A ScPsbP gene, protein and application with disease resistance

Abstract

The present invention provides a kind of disease-resistant ScPsbP Genes, proteins and applications, belonging to the field of biotechnology, and specifically discloses sugarcane ScPsbP The application of genes in improving disease resistance. PsbP Genes play an important role in plant response to pathogenic bacteria stress. ScPsbP A preliminary study was conducted on the disease resistance function of the gene, which laid a solid foundation for in-depth research on the function and mechanism of this gene in the process of sugarcane responding to pathogen stress.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and relates to plant transgenic biotechnology breeding, and in particular to a plant with disease resistance. ScPsbP Genes, proteins and applications. Background Art

[0002] sugar cane( Saccharum spp.), is the largest cash crop in terms of biomass, and can store extremely high concentrations of sucrose, which can account for 25% of fresh weight under suitable conditions. Sugarcane is mainly grown in tropical and subtropical regions. The global sugarcane planting area exceeds 24.9 million hectares, covering more than 80 countries. In my country, more than 85% of sugar comes from sugarcane. However, sugarcane production is attacked by a variety of pests and diseases, among which the most devastating is sugarcane smut, known as "cancer", which can cause a loss of more than 60% of sugarcane yield under favorable environmental conditions, and even more so for ratoon sugarcane. Against the background of global warming, the prevalence of fungal diseases and their pathogenicity will show an intensifying trend. At present, Guitang 42, Liucheng 05-136, Yunzhe 08-1609 and ROC22 have poor resistance to smut, resulting in increased disease stress in the sugarcane industry. Exploring key disease-resistant genes in sugarcane and using molecular breeding methods to cultivate new disease-resistant sugarcane germplasm are the most economical and effective measures to prevent and control sugarcane diseases.

[0003] Oxygen-evolving enhancer proteins (OEEs) are nuclear-encoded chloroplast proteins composed of three subunits (OEE1 [33 kDa, PsbO], OEE2 [23 kDa, PsbP] and OEE3 [16 kDa, PsbQ]) that bind to the periphery of photosystem II (PSII) on the thylakoid lumen side. After PsbP is synthesized on the cytoplasmic ribosomes, it is transported to the thylakoid lumen inside the chloroplast via a transit peptide. PsbP regulates the water splitting reaction and is essential for the core assembly and stability of PSII. Reduced PsbP expression leads to reduced photosynthesis. PsbP protein is expressed in sweet potato ( Ipomoea batatas ) accumulates under heat stress and is stabilized by IbOr, resulting in carotenoid accumulation and conferring tolerance to heat stress in transgenic sweet potato plants. Proteomic studies have shown that inoculation with downy mildew ( Plasmopara viticola ) After the PsbP protein was accumulated in large quantities, the above indicated that PsbP could participate in the process of plant response to abiotic stress and biotic stress. Summary of the invention

[0004] In view of the above problems, the present invention provides a disease-resistant ScPsbP Genes, proteins and applications.

[0005] A disease-resistant ScPsbP Gene, the ScPsbP The coding sequence of the gene is shown in SEQ ID NO:1.

[0006] A ScPsbP protein with disease resistance, the ScPsbP protein is the above ScPsbP The protein encoded by the gene has an amino acid sequence as shown in SEQ ID NO: 2;

[0007] The ScPsbP protein is a chloroplast protein.

[0008] A primer pair, characterized in that the primer pair is used for PCR amplification of the above ScPsbP Gene, the primer pair comprises:

[0009] ScPsbP -F:5'-CACAATGCCACACATACG-3';

[0010] ScPsbP -R:5'-TATGTACCGTAGGCTGCT-3';

[0011] Specifically, the PCR amplification is based on sugarcane ScPsbP The cDNA of the gene was used as a template for PCR amplification; ScPsbP The cDNA sequence of the gene is shown in SEQ ID NO: 3;

[0012] Among them, the reaction system of PCR amplification is:

[0013]

[0014] The reaction procedure for PCR amplification is:

[0015] .

[0016] A kind of ScPsbP A plasmid containing a gene ScPsbP The plasmid of the gene was constructed using the above ScPsbP The gene was connected to a cloning vector.

[0017] A kind of ScPsbP The gene expression vector is characterized in that the ScPsbP Gene expression vectors are constructed using ScPsbP The PCR amplification product of the gene was ligated with the overexpression vector pEarleyGate203, the subcellular localization vector pCAMBIA1300-GFP, or the vector pGBKT7 to obtain;

[0018] SaidScPsbP The coding sequence of the gene is shown in SEQ ID NO:1.

[0019] A kind of ScPsbP A strain comprising a gene ScPsbP The strain containing the gene is ScPsbP Agrobacterium tumefaciens or the like containing the gene ScPsbP Genetic yeast;

[0020] The inclusion ScPsbP The Agrobacterium tumefaciens containing the gene is ScPsbP The gene expression vector is introduced into Agrobacterium to obtain;

[0021] The method introduced into Agrobacterium ScPsbP Gene expression vectors are constructed using ScPsbP The PCR amplification product of the gene was ligated with the overexpression vector pEarleyGate203 or the subcellular localization vector pCAMBIA1300-GFP to obtain;

[0022] The inclusion ScPsbP The yeast containing the gene is used ScPsbP The gene expression vector is introduced into yeast to obtain;

[0023] The method introduced into the yeast comprises ScPsbP Gene expression vectors are constructed using ScPsbP The PCR amplification product of the gene was connected with the vector pGBKT7 to obtain.

