A sugarcane ScGSTU9 gene with stress resistance, protein and application

Abstract

The present invention provides a sugarcane with stress resistance function ScGSTU9 Genes, proteins and applications thereof, belonging to the field of biotechnology, and specifically disclosing sugarcane ScGSTU9 The application of genes in disease resistance, drought resistance, salt resistance and low temperature resistance. ScGSTU9 Preliminary research has been conducted on responses to biotic and abiotic stresses, laying a solid foundation for in-depth research on the function and mechanism of action of this gene in the process of sugarcane responding to pathogen stress and abiotic 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 sugarcane with stress resistance. ScGSTU9 Genes, proteins and applications. Background Art

[0002] Sugarcane smut is one of the most significant diseases causing reduced sugarcane yield and quality loss, severely impacting the sustainable and healthy development of the sugar industry. First reported in Natal, South Africa in 1877, the disease has since spread to sugarcane-growing countries including Central Africa, East Africa, West Africa, Indonesia, Central America, South America, Brazil, and Australia. Sugarcane smut was first documented in Guangdong Province in China, followed by multiple large-scale outbreaks in Taiwan. In recent years, the incidence of smut has steadily worsened in major sugarcane-producing regions such as Guangxi, Yunnan, Guangdong, and Hainan, with dryland and ratoon fields being particularly severe. The incidence of ratoon fields of susceptible varieties generally exceeds 50%, with rates reaching peaks of 80% to 90% in some severely affected areas, causing significant economic losses to the sugarcane industry. Regional outbreaks of sugarcane smut pose a serious threat to the yield stability and quality safety of the sugarcane industry. Developing a scientific and effective sugarcane smut prevention and control strategy is a key research priority for achieving high-quality development and improving the quality and efficiency of the sugarcane industry. It is of great significance to explore and identify sugarcane smut-resistant genes as much as possible and provide gene resources with application value for sugarcane smut-resistant biological breeding.

[0003] Glutathione S-transferases (GSTs) are a large family of multifunctional proteases. GST-encoded proteins possess two characteristic functional regions: the N-terminal and C-terminal domains. The N-terminal domain contains a glutathione-binding site (G-site), and the C-terminal domain contains an electrophilic substrate-binding site (H-site). The G-site is specific for glutathione, promoting the formation of glutathione-catalyzed active thioanions and is central to terrestrial plant regulation. The promoter regions of GST genes often contain multiple cis-acting elements, such as MYB binding sites, ABA-responsive elements, and stress-responsive elements, which play important roles in GST gene transcriptional regulation. GST genes not only play a role in plant growth and development but are also widely involved in plant responses to biotic and abiotic stresses. Summary of the Invention

[0004] In view of the above problems, the present invention provides a sugarcane with stress resistance function. ScGSTU9 Genes, proteins and applications.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] Sugarcane with stress resistance ScGSTU9gene, the ScGSTU9 The coding sequence of the gene is SEQ ID NO: 1;

[0007] described ScGSTU9 The gene is associated with at least one of the following abilities: disease resistance, drought resistance, salt resistance, and low temperature resistance.

[0008] A sugarcane ScGSTU9 protein with stress resistance function, wherein the ScGSTU9 protein is the above-mentioned ScGSTU9 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2.

[0009] A primer pair, the primer pair is used for PCR amplification of the above-mentioned ScGSTU9 gene, the primer pair comprising:

[0010] ScGSTU9 -F: 5'-CGTCGAACAGAAGCGAAAGC-3';

[0011] ScGSTU9 -R: 5'-TGACAGGCGGATCGAGATGA-3'.

[0012] A kind of ScGSTU9 A plasmid containing a gene ScGSTU9 The plasmid of the gene is the use of the above-mentioned stress resistance function ScGSTU9 The gene was connected to a cloning vector.

[0013] A kind of ScGSTU9 A recombinant vector of a gene, comprising ScGSTU9 The recombinant vector of the gene is a ScGSTU9 The PCR amplification product of the gene or ScGSTU9 The plasmid of the gene is connected to the vector to obtain;

[0014] described ScGSTU9 The coding sequence of the gene is shown in SEQ ID NO: 1.

[0015] A kind of ScGSTU9 strain containing a gene ScGSTU9 The strain containing the gene ScGSTU9 Agrobacterium containing genes ScGSTU9 Yeast containing the gene or the ScGSTU9 Gene of Escherichia coli;

[0016] Said inclusion ScGSTU9 The Agrobacterium tumefaciens gene is the one mentioned above containing ScGSTU9 The recombinant vector of the gene is transformed into Agrobacterium to obtain;

[0017] Said inclusion ScGSTU9The yeast strain containing the gene is ScGSTU9 The recombinant vector of the gene is transformed into yeast;

[0018] Said inclusion ScGSTU9 The E. coli containing the gene is used ScGSTU9 The recombinant vector of the gene or the above-mentioned ScGSTU9 The plasmid containing the gene was transformed into Escherichia coli.

