and StRTP5a / b i-7 (Fig. 1 Figure 6(See Figure B). Transgenic potato lines transformed with pART27-GFP-StRTP5a and pART27-GFP-StRTP5b vectors (named StRTP5a OE-1~StRTP5a OE-5 and StRTP5b OE-1~StRTP5b OE-5, respectively) showed accumulation of the corresponding target protein in both lines after PCR and Western blotting analysis, as indicated by PCR and Western blotting. Furthermore, the accumulation of the target protein was relatively high in the StRTP5aOE-3, StRTP5aOE-4, StRTP5b OE-1, and StRTP5b OE-4 lines. Figure 6 (C and D in the middle).

[0215] Further observation was conducted on the growth phenotypes of RNAi lines StRTP5a / b i-2 and StRTP5a / b i-7 and overexpression lines StRTP5aOE-3, StRTP5aOE-4, StRTP5b OE-1, and StRTP5b OE-4 in soil seedlings approximately 10 days old. The results showed no significant difference in growth phenotype between lines simultaneously silenced and separately overexpressed StRTP5a and StRTP5b compared to wild-type Désirée. Figure 7 The potato lines were plantlets that had grown for 10 days after being transplanted from tissue culture seedlings into soil. Therefore, the potato lines StRTP5a / bi-2 and StRTP5a / bi-7, which showed the most obvious simultaneous silencing of the genes StRTP5a and StRTP5b, as well as the overexpression lines StRTP5a OE-3, StRTP5a OE-4, StRTP5b OE-1, and StRTP5b OE-4, were selected for subsequent experimental analysis.

[0216] 2.4 StRTP5a and StRTP5b negatively regulate potato resistance to late blight

[0217] To investigate whether StRTP5a and StRTP5b play a role in the interaction between potato and Phytophthora blight, the RNAi gene-silenced lines (StRTP5a / bi-2 and StRTP5a / bi-7, i.e., StRTP5a&bi-2 and StRTP5a&bi-7), the StRTP5a overexpression lines (StRTP5a OE-3 and StRTP5a OE-4), and the StRTP5b overexpression lines (StRTP5b OE-1 and StRTP5b OE-4) were inoculated with Phytophthora blight using the detached leaf method. Resistance analysis showed that, compared with the Désirée control, potato lines that simultaneously silenced both StRTP5a and StRTP5b had significantly smaller lesion areas on their leaves, while potato lines that overexpressed StRTP5a and StRTP5b respectively had significantly larger lesion areas on their leaves. Figure 8 (A and B), and biomass analysis of pathogen colonization also showed that potato lines with StRTP5a / b silenced had significantly reduced Phytophthora colonization, while potato leaves overexpressing StRTP5a and StRTP5b, respectively, had more Phytophthora colonization. Figure 8 (C). This result indicates that the homologous genes StRTP5a and StRTP5b negatively regulate potato resistance to Phytophthora in a functionally redundant manner.

[0218] 2.5 StRTP5a and StRTP5b negatively regulate potato resistance to early blight

[0219] To investigate whether StRTP5a and StRTP5b also regulate potato resistance to other potato pathogens, RNAi gene-silenced lines (StRTP5a / bi-2 and StRTP5a / bi-7), StRTP5a overexpression lines (StRTP5a OE-3 and StRTP5a OE-4), and StRTP5b overexpression lines (StRTP5b OE-1 and StRTP5b OE-4) were inoculated with *Alternaria solani*, the pathogen of early blight of potato, using the in vitro leaf inoculation method. Analysis of the inoculation results showed that, compared with the Désirée control, potato lines that simultaneously silenced both StRTP5a and StRTP5b were more resistant to *Alternaria solani*, while potato lines that overexpressed StRTP5a and StRTP5b were more susceptible to *Alternaria solani* infection. Figure 9 This result indicates that the homologous genes StRTP5a and StRTP5b also negatively regulate resistance to early blight of potato in a functionally redundant manner.

[0220] The above results show that StRTP5a and StRTP5b negatively regulate the resistance to potato late blight and early blight in a functional redundant manner, and imply that the genes StRTP5a and StRTP5b have the potential to be used for creating potato broad-spectrum disease-resistant materials.

[0221] The present application uses the agrobacterium-mediated potato genetic transformation technology to obtain transgenic lines overexpressing and silencing the genes StRTP5a and StRTP5b respectively, and then tests the resistance of the obtained transgenic lines to the pathogenic P. infestans and other potato pathogens (early blight pathogens), which can provide materials and reference information for further analyzing the mechanism of the genes StRTP5a and StRTP5b in the potato late blight resistance.

[0222] The above-described embodiments are part of the embodiments of the present application, rather than all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but to represent selected embodiments of the application. All other embodiments obtained by persons of ordinary skill in the art based on the related deductions and substitutions under the condition of the concept of the present application, without making creative efforts, belong to the scope of protection of the present application.

Claims

1. Application of potato late blight pathogen responsive gene StRTP5 in regulating potato disease resistance, wherein the potato late blight pathogen responsive gene StRTP5 comprises a gene StRTP5a and a gene StRTP5b, characterized in that, the nucleotide sequence of the gene StRTP5a is shown as SEQ ID NO: 1; and the nucleotide sequence of the gene StRTP5b is shown as SEQ ID NO:

2. The regulation of potato disease resistance comprises: a) overexpression of the gene StRTP5a and the gene StRTP5b to enhance the susceptibility of potato to late blight and early blight; and / or 2. Use according to claim 1, characterized in that, b) silencing of the gene StRTP5a and the gene StRTP5b to enhance the resistance of potato to late blight and early blight. The amino acid sequence of the protein encoded by the gene StRTP5a is shown as SEQ ID NO:

3. The amino acid sequence of the protein encoded by the gene StRTP5b is shown as SEQ ID NO:

4.

3. Use according to claim 1, characterized in that, The pathogen of the late blight is Phytophthora infestans.

4. Use according to claim 1, characterized in that, The pathogen of the early blight is Alternaria solani.

5. The use according to claim 1, characterized in that, 7. A potato gene silencing strain, characterized in that, 6. Use according to claim 1, characterized in that, The potato gene silencing strain is obtained by silencing potato late blight pathogen responsive gene StRTP5; The potato late blight pathogen responsive gene StRTP5 comprises a gene StRTP5a and a gene StRTP5b; The nucleotide sequence of the gene StRTP5a is shown as SEQ ID NO: 1; The nucleotide sequence of the gene StRTP5b is shown as SEQ ID NO: 2; Silencing of the gene StRTP5a and the gene StRTP5b enhances the resistance of potato to late blight and early blight.

8. An expression vector, characterized in that, The expression vector contains potato late blight pathogen responsive gene StRTP5; The potato late blight pathogen responsive gene StRTP5 comprises a gene StRTP5a and a gene StRTP5b; The nucleotide sequence of the gene StRTP5a is shown as SEQ ID NO: 1; The nucleotide sequence of the gene StRTP5b is shown as SEQ ID NO:

2. The expression vector of claim 8 is introduced into potato to silence the gene StRTP5a and the gene StRTP5b.

10. A method for breeding potato varieties resistant to Phytophthora infestans and Alternaria solani, characterized in that, 9. A method for breeding potato varieties resistant to late and early blight, characterized in that, The method comprises the step of silencing the gene StRTP5a and the gene StRTP5b; The nucleotide sequence of the gene StRTP5a is shown as SEQ ID NO: 1; The nucleotide sequence of the gene StRTP5b is shown as SEQ ID NO:

2. ​ ​

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