[0024] One of the above ScPsbP The application of genes in improving disease resistance is ScPsbP Genes are used in sugarcane or Nicotiana benthamiana to improve disease resistance.

[0025] Furthermore, the application is overexpression ScPsbP genes to improve disease resistance;

[0026] Specifically, the application is to use ScPsbP Agrobacterium tumefaciens with the gene was used to transform sugarcane or Nicotiana benthamiana to improve disease resistance.

[0027] A plant breeding method, by ScPsbP Gene overexpression to obtain disease-resistant plants;

[0028] Said ScPsbP The coding sequence of the gene is shown in SEQ ID NO:1.

[0029] A planting method for improving disease resistance, the planting method comprising: ScPsbPGene overexpression in plants to improve disease resistance;

[0030] Said ScPsbP The coding sequence of the gene is shown in SEQ ID NO:1.

[0031] A disease-resistant ScPsbP The beneficial effects of genes, proteins and applications are:

[0032] PsbP Genes play an important role in plant response to pathogenic bacteria stress. ScPsbP The preliminary study on the disease resistance function of β-lactamase has laid a solid foundation for in-depth study of the function and mechanism of this gene in the process of sugarcane responding to pathogenic bacteria stress.

[0033] The present invention is to treat sugarcane ScPsbP The study of genes is helpful to understand the disease resistance mechanism of sugarcane and provides a theoretical basis and reference for inducing plants to have disease resistance by using genetic engineering technology.

[0034] The present invention comprises ScPsbP Plant expression recombinant vector and containing ScPsbP Gene-transfected Agrobacterium tumefaciens ScPsbP The gene was introduced into heterologous Nicotiana benthamiana and analyzed ScPsbP The disease resistance function of the gene in Nicotiana benthamiana; the results showed that ScPsbP After the gene is overexpressed, it has obvious disease resistance function; therefore, the ScPsbP The gene has important application value not only in improving the disease resistance of native sugarcane, but also in heterologous Nicotiana benthamiana.

[0035] The present invention is to treat sugarcane ScPsbP Gene research found ScPsbP The gene is expressed in different tissues of sugarcane, with the highest expression in sugarcane +1 leaves, followed by young leaves. It is a chloroplast protein, indicating that sugarcane ScPsbP The genes may play a role in different sugarcane tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The sugarcane in Example 1 of the present invention ScPsbP Gene expression pattern results diagram; Figure 1 Figure A in ScPsbP The expression pattern of genes in different tissues of sugarcane. ScPsbP The expression pattern of genes under salicylic acid stress, Figure C ScPsbP The expression pattern of genes under methyl jasmonate stress, Figure D ScPsbPThe expression pattern of genes in the interaction between smut-resistant sugarcane varieties and smut fungi, Figure E ScPsbP Expression patterns of genes in the interaction between sugarcane varieties susceptible to smut and smut pathogens; among them, the smut-resistant sugarcane varieties are YT93-159 and GT29; the smut-susceptible sugarcane varieties are ROC22 and LC03-182; GAPDH The gene was used as the internal reference gene; the error bars represent the standard deviation; the significant difference between Figure A, Figure B and Figure C was tested by one-way analysis of variance (P<0.01); the significant difference between Figure D and Figure E was tested by two-way analysis of variance (P<0.01);

[0037] Figure 2 is the bioinformatics analysis result of ScPsbP in Example 3 of the present invention; Figure 2 Figure A shows the conserved domain of ScPsbP; Figure B shows the comparison of homologous sequences of ScPsbP with other species; Figure C shows the phylogenetic tree of homologous sequences of ScPsbP with other species;

[0038] Figure 3 This is a diagram of the recombinant plasmid pCAMBIA1300-ScPsbP-GFP vector in Example 4 of the present invention;

[0039] Figure 4 This is the subcellular localization analysis of the sugarcane ScPsbP protein in Example 4 of the present invention; wherein, 35S:GFP represents the empty pCAMBIA1300-GFP, 35S:ScPsbP:GFP represents the fusion expression vector pCAMBIA1300-ScPsbP-GFP; the direction indicated by the white arrow is the chloroplast; after transient expression in tobacco for 2 days, green fluorescence (green fluorescent protein, GFP), chloroplast autofluorescence (Chloroplast), visible light (Visible) and fused image (Merged) were captured on a laser confocal microscope; 35S:ScPsbP:GFP (1) represents the tobacco leaf with the back side facing up and the photo taken; 35S:ScPsbP:GFP (2) represents the tobacco leaf with the front side facing up and the photo taken; Bars=25 μm;

[0040] Figure 5 This is a diagram of the recombinant plasmid pGBKT7-ScPsbP vector in Example 4 of the present invention;

[0041] Figure 6This is the analysis of the transcriptional self-activation activity of the sugarcane ScPsbP protein in Example 4 of the present invention; wherein the positive control is pGBKT7-53+pGADT7-T; the negative control is pGADT7-T; -T corresponds to SD / -Trp medium; -T / X corresponds to SD / -Trp / X-α-Gal (40 μg / mL) medium; -T / X / A corresponds to SD / -Trp+X-α-Gal (40 μg / mL)+AbA (200 ng / mL) medium;

[0042] Figure 7 The fusion expression vector pEarleyGate203- ScPsbP Carrier map;