[0019] One of the above ScGSTU9 Application of a gene in improving disease resistance, wherein the gene is overexpressed in sugarcane or tobacco ScGSTU9 genes to improve disease resistance.

[0020] One of the above ScGSTU9 Application of genes in resistance to abiotic stress, said application being overexpression in sugarcane ScGSTU9 Genes to improve at least one of drought resistance, salt resistance, and low temperature resistance;

[0021] Alternatively, the application is overexpression in E. coli ​ Genes to improve at least one of drought resistance, salt resistance and low temperature resistance.

[0022] A plant breeding method for disease resistance by overexpressing a gene in sugarcane ​ genes to produce disease-resistant sugarcane;

[0023] Alternatively, by overexpression in tobacco ​ genes to produce tobacco with disease resistance;

[0024] described ​ The coding sequence of the gene is shown in SEQ ID NO: 1.

[0025] A breeding approach for abiotic stress resistance by overexpressing ​ Genes to obtain sugarcane with at least one of drought resistance, salt resistance and low temperature resistance;

[0026] Alternatively, by overexpression in E. coli ​ Genes for obtaining Escherichia coli with at least one of the ability to resist drought, salt and low temperature;

[0027] described ​ The coding sequence of the gene is shown in SEQ ID NO: 1.

[0028] Sugarcane with stress resistance of the present invention ​ The beneficial effects of genes, proteins and applications are:

[0029] The present invention is ​The preliminary research on the response to biotic and abiotic stresses has laid a solid foundation for in-depth study of the function and mechanism of this gene in the sugarcane response to pathogenic and abiotic stresses.

[0030] The present invention is carried out by ​ Research on genes helps to understand the mechanisms of sugarcane's disease resistance, drought resistance, salt resistance and low temperature resistance, and provides a theoretical basis and reference for inducing plants to have disease resistance, drought resistance, salt resistance and low temperature resistance using genetic engineering technology;

[0031] The present invention is constructed by ​ Recombinant vector and containing ​ Agrobacterium tumefaciens successfully ​ The gene was introduced into heterologous Nicotiana benthamiana and analyzed ​ The results showed that the gene has disease resistance function in Nicotiana benthamiana by overexpression ​ , can improve disease resistance; therefore, the present invention discloses ​ The gene has important application value not only in improving the disease resistance of native sugarcane, but also in heterologous Nicotiana benthamiana.

[0032] The present invention is constructed by ​ Genetic analysis of Escherichia coli ​ The drought resistance, salt resistance and low temperature resistance of the gene in Escherichia coli; the results showed that overexpression ​ Can improve the tolerance of Escherichia coli to D-mannitol, salt and low temperature stress, that is, overexpression ​ Can improve drought resistance, salt resistance and low temperature resistance; therefore, the invention discloses ​ Genes not only have important application value in improving disease resistance, but can also be used to improve drought resistance, salt resistance and low temperature resistance;

[0033] The present invention is carried out by ​ Gene research revealed ​ The gene was expressed in different tissues of sugarcane, with the highest expression in new sugarcane pulp and the lowest expression in old leaves, indicating that ​ Genes play different functions in the growth and development of sugarcane. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] ​ The sugarcane in Example 1 of the present invention ​ Gene cloning and sequence analysis; In Figure A, M represents a 2000 bp DNA marker. ​ Represents PCR amplification products; the green box and red box sequences in Figure B represent the conserved domains of GST_N_Tau and GST_C_Tau, respectively. Indicates the stop codon; Figure C shows the predicted tertiary structure of ScGSTU9 protein; Figure D shows the predicted conserved domain of ScGSTU9 protein;

[0035] ​ In Example 1 of the present invention ​ The expression pattern of genes; Figure A is ​ The expression pattern of genes in the interaction between smut-resistant sugarcane varieties and smut fungi; Figure B is ​ Gene expression patterns in different sugarcane tissues; Figure C ​ The expression of gene under salicylic acid stress; Figure D shows the expression of ScGSTU9 gene under methyl jasmonate stress; sugarcane ​ The gene was used as an internal reference gene; all data points are expressed as mean ± standard error (n = 3), and different letters indicate significant differences calculated by one-way analysis of variance (P < 0.01);

[0036] ​ The subcellular localization vector plasmid pFAST-R05- ​ Carrier map;