[0043] Figure 8 is the transient overexpression in Example 5 of the present invention ScPsbP The results of disease resistance identification of tobacco leaves after inoculation with Phytophthora; Figure 8 Figure A in the middle shows transient overexpression under UV ScPsbP The phenotype of tobacco leaves after inoculation with Phytophthora, where 35S:00 represents the overexpression vector pEarleyGate203; 35S:ScPsbP represents the fusion expression vector pEarleyGate203- ScPsbP ; Figure B shows transient overexpression ScPsbP DAB staining of tobacco leaves after inoculation with Phytophthora, where 35S:00 represents the overexpression vector pEarleyGate203; 35S:ScPsbP represents the fusion expression vector pEarleyGate203- ScPsbP ; Figure C shows transient overexpression ScPsbP Detection of immune marker-related genes in tobacco leaves after inoculation with Phytophthora NbEF1-α Genes were used as internal reference genes; Figure 8 The leaves were photographed 2 days after inoculation with Phytophthora; error bars represent standard deviations; and two-way analysis of variance was used to test for significant differences (P<0.01). DETAILED DESCRIPTION

[0044] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0045] In addition, if no specific techniques or conditions are specified in the specific examples disclosed below, the techniques or conditions described in the literature in this field (for example, refer to "Molecular Cloning Experiment Guide" written by Sambrook et al., translated by Huang Peitang et al., third edition, Science Press) or the product instructions are used. If the manufacturer of the reagents used is not specified, they are all conventional products that can be purchased.

[0046] Plant materials: Smut-resistant sugarcane varieties (GT29 and YT93-159), smut-susceptible sugarcane varieties (LC03-182 and ROC22), and wild-type Nicotiana benthamiana were provided by Fujian Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs, Fujian Agriculture and Forestry University.

[0047] Wild-type Nicotiana benthamiana (WT) was grown in an incubator at 25°C with a light condition of 16 h / 8 h (light / dark).

[0048] Vectors: pCAMBIA1300-GFP, pGBKT7-53, pGBKT7-T, pGBKT7 and pEarleyGate203.

[0049] Strains: Escherichia coli strain DH5α, yeast strain Y2Hgold and Agrobacterium strain GV3101.

[0050] Main reagents: High-fidelity enzyme (2×Phanta Flash Master Mix, #P510), blue universal dye qPCR detection kit (ChamQ Blue Universal SYBR qPCR Master Mix, #Q312) were purchased from Nanjing Novozyme Biotechnology Co., Ltd.; Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (#11141ES60) and acetosyringone (Acetosyringone, AS, #60326ES03) were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; MagZolTM Reagent (#R4801-01), agarose gel DNA recovery kit (#D2110-03) and plasmid extraction kit (#P1001-03C) were purchased from Guangzhou Meiji Biotechnology Co., Ltd.; Gateway™ BP Clonase™ II enzyme mixture (#11789020) Gateway™ LR Clonase™ II enzyme mixture (#11791020), restriction enzymes FastDigest KpnI (#FD0524), FastDigest XbaI (FD0684), FastDigestEcoRI (#FD0274), and FastDigest BamHI (#FD0054) were purchased from Thermo Fisher Scientific; 2-(N-morpholino)ethanesulfonic acid monohydrate (MES, #A610341) and magnesium chloride hexahydrate (MgCl2, #A610328) were purchased from Sangon Biotech (Shanghai) Co., Ltd.; SD / -Trp with Agar (#PM2252), SD / -Trp Broth (PM2251), Aureobasidin A (#CA2332G), and X-α-gal solution (X-α-gal Solution, #SL0940) was purchased from Beijing Coolaibo Technology Co., Ltd.; 3,3'-Diaminobenzidine Tetrahydrochloride (DAB, #D807420) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0051] All primers involved in the present invention were designed using SnapGene, Primer Premier 5 and CE Design software, and sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0052] Example 1 ScPsbP Gene expression pattern

[0053] 1) RNA extraction and RT-qPCR analysis

[0054] RNA was extracted from sugarcane (GT29, YT93-159, LC03-182 and ROC22) and Nicotiana benthamiana plants according to MagZolTM Reagent (#R4801-01), and then reverse transcribed and synthesized into cDNA according to the instructions of Hifair® Ⅲ 1st Strand cDNA SynthesisSuperMix for qPCR (gDNA digester plus) (#11141ES60) for subsequent gene expression detection template. ScPsbP The cDNA sequence of the gene is shown in SEQ ID NO:3.

[0055] Designed with Primer Premier 5 software ScWRKY2 Specific quantitative primers (primer pair: Q- ScPsbP -F / R), and the reference gene of sugarcane was glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase, GAPDH ) (Primer pair: GAPDH -F / R), the internal reference gene of Nicotiana benthamiana is NbEF-1α (Primer pairs: NbEF- 1α -F / R), see Table 1 for details.

[0056] Table 1 Primers for RT-qPCR analysis

[0057]

[0058] in, ScPsbP The gene was transiently overexpressed in tobacco. After inoculation with pathogens, the immune marker-related genes were detected. NbEF-1α As an internal reference gene, it is used for the subsequent calculation of gene expression.

[0059] 2) Analysis of gene expression patterns

[0060] To determine ScPsbP The expression characteristics of genes in sugarcane tissues were detected using the above-mentioned RT-qPCR technology ScPsbPGene expression in different tissues of sugarcane.

[0061] Healthy plants of mature sugarcane cultivar ROC22 were selected, and six tissues including young leaves (newest leaves), +1 leaves (first fully expanded leaves), leaf sheaths, pith, skin and buds were collected. RNA was extracted and then cDNA was synthesized to detect the tissue specificity of the target gene.

[0062] Sugarcane cDNA samples at different time points after treatment with exogenous hormones salicylic acid (SA) and methyl jasmonate (MeJA) were used as templates to detect the expression of target genes under hormone stress. The specific treatment method is referred to "Identification and characterization of WAK gene family in Saccharum and the negative roles of ScWAK1 under the pathogen stress" [J]. (Wang D, Qin L, Wu M, et al. International Journal of Biological Macromolecules, 2023, 224: 1-19).

[0063] Sugarcane (GT29, YT93-159, LC03-182 and ROC22) buds were inoculated with smut fungus according to the method described in "Transcriptome and proteome research on sugarcane response to smut fungus infection and mining of resistance-related genes" (Su Yachun et al., Fujian Agriculture and Forestry University, 2014), and bud samples were collected 0 d, 1 d, 2 d, 3 d and 5 d after inoculation. Specifically, 10-month-old healthy plants were selected from the field, cut into single bud segments, and placed in a 32°C incubator for germination. After the buds grew to 2-3 cm, buds with consistent growth were selected and inoculated with a mixed spore suspension of smut fungus (5×10 6 Each time point was set up with 3 biological replicates, and 3 sugarcane buds were taken from each replicate. The treated samples were quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator for later use. ScPsbP Detection of gene expression.

[0064] The cDNA of sugarcane with different genotypes inoculated with Ustilago smut was used as template to detect the expression of target genes under the stress of Ustilago smut. GAPDHThe internal reference was selected according to "Selection of internal reference genes in quantitative PCR analysis of sugarcane gene expression" [J]. (Que Youxiong, Xu Liping, Xu Jingsheng, et al. Journal of Tropical Crops, 2009, 30(03): 274-278.). The gene expression was detected according to the instructions of the blue universal dye qPCR detection kit (ChamQ Blue Universal SYBR qPCR Master Mix, #Q312). The data were obtained using 2 -ΔΔCt The data were processed according to the method of "Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta DeltaC(T)) Method" [J]. (Livak KJ, Schmittgen T D. Methods, 2001, 25(4): 402-408.), and the graphs were drawn using GraphPad Prism 10 software. The specific results are shown in Figure 1 .

[0065] Depend on Figure 1 As can be seen from Figure A, ScPsbP The gene expression level was highest in the first fully expanded leaf (i.e., +1 leaf), followed by young leaves, and lowest in cane pulp and buds. ScPsbP The expression levels of the gene in +1 leaf were 24.31 and 23.95 times that in sugarcane pith and sugarcane bud, respectively.

[0066] Depend on Figure 1 As can be seen in Figure B, after 3 h of exogenous hormone salicylic acid (SA) stress, ScPsbP The gene began to be induced to express, and the expression level reached the highest level at 12 h of stress, which was 7.07 times that of the 0 h control.

[0067] Depend on Figure 1 As can be seen in Figure C, ScPsbP The expression pattern of genes under methyl jasmonate (MeJA) stress was different from that under SA. ScPsbP The gene was significantly induced to up-regulate expression, and at 24 h, ScPsbP The expression began to be downregulated, and the expression level at 12 h was 26.46 times that of the 0 h control.

[0068] Depend on Figure 1 As can be seen from Figures D and E, ScPsbPThe expression levels of the gene in the smut-resistant sugarcane varieties YT93-159 and GT29 showed an overall upward expression trend, reaching the highest expression levels at 2 days, which were 8.09 and 9.44 times that of the 0-day control, respectively. ScPsbP The gene began to down-regulate expression. In the sugarcane varieties ROC22 and LC03-182 susceptible to smut, ScPsbP The gene expression levels were low overall, especially in LC03-182. ScPsbP The gene expression was significantly suppressed.

[0069] Example 2 ScPsbP Gene cloning

[0070] 1) Based on the genome annotation information of sugarcane cultivar R570, cloning primers were designed using SnapGene software ScPsbP -F / R, see the table below.

[0071] Table 2 Cloning primers ScPsbP-F / R

[0072]

[0073] 2) Using sugarcane leaf cDNA as a template, PCR amplification was performed according to the instructions of the high-fidelity enzyme (2×Phanta Flash Master Mix, #P510). The specific PCR amplification reaction system and reaction procedure are as follows:

[0074] Table 3 PCR reaction system

[0075]

[0076] Table 4 PCR reaction procedure

[0077]

[0078] 3) Perform electrophoresis on the PCR product using 1.5% agarose gel and recover the target band (i.e. ScPsbP Gene);

[0079] in, ScPsbP The coding sequence of the gene is shown in SEQ ID NO: 1, and its open reading frame (ORF) is 750 bp, i.e. ScPDC1 The open reading frame of the gene is shown in SEQ ID NO:4.

[0080] 4) Connect the recovered product to the cloning vector pEASY®-Blunt Zero. For the specific connection method, refer to the instructions of the pEASY®-Blunt Zero Cloning Kit;

[0081] 5) Transform the ligation product into E. coli strain DH5α and spread on LB (Kan + 50 mg / L) plates;

[0082] 6) Culture in a 37°C incubator overnight, pick the colonies on the plate, identify them by PCR, select the colonies with the correct bands, and send them to Sangon Biotech (Shanghai) Co., Ltd. for sequencing;

[0083] 7) Use a plasmid extraction kit to extract plasmids from the colonies that have been sequenced correctly. The resulting plasmid is ScPsbP The inserted fragments were stored at -20°C for subsequent experiments.