[0037] ​ This is the analysis of the subcellular localization and transcriptional autoactivation activity of the sugarcane ScGSTU9 protein in Example 2 of the present invention; wherein, Figure A shows the subcellular localization analysis of the sugarcane ScGSTU9 protein, Visible represents the cell outline under bright field, GFP and ER-RFP represent images of epidermal cells captured using green fluorescence and red fluorescence, respectively, Merged represents the superposition of the first three images, and Bar = 25 μm; Figure B shows the analysis of the transcriptional autoactivation activity of the sugarcane ScGSTU9 protein, pGBKT7-p53+pGADT7-T represents the positive control, pGBKT7-lam+pGADT7-T represents the negative control, DDO represents the SD / -Leu / -Trp solid culture medium lacking leucine and tryptophan; QDO represents the SD / -Ade / -His / -Leu / -Trp solid culture medium lacking leucine, tryptophan, histidine, and adenine;

[0038] ​ The recombinant vector pGBKT7- ​ Carrier map;

[0039] ​ The recombinant plasmid pET28a- ​ Carrier map;

[0040] ​This is the prokaryotic expression of the sugarcane ScGSTU9 recombinant protein in Example 3 of the present invention in Escherichia coli Tssetta cells; wherein M represents a protein marker; 1 represents an uninduced Tssetta empty bacterium; 2 represents a Tssetta empty bacterium induced for 10 hours; 3 represents an uninduced pET28a empty vector; 4 represents pET28a induced for 10 hours; 5 represents an uninduced pET28a-ScGSTU9 protein; 6 to 11 represent pET28a-ScGSTU9 proteins induced for 2 hours, 4 hours, 6 hours, 8 hours, and 10 hours, respectively;

[0041] ​ This is the plate stress verification of the sugarcane ScGSTU9 prokaryotic recombinant protein in Example 3 of the present invention under abiotic factor treatment; wherein, Figure A is the 4°C low temperature stress verification, Figure B is the D-mannitol stress verification, and Figure C is the sodium chloride stress verification; 10 -3 , 10 -4 Indicates that the bacterial solution concentration is diluted 10 -3 and 10 -4 times, with 4 replicates for each concentration point;

[0042] ​ The recombinant plasmid pEarleyGate-203- ​ Carrier map;

[0043] ​ is the transient overexpression in Example 4 of the present invention ​ Identification of resistance of Nicotiana benthamiana to tobacco Fusarium oxysporum inoculation; Figure A shows transient overexpression ​ Phenotype and DAB staining of tobacco leaves after inoculation with Fusarium spp. 35S::00 represents leaves of Nicotiana benthamiana transiently overexpressing empty pEarleyGate 203, and 35S::ScGSTU9 represents leaves of Nicotiana benthamiana transiently overexpressing pEarleyGate 203-ScGSTU9; Figure B shows leaves of Nicotiana benthamiana transiently overexpressing ​ Detection of immune marker-related genes in tobacco leaves after inoculation with Fusarium spp. ​ The gene was used as the internal reference gene, and the error bars represent the standard deviation; the significance of the difference was tested using the one-way analysis of variance method ( P <0.01). DETAILED DESCRIPTION

[0044] The technical solutions in the embodiments of the present invention are described clearly and completely below. Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The present invention is further described in detail below in conjunction with specific embodiments to facilitate understanding by those skilled in the art.

[0045] In the examples disclosed below, if specific techniques or conditions are not specified, the experiments were performed according to those described in literature in the field (e.g., Molecular Cloning: A Laboratory Manual, 3rd edition, by Sambrook et al., translated by Huang Peitang et al., Science Press) or according to the product instructions. Reagents used without manufacturer's information are commercially available.

[0046] Plant materials: Smut-resistant sugarcane varieties (YT93-159), smut-susceptible sugarcane varieties (ROC22), and wild-type Nicotiana benthamiana were provided by the Fujian Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs, Fujian Agriculture and Forestry University. Wild-type Nicotiana benthamiana (WT) was grown in an incubator at 25°C under a 16 h / 8 h light / dark cycle.

[0047] Vectors: pDONR-207, pFAST-R05-GFP, pET28a, pGBKT7-53, pGBKT7-Lam, pGBKT7-T, pGBKT7, and pEarleyGate203.

[0048] Strains: Escherichia coli strain DH5α, Escherichia coli Tssetta (DE3), yeast strain Y2Hgold and Agrobacterium strain GV3101.