[0084] Example 3 ScPsbP Gene cloning and sequence analysis

[0085] 1) Bioinformatics analysis

[0086] The domain structure of ScPsbP protein was predicted using the NCBI Conserved Domain Search online website (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi). The homologous sequences of candidate interacting proteins in other species were retrieved using the BLAST tool on the NCBI website, and multiple sequence alignment was performed using DANMAN software. The physicochemical properties of candidate interacting proteins were predicted using the ProtParam tool website (https: / / web.expasy.org / protparam / ). Subcellular localization was predicted using Plant-mPLoc (http: / / www.csbio.sjtu.edu.cn / bioinf / plant-multi / ). A phylogenetic tree was constructed using IQ-TREE in PhyloSuite software.

[0087] Using sugarcane leaf cDNA as template, we cloned ScPsbP The CDS sequence of the gene was obtained and the nucleic acid sequence was translated into a protein sequence (i.e., ScPsbP protein). The amino acid sequence of the ScPsbP protein is shown in SEQ ID NO: 2. ScPsbP To determine the biological function of a gene, we first analyze its sequence characteristics. ScPsbPThe ORF length of the gene is 750 bp, encoding 249 amino acids. The molecular weight of the protein is 26.61 kDa, and the isoelectric point is 5.51, which is less than 7, so it is speculated to be an acidic protein. In addition, the instability coefficient, fat coefficient and average hydrophobicity of the protein are 49.97, 79.28 and -0.214, respectively. The instability coefficient of the ScPsbP protein is greater than 40 and the average hydrophobicity is less than 0, so it is speculated that the protein is an unstable hydrophilic protein. The subcellular localization prediction results show that ScPsbP is located in the chloroplast.

[0088] The results of the conserved domain analysis showed that the ScPsbP protein had a PsbP domain at positions 86 to 248 (e.g. Figure 2 ScPsbP was retrieved by BLAST tool of NCBI and had high homology with sorghum XP_021321129.1 and XP_021321130.1 proteins, maize NP_001358948.1 protein, rice NP_001409559.1 protein, wheat KAF7101046.1 protein, barley XP_044957774.1 protein, pineapple XP_020110542.1 protein and Arabidopsis NP_565131.1 protein (as shown in Figure A). Figure 2 The phylogenetic tree of ScPsbP protein and the above 8 proteins was constructed, and the results showed that ScPsbP protein had the highest homology with XP_021321130.1 and XP_021321129.1 proteins in sorghum, which were 86.11% and 81.85%, respectively (see Figure 2). Figure 2 (as shown in Figure C).

[0089] Example 4 ScPsbP protein is localized in chloroplasts and has no transcriptional autoactivation activity

[0090] 1) Subcellular localization analysis

[0091] Subcellular localization prediction showed that ScPsbP protein was localized in chloroplasts. In order to verify the subcellular localization of ScPsbP, the ScPsbP protein was constructed into an expression vector pCAMBIA1300-GFP (referred to as 35S:GFP) with a GFP fluorescent tag to form a fusion expression vector pCAMBIA1300-ScPsbP-GFP (referred to as 35S:ScPsbP:GFP), as follows:

[0092] (1) Select KpnI and XbaI restriction sites to linearize the pCAMBIA1300-GFP vector to obtain a linearized vector;

[0093] (2) Use CE Design software to design the pCAMBIA1300-GFP vector linker primer 1300-ScPsbP-F / R, see the table below:

[0094] Table 5 pCAMBIA1300-GFP vector linker primer 1300-ScPsbP-F / R

[0095]

[0096] (3) Set up the one-step cloning reaction system according to the instructions of the non-ligase-dependent single-fragment one-step cloning kit (ClonExpress II One Step Cloning Kit, #C112-01) (see Table 6) and react at 37°C for 30 min;

[0097] Table 6 One-step cloning reaction system

[0098]

[0099] (4) The ligation product obtained above was transformed into Escherichia coli DH5α and cultured on LB solid medium (kanamycin 50 mg / L) in a 37°C incubator overnight;

[0100] (5) Pick single clones for PCR detection and sequencing analysis (sequencing by Sangon Biotech (Shanghai) Co., Ltd.);

[0101] (6) Extract the plasmid from the bacterial suspension with the correct sequencing results according to the instructions of the plasmid extraction kit (#P1001-03C) to obtain the subcellular localization recombinant vector pCAMBIA1300-ScPsbP-GFP, as shown in Figure 3 shown.

[0102] (7) Transient expression method: The subcellular localization vector pCAMBIA1300-GFP (referred to as 35S:GFP) and the fusion expression vector pCAMBIA1300-ScPsbP-GFP (referred to as 35S:ScPsbP:GFP) were transformed into Agrobacterium strain GV3101 by freeze-thaw method and spread on LB resistance plates (kanamycin 50 µg / mL and rifampicin 50 µg / mL). After culturing in a 28°C incubator for 2 days, single clones on the plates were picked and placed in 1 mL LB liquid medium (kanamycin 50 µg / mL and rifampicin 50 µg / mL) and cultured overnight. 200 µL of the positive bacterial solution after PCR detection was selected and added to a new 10 mL LB liquid medium (kanamycin 50 µg / mL and rifampicin 50 µg / mL), and cultured overnight in a 28°C shaker at 250 rpm. The cells were collected by centrifugation and inoculated with infection solution (10 mM MES, 10 mM MgCl2 , 200 µM acetosyringone) to wash the cells twice, and then adjust the OD 600 ≈0.6, and injected into the back of tobacco leaves with a 1 mL sterile syringe. After culturing at room temperature for 2 days, cut 1 cm 2 Slides were made from tobacco leaves and observed and photographed under a laser confocal microscope (Leica TCS SP8, Leica Microsystems (Shanghai) Trading Co., Ltd.).

[0103] like Figure 4 As shown in the figure, 35S:GFP and 35S:ScPsbP:GFP were transformed into Agrobacterium strain GV3101 respectively. After transient overexpression in tobacco leaves for 2 days, it was found that the control group 35S:GFP was expressed in the cell membrane, nucleus and cytoplasm, but there was no overlap with the chloroplast autofluorescence. However, the experimental group 35S:ScPsbP:GFP can be observed as a dot-shaped green fluorescence, which can overlap with the chloroplast autofluorescence. In order to confirm whether the ScPsbP protein is located in the chloroplast, the tobacco leaves are facing up to observe the fluorescence, and most of the GFP fluorescence can be more clearly seen overlapping with the chloroplast autofluorescence RFP. The above results prove that ScPsbP is located in the chloroplast.

[0104] 2) Analysis of transcriptional autoactivation activity

[0105] In order to detect whether ScPsbP has transcriptional autoactivation activity, ScPsbP The gene was constructed into the vector pGBKT7 according to the method of subcellular localization analysis to form the recombinant vector pGBKT7-ScPsbP, as Figure 5 As shown, the detection primer is BD- ScPsbP -F / R, see the table below:

[0106] Table 7 Detection Primer BD- ScPsbP -F / R

[0107]

[0108] pGBKT7-53 and pGADT7-T were used as positive controls, pGBKT7 was used as a negative control, and the recombinant vector pGBKT7-ScPsbP was used as the experimental group. The positive control, negative control, and experimental group pGBKT7-ScPsbP were transformed into the yeast strain Y2HGold. On the -T screening plate, all combinations grew normally, indicating that they were successfully transformed into the yeast strain, as follows:

[0109] The constructed recombinant vector pGBKT7-ScPsbP was transformed into the yeast strain Y2HGold, spread on SD / Trp (-T) screening plates, and cultured in a 28°C incubator for 3 days. Single clones were picked and placed in -T liquid medium, and cultured overnight at 28°C in a shaker at 250 rpm. 10 µL of the bacterial solution was diluted in 90 µL of ddHO 2 O, and then diluted 10-fold in 3 gradients, finally obtaining 4 gradients of yeast solution (10 -1 , 10 -2 , 10 -3 and 10 -4 ). Take 10 μL of each of the above bacterial solutions and drop them on SD / Trp (marked as -T), SD / -Trp / X-α-Gal (40 μg / mL) (marked as -T / X) and SD / -Trp / X-α-Gal (40 μg / mL) / AbA (200 ng / mL) (marked as -T / X / A) medium, and observe the growth after inverted culture at 28°C for 3 days. Among them, the positive control is pGBKT7-53 and pGADT7-T co-transformed into the Y2HGold yeast strain, and the negative control is pGADT7-T alone transformed into the Y2HGold yeast strain.

[0110] The formula of SD / Trp medium is as follows: yeast nitrogen base (YNB), 6700 mg / L; glucose, 20000 mg / L; adenine sulfate, 40 mg / L; L-arginine hydrochloride, 20 mg / L; L-aspartic acid, 100 mg / L; L-glutamic acid, 100 mg / L; L-histidine, 20 mg / L; L-leucine, 60 mg / L; L-lysine, 30 mg / L; L-methionine, 20 mg / L; L-phenylalanine, 50 mg / L; L-serine, 375 mg / L; L-threonine, 200 mg / L; L-tyrosine, 30 mg / L; L-valine, 150 mg / L; uracil, 20 mg / L.

[0111] The formula of SD / -Trp / X-α-Gal (40 μg / mL) medium is as follows: yeast nitrogen basal (YNB), 6700 mg / L; glucose, 20000 mg / L; adenine sulfate, 40 mg / L; L-arginine hydrochloride, 20 mg / L; L-aspartic acid, 100 mg / L; L-glutamic acid, 100 mg / L; L-histidine, 20 mg / L; L-leucine, 60 mg / L; L-lysine, 30 mg / L; L-methionine, 20 mg / L; L-phenylalanine, 50 mg / L; L-serine, 375 mg / L; L-threonine, 200 mg / L; L-tyrosine, 30 mg / L; L-valine, 150 mg / L; uracil, 20 mg / L; X-α-Gal, 40 μg / mL.

[0112] The formula of SD / -Trp+X-α-Gal (40 μg / mL)+AbA (200 ng / mL) medium is as follows: yeast nitrogen base (YNB), 6700 mg / L; glucose, 20000 mg / L; adenine sulfate, 40 mg / L; L-arginine hydrochloride, 20 mg / L; L-aspartic acid, 100 mg / L; L-glutamic acid, 100 mg / L; L-histidine, 20 mg / L; L-leucine, 60 mg / L; L-lysine, 30 mg / L; L-methionine, 20 mg / L; L-phenylalanine, 50 mg / L; L-serine, 375 mg / L; L-threonine, 200 mg / L; L-tyrosine, 30 mg / L; L-valine, 150 mg / L; uracil, 20 mg / L; X-α-Gal, 40 μg / mL; aurobacitin, 200 ng / mL.

[0113] Depend on Figure 6 It can be seen that on the -T / X screening plate, only the colonies of the positive control group turned blue, while the colonies of the negative group and the experimental group did not change in color. On the -T / X / A screening plate, only the positive control could grow normally and the colonies turned blue, while the negative group and the experimental group could not grow normally. The above results show that ScPsbP The gene does not have transcriptional autoactivation activity.