[0049] Main reagents: High-fidelity enzyme (2×Phanta Flash Master Mix, #P510), rapid PCR-Taq enzyme (2×Rapid Taq Master Mix, #P222), and blue universal dye qPCR detection kit (ChamQ BlueUniversal SYBR qPCR Master Mix, #Q312) were purchased from Nanjing Novozymes Biotechnology Co., Ltd.; Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (#11141ES60) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; MagZol™ Reagent (#R4801-01), HiPure Gel Pure DNA Kit (#D2110-03), and Plasmid Extraction Kit (#P1001-03C) were purchased from Guangzhou Meiji Biotechnology Co., Ltd.; Gateway™ BP Clonase™ II Enzyme Mix (#11789020) and Gateway™ LRClonase™ II enzyme mixture (#11791020), restriction endonucleases FastDigest HindIII (#FD0504), FastDigest EcoRI (#FD0274), and FastDigest BamHI (#FD0054) were purchased from Thermo Fisher Scientific; 2-(N-morpholino)ethanesulfonic acid (MES, #A610341), magnesium chloride hexahydrate (MgCl2, #A610328), and 3,3'-diaminobenzidine tetrahydrochloride (DAB, #A600140) were purchased from Sangon Biotech (Shanghai) Co., Ltd.; acetosyringone (AS, #CA1061), SD / -Trp / -Leu withAgar (#PM2252) and SD / -Trp / -Leu Broth (PM2251) were purchased from Beijing Coolaibo Technology Co., Ltd.; Coomassie Brilliant Blue Fast Staining Solution (#P0017) was purchased from Beyotime Biotechnology (Shanghai) Co., Ltd.The Blunt Zero Cloning Kit (pEASY®-Blunt Zero Cloning Kit, #CB501), the Basic Seamless Cloning and Assembly Kit (pEASY®-CU201), and DNA markers (#BM111, #BM161) were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0050] All primers involved in the present invention were designed using SnapGene, Primer Premier 5 and CE Design software and sent to SunAsia Biotech Co., Ltd. for synthesis.

[0051] Example 1 ​ Gene bioinformatics analysis and expression pattern analysis

[0052] 1) Material handling

[0053] Healthy and consistent growth of the disease-resistant sugarcane variety YT93-159 was selected. Tissue samples were collected from different parts of the plant, including young leaves, +1 leaves, old leaves, leaf sheaths, husks, new pulp, senescent pulp, and buds. Three biological replicates were set for each sample group. Exogenous hormone treatments were used: Tissue cultured seedlings of the susceptible sugarcane variety ROC22, 4-5 leaf age with consistent growth trends, were sprayed with 5 mM salicylic acid (SA) and 25 μM methyl jasmonate (MeJA). The seedlings were then hydroponically cultured at 28°C under a 16-hour light / 8-hour dark cycle. Leaves from individual plants were collected at 0, 3, 12, and 24 hours after treatment. Sugarcane (YT93-159 and ROC22) buds were inoculated with Ustilago smut according to the method of Su Yachun et al. (Transcriptome and Proteome Study of Sugarcane Response to Ustilago smut Infection and Discovery of Resistance-Related Genes). Bud samples were collected 0, 1, 2, 4, and 5 days after inoculation. Specifically, healthy, 10-month-old plants were selected from the field, cut into single bud segments, and placed in a 32°C incubator for germination. After buds grew to 1-2 cm, buds of uniform growth were selected and inoculated with a mixed spore suspension of Ustilago smut (5×10 6 / mL, containing 0.01% Tween-20, v / v ). Three biological replicates were set up at each time point, and three sugarcane buds were taken for each replicate. All treated samples were quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator for subsequent use. ​ Detection of gene expression.

[0054] 2) RNA extraction and reverse transcription

[0055] RNA was extracted from sugarcane and Nicotiana benthamiana plants using MagZol™ Reagent (#R4801-01, Meiji). RNA was then reverse transcribed and synthesized into cDNA according to the instructions of the HRbio™ Ⅲ 1st Strand cDNA Synthesis Kit (with gDNA Removal) (#HRF0462) and the Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (#11141ES60). RNA and cDNA were used as templates for subsequent gene cloning and gene expression detection, respectively. The RNA and cDNA were stored at −80°C until use.

[0056] 3) Gene cloning

[0057] The Primer Premier 5 software was used to design the genome of sugarcane cultivar R570 based on its annotation information. ​ Gene cloning primers ( ​ -F / R), the PCR system and PCR reaction program were configured according to the instructions of the high-fidelity enzyme (2× Phanta Flash Master Mix, #P510). The PCR program was as follows: 98°C pre-denaturation for 30 seconds; 98°C denaturation for 10 seconds, 65°C gradient ramp (0.5°C decrease per cycle) annealing for 5 seconds, 72°C extension for 15 seconds, 35 cycles; and a final extension at 72°C for 5 minutes. The PCR product was detected by 1.5% agarose gel electrophoresis and purified to obtain the target band (i.e. ​ Gene); Among them, the gel electrophoresis test results are as follows ​ As shown in Figure A, ​ The coding sequence of the gene and ​ The open reading frame of the gene is SEQ ID NO: 1, and its open reading frame (ORF) is 702 bp; ​ The gene coding sequence and its upstream and downstream sequences are shown in SEQ ID NO: 3 (PCR amplification primers were designed based on the upstream and downstream sequences) ​ -F / R); ​ The cDNA sequence of the gene is shown in SEQ ID NO: 4. The target band was ligated to the pEASY®-Blunt Zero cloning vector and transformed into E. coli DH5α. Single colonies were selected for PCR and sequencing verification. Plasmids were extracted from correctly sequenced colonies using a plasmid extraction kit and stored at -20°C for subsequent experiments.

[0058] Table 1 PCR amplification primers

[0059]

[0060] Table 2 PCR reaction system

[0061]

[0062] 4) Bioinformatics analysis

[0063] Using sugarcane leaf cDNA as template, we cloned ​ In order to explore the CDS sequence of the gene ​ To determine the biological function of genes, we first analyzed their sequence characteristics. We used the NCBI Conserved Domain Search (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) and NCBI-ORF finder (https: / / www.ncbi.nlm.nih.gov / orffinder / ) online analysis platforms to analyze the ​ The open reading frames and conserved protein domains of the gene sequences were analyzed using the ProtParam tool on the ExPASy online website (https: / / web.expasy.org / protparam / ). ​ Physicochemical properties of the gene-encoded protein were determined. Protein secondary and tertiary structures were predicted using the SOPMA (https: / / npsa.lyon.inserm.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_sopma.html) and SWISS-MODEL tools, respectively. Protein transmembrane domains were predicted using the online website TMHMM-2.0 (https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ).

[0064] ​ The ORF of the gene is 702 bp in length and encodes 233 amino acids. The amino acid sequence of the ScGSTU9 protein is shown in SEQ ID NO: 2, which contains the GST_N_Tau and GST_C_Tau domains (see ​ The results of protein physicochemical property analysis showed that the molecular formula of ScGSTU9 protein was C 1149 H 1805 N 309 O 323S4 has a molecular weight of 25.24 kDa and an isoelectric point of 6.17, which is less than 7.0, suggesting it is an acidic protein. Furthermore, its instability coefficient and average hydrophobicity are 27.62 and 0.102, respectively. ScGSTU9, with an instability coefficient less than 40 and an average hydrophobicity greater than 0, is considered a stable, hydrophobic protein. Subcellular localization prediction indicates that ScGSTU9 is localized in the cytoplasm. The primary secondary structure components of ScGSTU9 are α-helices, extended strands, and random coils, with α-helices comprising the largest proportion, at 54.08%. ScGSTU9 is predicted to have a transmembrane domain.

[0065] 5) ​ Gene expression patterns

[0066] RT-qPCR primers for ScGSTU9 gene (Q- ​ -F / R). The internal reference gene was glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase, ​ The DNA was analyzed using the Applied Biosystems QuantStudio 3 real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). ​ The tissue expression characteristics of the gene and its expression levels under the stress of smut fungus, SA and MeJA were detected by RT-qPCR. The RT-qPCR reaction system and RT-qPCR program were prepared according to the instructions of 2×ChamQ Universal SYBR qPCR MasterMix. The RT-qPCR amplification program was as follows: 95℃, 30s; 95℃, 10s, 60℃, 30s, 40 cycles. -△△Ct The relative gene expression levels were normalized using the GraphPad Prism 10.1.2 software, and SPSS software was used for one-way analysis of variance to calculate the significant differences (P < 0.01).

[0067] Table 3 RT-qPCR analysis and identification primers

[0068]

[0069] Table 4 RT-qPCR reaction system

[0070]

[0071] RT-qPCR analysis ​Gene expression levels in the interaction between different resistant sugarcane varieties and smut fungus ( ​ Figure A). In the smut-resistant sugarcane variety YT93-159, compared with the control, ​ The expression level increased significantly at 1-4 dpi (daypost inoculation) and decreased at 5 dpi. In the smut-susceptible sugarcane variety ROC22, the expression level of ScGSTU9 did not show significant differences after inoculation with smut fungus, indicating that ScGSTU9 can be induced to express by smut fungus stress and that its expression patterns vary in different sugarcane varieties during the interaction with smut fungus.