[0114] Example 5 ScPsbP Genes positively regulate resistance to pathogens

[0115] With the help of the overexpression vector pEarleyGate203 (referred to as 35S::00), ScPsbPThe stop codon of the gene was removed and the vector was constructed into the overexpression vector pEarleyGate203 through the Gateway vector construction method to form a fusion expression vector pEarleyGate203- ScPsbP (abbreviated as 35S:ScPsbP), such as Figure 7 As shown, the details are as follows:

[0116] The obtained in Example 2 ScPsbP The primer for amplification of the insert fragment is G- ScPsbP -F / R, see Table 8. Refer to the instructions of Gateway™ BP Clonase™ II Enzyme Mix (#11789020) and clone the obtained ScPsbP Insert the fragment into the entry vector pDONR221 to form the fusion vector pDONR221- ScPsbP Then, refer to the instructions of Gateway™ LRClonase™ II enzyme mixture (#11791020) to transfer the fusion vector pDONR221- ScPsbP The recombinant plasmid pEarleyGate203- ScPsbP The LR reaction system is shown in Table 9.

[0117] Table 8 ScPsbP The primer for amplification of the insert fragment is G- ScPsbP -F / R

[0118]

[0119] Table 9 LR reaction system

[0120]

[0121] Furthermore, 35S:00 (control group) and 35S:ScPsbP (experimental group) were transiently overexpressed in tobacco leaves using the Agrobacterium-mediated transformation system. Two days after transient overexpression, tobacco leaves were inoculated with Phytophthora nicotianae in vitro.

[0122] According to the transient overexpression method described in step (7) of the subcellular localization analysis of Example 4, 35S:00 and 35S:ScPsbP were transiently overexpressed in tobacco leaves using the Agrobacterium-mediated tobacco transient expression method. Referring to the method described in "Phytophthora infection signals-induced translocation of NAC089 is requiredfor endoplasmic reticulum stress response-mediated plant immunity" [J]. (Ai G, Zhu H, Fu X, et al. The Plant Journal, 2021, 108(1): 67-80), the transient overexpression vector pEarleyGate203 (35S:00) and the fusion expression vector pEarleyGate203- ScPsbP Benthellae plants with 35S:ScPsbP were inoculated with Phytophthora nicotianae ( Phytophthora nicotianae ). Two days after inoculation with tobacco phytophthora, the tobacco leaves were stained with DAB and photographed under UV light, and the lesion area was calculated using ImageJ 1.53 software. The specific steps of DAB staining are as follows: dissolve DAB powder at a ratio of 1:1000 (DAB powder: sterile water), soak the tobacco leaves in the DAB aqueous solution, let stand for 8 hours in the dark, then decolorize with anhydrous ethanol, and take pictures. The RNA of tobacco leaves at 0d and 2d after inoculation with phytophthora was extracted and cDNA was synthesized. Afterwards, the HR marker gene was detected according to the method in "Sugarcane transcription factorScWRKY4 negatively regulates resistance to pathogen infection through the JAsignaling pathway" [J]. (Wang D, Wang W, Zang S, et al. The Crop Journal, 2024,12(1): 164-176) NbHIN1 and NbHSR515 )、JA synthesis related genes( NbDEF1 and NbLOX1 ) and ROS scavenging-related genes ( NbCAT1 and NbGST1 ) expression levels, and the detection primers are shown in Table 10.

[0123] Table 10 Detection primers

[0124]

[0125] Transient overexpression in tobacco leaves ScPsbP After the gene was inoculated with tobacco phytophthora in vitro for 2 days, the dead cell area of ​​tobacco leaves was observed under UV. Figure 8 As shown in Figure A, in the control group 35S:00, the disease severity of the leaves was significantly more serious than that in the experimental group 35S:ScPsbP.

[0126] DAB staining results, see Figure 8 As shown in Figure B, the diseased area of ​​the leaves in the control group 35S:00 appeared darker brown, while the color in the experimental group 35S:ScPsbP was lighter.

[0127] At the same time, the expression levels of immune-related marker genes in tobacco leaves of the control group and the experimental group were detected 0 and 2 days after inoculation with pathogens. Figure 8 In Figure C, we can see that ScPsbP After transient overexpression of the gene in tobacco, it significantly induced JA synthesis-related genes ( NbDEF1 and NbLOX1 ) and ROS scavenging-related genes ( NbCAT1 and NbGST1 ) were up-regulated. NbCAT1 The expression level of the gene was the highest, 2.35 times that of the control group. Two days after inoculation with tobacco phytophthora, the HR marker gene ( NbHIN1 and NbHSR515 )、JA synthesis related genes( NbDEF1 and NbLOX1 ) and ROS scavenging-related genes ( NbCAT1 and NbGST1 ) were significantly upregulated. The above results showed ScPsbP The gene may positively regulate the expression of HR marker genes, JA synthesis pathway-related genes, and ROS clearance-related genes. ScPsbP Gene resistance to pathogens. It can also be seen from the above that by heterologous overexpression in Nicotiana benthamiana ScPsbP Genes can improve the disease resistance of Nicotiana benthamiana.

[0128] Furthermore, the fusion expression vector pEarleyGate203- ScPsbP (abbreviated as 35S:ScPsbP) overexpression in sugarcane ScPsbP Gene to increase overexpression ScPsbP Genetically modified sugarcane plants for disease resistance.

[0129] Furthermore, by subjecting plants (such as sugarcane or tobacco) to ScPsbPGene overexpression results in disease-resistant plants (such as disease-resistant sugarcane plants or disease-resistant tobacco plants).