[0072] To determine the expression characteristics of ScGSTU9 in sugarcane tissues, RT-qPCR was used to detect the expression levels of ScGSTU9 in new leaves, +1 leaves (the first fully expanded leaf on the sugarcane plant), old leaves, leaf sheaths, sugarcane peels, new sugarcane pulp, senescent sugarcane pulp, and sugarcane buds of the sugarcane cultivar YT93-159 (see ​ The results show that ​ The gene is expressed in different tissues of sugarcane, including ​ The expression level in new sugarcane pulp was the highest, and the expression level in old leaves was the lowest. The expression level in new sugarcane pulp was 29.17 times that in old leaves. ​ Genes may play different functions in the growth and development of sugarcane. ​ The expression level of ScGSTU9 did not change significantly under the stress of exogenous hormone MeJA, but increased significantly at 3 h after SA stress, which was 13.38 times that of the control group, indicating that ScGSTU9 is induced by hormone signals ( ​ C and D in Figure 5).

[0073] Example 2 ScGSTU9 protein is localized in the cytoplasm and endoplasmic reticulum and has no transcriptional autoactivation activity

[0074] 1) Subcellular localization analysis

[0075] The ScGSTU9 gene entry vector primers Gate- ​ -F / R (Table 5), using the plasmid obtained in step 3) of Example 1 as a template, PCR amplification was performed. ​ The gene was connected to the entry vector pDONR-207, and the positive recombinant entry vector plasmid was named pDONR-207- ​ Then the target gene was constructed into the subcellular localization vector pFAST-R05- ​ The obtained positive recombinant subcellular localization vector plasmid was named pFAST-R05- ​ ( ​). BP and LR reaction systems are shown in Table 6 and Table 7. ​ Transform Agrobacterium GV3101, pick a single clone and test it by PCR. After successful transformation, expand it. Then use the infection solution containing 10mM MES (2-(N-morpholino)ethanesulfonic acid monohydrate) and 10mM MgCl2 to adjust the concentration of the bacterial solution to OD 600 ≈0.5, then added with 200 mM acetosyringone and allowed to stand in the dark for 2–3 hours. The mixture was then mixed in equal proportions with Agrobacterium tumefaciens GV3101 harboring the plant expression vector (ER-rk CD3-959) and injected into leaves of six-leaf-old Nicotiana benthamiana plants. Agrobacterium tumefaciens GV3101 harboring an empty pFAST-R05-GFP vector served as a control. Following injection, the cells were cultured at 28°C under a 16-hour light / 8-hour dark cycle for 2–3 days. Subcellular localization was then observed using a LEICA TCS SP8 laser confocal microscope (Leica, Wetzlar, Germany).

[0076] Table 5. List of Gateway entry vector primer sequences

[0077]

[0078] Table 6 BP reaction system

[0079]

[0080] Table 7 LR reaction system

[0081]

[0082] pFAST-R05- ​ For the experimental group, the empty vector pFAST-R05- ​ As a negative control, transient expression was performed in Nicotiana benthamiana leaves by Agrobacterium-mediated method. The results showed that there was no green fluorescence in the plasma membrane, cytoplasm and nucleus of Nicotiana benthamiana leaf cells in the control group. However, the experimental group pFAST-R05- ​ Green fluorescence was observed in the cytoplasm of the epidermal cells of Nicotiana benthamiana leaves, and ER-rk CD3-959 was used as an endoplasmic reticulum marker. The fusion field showed that the green and red colors in the three experimental groups overlapped, indicating that sugarcane ​ Cytoplasmic and endoplasmic reticulum localized proteins ( ​ Figure A in the figure).

[0083] 2) Analysis of transcriptional autoactivation activity

[0084] according to ​ The ORF sequence of the gene and the map of the pGBKT7 vector were used to design a primer pair (BD-​ -F / R) (Table 8), using the plasmid obtained in step 3) of Example 1 as a template, PCR amplification and gel recovery purification were performed. The linearized vector was ligated with the gel-recovered target fragment using the ClonExpress II One Step Cloning Kit (Novozymes, Nanjing, China), and transformed into DH5α competent cells. After bacterial liquid PCR and sequencing, the recombinant vector pGBKT7- ​ ( ​ ).

[0085] Table 8 BD- ​ Primer pairs

[0086]

[0087] According to the instructions of Y2HGold Chemically Competent Cell, the constructed recombinant vector pGBKT7- ​ Co-transform the empty plasmid pGADT7 into yeast Y2HGold competent cells, spread on SD / -Trp / -Leu (DDO) selection plates, and culture in a 28°C incubator for 3 days. After colonies grow, pick a single colony and culture it in -Trp / -Leu liquid medium at 28°C in a shaker at 250 rpm overnight. Centrifuge the bacterial solution at the same concentration at 5000 rpm to collect the cells, and dilute the cells to 10 -1 , 10 -2 , 10 -3 and 10 -4 10 µL of each of the four concentration gradients was spotted onto SD / -Leu / -Trp solid medium (DDO) and SD / -Ade / -His / -Leu / -Trp solid medium (QDO) plates. The plates were incubated upside down at 28°C for 2–3 days. The resulting colonies were photographed and their growth was used to assess the transcriptional autoactivation of the target protein. A positive control consisted of co-transformation of pGBKT7-53 and pGADT7-T into the Y2HGold yeast strain, and a negative control consisted of co-transformation of pGBKT7-lam and pGADT7-T into the Y2HGold yeast strain.

[0088] The formula of SD / -Trp / -Leu (DDO) solid 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-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, and agar 20 g.

[0089] The formula of -Trp / -Leu liquid 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-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, and uracil 20 mg / L.

[0090] SD / -Ade / -His / -Leu / -Trp solid medium formula: yeast nitrogen base (YNB) 6700 mg / L, glucose 20000 mg / L, L-arginine hydrochloride 20 mg / L, L-aspartic acid 100 mg / L, L-glutamic acid 100 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, and uracil 20 mg / L, 20 g agar.

[0091] To detect ​ Whether it has transcriptional self-activation activity, ​ The construct was inserted into the vector pGBKT7 to form the expression vector pGBKT7-ScGSTU9. ​ The self-activation activity of ​ Figure B in the figure shows that on the DDO plate, the positive control pPGBKT7-53+pGADT7-T, the negative control pPGBKT7-lam+pGADT7-T and the experimental group pGBKT7- ​+pGADT7 can grow normally, indicating that the recombinant vector has been successfully transformed into Y2HGold yeast. The growth of the colonies showed a consistent trend with the gradient of the diluted liquid culture, indicating that the ScGSTU9 protein binds to GAL4-BD and expresses the missing tryptophan in the culture medium (where GAL4-BD is the DNA binding domain of the yeast transcription factor GAL4; pGADT7 is a yeast expression vector containing GAL4-AD, the transcription activation domain); on QDO plates, the positive control pPGBKT7-53+pGADT7-T had colonies growing, while the negative control pPGBKT7-lam+pGADT7-T and the experimental group pGBKT7- ​ +pGADT7 had no colony growth, indicating that ScGSTU9 protein had no self-activation activity and could not activate the expression of the reporter gene ADE2. ​ It does not have transcriptional autoactivation activity.

[0092] Example 3 Prokaryotic expression and plate stress analysis

[0093] according to ​ Gene sequence and prokaryotic expression vector pET28a sequence information, using CE Design software to design specific amplification primers pET28a- ​ -F / R (Table 9), using the target gene cloning plasmid as a template, PCR amplification and gel recovery purification were performed to obtain the target fragment recovered by gel. The PCR primer and the prokaryotic expression vector pET28a were digested with BamHI and HindIII respectively. The linearized vector was ligated with the target fragment recovered by gel recovery using the ClonExpress II One Step Cloning Kit homologous recombination kit, and DH5α competent cells were transformed. After correct bacterial liquid PCR and sequencing, the recombinant plasmid pET28a- ​ (See ​ ). Use freeze-thaw method to transfer pET28a- ​ Transform E. coli Tssetta (DE3), culture, select the correct positive clones for propagation. 600 = 0.6~0.7, pET28a- ​ -Tssetta bacterial suspension, add isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 1.0 mmol / L, place at 37 ° C, 200 rpm shaker to induce the target protein, take a sample every 2 hours, and then use 4-20% FastPAGE TMProtein precast gels (Qingke Biotechnology, Beijing, China) were run on electrophoresis to observe whether the target protein was induced to express. Meanwhile, the prokaryotic expression vector pET28a was used as a control, and the pET28a-Tssetta bacterial culture was cultured using the same method.

[0094] Table 9 pET28a-ScGSTU9 primer pairs

[0095]

[0096] SDS-PAGE detection results showed that the ScGSTU9 recombinant protein could be induced to express under the conditions of 1mM IPTG and 37℃. ​ As shown in the red box, ScGSTU9 has a distinct specific band above 25 kDa, which contains the 6×His tag in the 0.84 kDa vector. In addition, as the IPTG induction time increases, the fusion protein accumulates ( ​ ).