[0130] At the same time, by planting overexpression ScPsbP Plants with genes (such as overexpression ScPsbP Sugarcane or overexpression of genes ScPsbP Genetic tobacco) can improve the disease resistance of the plant during the planting process.

[0131] In summary, PsbP protein is a chloroplast protein that binds to the periphery of photosystem II (PSII) on the thylakoid lumen and participates in photosynthesis. PsbP can respond to abiotic and biotic stresses. RXLR31154 It can stabilize PsbP and reduce H 2 O 2 accumulation, activation 1 O 2 Signaling pathways promote the occurrence of plant host diseases. In the present invention, a ScPsbP The protein has a molecular weight of 26.61 kDa and contains a PsbP domain at 86-248 aa. Figure 2 The highest homology between ScPsbP protein and XP_021321130.1 protein in sorghum is 86.11% ( Figure 2 However, no reports on the disease resistance-related functions of PsbP have been found in sorghum. ScPsbP The gene was expressed at the highest level in sugarcane +1 leaves, followed by young leaves ( Figure 1 This is consistent with its nature as a chloroplast protein. Similarly, cassava ( Manihot esculenta )middle PsbP The gene was expressed at the highest level in mature leaves of cassava, followed by young leaves. MePPD3 was also located in chloroplasts.

[0132] When tobacco is infected with tobacco mosaic virus (TMV), PsbP The accumulation in tobacco leaves was significantly reduced. PsbP Reduced the accumulation of alfalfa mosaic virus (AMV), silenced PsbP It can increase the infection of rice stripe virus (RSV) and promote the accumulation of tobacco viruses. Transient silencing in Nicotiana benthamiana plants PsbP Genes enhance pathogenicity and accumulation of viral DNA in Nicotiana benthamiana. PsbP Overexpression ofPsbP Participates in the production of defense responses during geminivirus infection. ScPsbP The gene was induced to be up-regulated in sugarcane varieties resistant to smut, but down-regulated in sugarcane varieties susceptible to smut ( Figure 1 D and E in the figure). Photosynthesis in chloroplasts is the main way for plants to produce ROS, and ROS plays a dual role in plants, being a key regulator of growth, development and defense pathways, and a toxic byproduct of aerobic metabolism. When infected by pathogens, ROS regulates the rapid accumulation of JA in the plant systemic acquired resistance pathway. ScPsbP The gene was transiently overexpressed in tobacco leaves. After inoculation with tobacco phytophthora, the lesions in 35S:00 leaves were significantly more severe than those in 35S:ScPsbP, and it was able to significantly induce the up-regulation of HR marker genes, genes related to the JA synthesis pathway, and genes related to the ROS scavenging enzyme system ( Figure 8 Figure C). ScPsbP Genes that positively regulate plant resistance to pathogens. In contrast, overexpression VpPsbP Increased sensitivity to grape downy mildew and pepper phytophthora, silenced Nicotiana benthamiana PsbP Homologous genes NbPsbPs PsbP reduced the colonization of Phytophthora capsici, indicating that it is a susceptibility factor. In wheat, another member of the OEEs encoding PSII PsbO The transcript level in wheat was reduced, leading to the Puccinia striiformis )'s spore production decreased.

[0133] In summary, PsbP Genes play an important role in plant response to pathogenic bacteria stress. ScPsbP A preliminary study was conducted on the disease resistance function of the gene, which laid a solid foundation for in-depth research on the function and mechanism of this gene in the process of sugarcane responding to pathogen stress.

[0134] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, rather than all the embodiments. A person skilled in the art can also obtain other embodiments based on the embodiments without inventiveness, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A disease-resistant ScPsbP A gene characterized by Said ScPsbP The coding sequence of the gene is shown in SEQ ID NO:

1.

2. A ScPsbP protein with disease resistance, characterized in that: The ScPsbP protein is the one described in claim 1 ScPsbP The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

3. A type of ScPsbP A plasmid containing a gene, characterized in that The inclusion ScPsbP The plasmid of the gene is obtained by using the ScPsbP The gene was connected to a cloning vector.

4. A type of ScPsbP A gene expression vector, characterized in that The inclusion ScPsbP Gene expression vectors are constructed using ScPsbP The PCR amplification product of the gene is connected with the vector to obtain; Said ScPsbP The coding sequence of the gene is shown in SEQ ID NO:

1.

5. A type of ScPsbP A strain of gene, characterized in that The inclusion ScPsbP The strain containing the gene is ScPsbP Agrobacterium tumefaciens or the like containing the gene ScPsbP Genetic yeast; The inclusion ScPsbP The Agrobacterium containing the gene is used as claimed in claim 4 ScPsbP The gene expression vector is introduced into Agrobacterium to obtain; The inclusion ScPsbP The yeast containing the gene is used as claimed in claim 4 ScPsbP The gene expression vector is introduced into yeast to obtain.

6. A method according to claim 1 ScPsbP The application of genes in improving disease resistance is characterized in that: The application is to overexpress in Nicotiana benthamiana ScPsbP Genes to improve resistance to tobacco phytophthora.

7. A plant breeding method, characterized in that: By ScPsbP Gene overexpression to obtain resistance to tobacco phytophthora; Said ScPsbP The coding sequence of the gene is shown in SEQ ID NO:

1.

8. A planting method for improving disease resistance, characterized in that: The planting method is planting ScPsbP Gene overexpression in Nicotiana benthamiana to improve resistance to tobacco phytophthora; Said ScPsbP The coding sequence of the gene is shown in SEQ ID NO:1.

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