[0097] After the target protein was induced, the spot test method was used according to the method of "The alcoholdehydrogenase gene family in sugarcane and its involvement in cold stressregulation"[J]. Su WH, Ren YJ, Wang DJ, ​ BMC Genomics, 2020, 21(1):521. ​ Stress response of genes in E. coli. ​ -Tssetta bacterial solution and pET28a-Tssetta bacterial solution (control) were expanded to OD 600 When the concentration of the culture medium was about 0.6, 1 mM IPTG was added and the culture was shaken at 37°C and 200 rpm. After induction for 10 h, the culture was diluted to the same concentration with LB medium (containing 50 μg / mL kanamycin) and then diluted to 10 -3 , 10 -4 10 μL of bacterial solution was spotted on LB (containing kanamycin resistance) solid medium, and treated at 4°C in the dark for 0, 3, 6, and 12 days, then placed in a 37°C incubator for overnight culture. When the plaque grew to a certain extent, the photo was taken. In addition, in order to explore the effects of other abiotic stresses on the ​ For gene expression in Escherichia coli, 10 μL of bacterial solution was spotted on LB plates containing different concentrations of NaCl (150 mM, 300 mM, 450 mM) and D-mannitol (150 mM, 300 mM, 500 mM) and cultured at 37°C overnight.

[0098] Determination of sugarcane under different abiotic stresses (4℃ low temperature, NaCl, D-mannitol) ​ Gene stress tolerance in E. coli cells, the results are as follows ​ As shown. Under 4℃ low temperature treatment conditions, the recombinant cells pET28a- ​ -Tssetta was almost unaffected in terms of growth rate, while the colony number and growth rate of the control group (pET28a-Tssetta) were significantly inhibited after 6 days of low temperature treatment; under D-mannitol stress, the recombinant cells pET28a- ​ -Tssetta grew faster; under NaCl stress, the growth rate of the control group was significantly inhibited, and the recombinant cells pET28a- ​ -Tssetta grows faster. The above results show that the recombinant cells pET28a- ​ -Tssetta can respond positively to 4℃ low temperature, NaCl and D-mannitol stress. Since mannitol is a cheap, non-toxic free radical scavenger, often used as an osmotic agent and a commonly used dehydrating agent in clinical practice, its properties can be used to create different degrees of drought for plants. This paper constructs a D-mannitol stress experiment to verify the recombinant cell pET28a- ​ -Tssetta positive response to drought environment. In summary, it shows that overexpression ​ It can improve the tolerance of Escherichia coli to low temperature stress, salt stress and D-mannitol stress, that is, overexpression ​ It can improve resistance to low temperature, salt and drought.

[0099] Example 4 ScGSTU9 Genes positively regulating resistance to pathogens

[0100] Designed by using Gateway vector construction method ScGSTU9 Gene primer Gate- ScGSTU9 -F / R (Table 5), ScGSTU9 The gene was constructed into the overexpression vector pEarleyGate203 through LR reaction to form the fusion expression vector pEarleyGate-203- ScGSTU9 ( Figure 9 ). Select Nicotiana benthamiana plants with 5 to 7 leaf ages and uniform growth, and use the Agrobacterium-mediated method in step 1) subcellular localization analysis of Example 2 to insert the empty vector pEarleyGate-203 (35S::00) and the fusion expression vector pEarleyGate-203- ScGSTU9 Suspension Night (OD 600=0.8) was injected into Nicotiana benthamiana leaves for transient overexpression. After 2 days of transient expression, the fungal pathogen Fusarium solani var. Fusarium solani var. coeruleum ) Dilute the collected pathogenic bacteria to OD 600 = 0.8, inoculated into Nicotiana benthamiana leaves transiently overexpressing the pathogen for two days, and tracked leaf phenotypic changes. Leaves were harvested two and nine days after inoculation (when necrosis symptoms appeared) for total RNA extraction, phenotype observation, and DAB staining. The extent and range of leaf necrosis were assessed by photographing the diseased area. Three biological replicates were used.

[0101] DAB staining method: Nine days after inoculation with the pathogen, leaves were stained with DAB solution (the concentration of the solution was 1 mg / mL), kept in the dark overnight, and then heated in a 95% ethanol water bath. The depth and area of ​​the brown color on the leaves were observed and photographed. RT-qPCR was used to detect the expression levels of some tobacco immune-related marker genes in Nicotiana benthamiana. These genes include genes related to the salicylic acid synthesis pathway described in "Transgenic plants with enhanced resistance to the fungal pathogen rhizoctonia solani" [J]. Brogue K, Chet I, Holliday M, et al. Science, 1991, 254(5035): 1194-1197. NbNPR1 、 NbPR2 and NbPR3 , "Transgenic tobacco expressing the hrpN(EP) gene from Erwinia pyrifoliae Genes related to the jasmonic acid synthesis pathway in "[J] triggers defense responses against botrytis cinerea". Sohn SI, Kim YH, Kim BR, et al. Mol Cells, 2007, 24(2): 232-239 NbDEF1 and NbLOX1The role of ethylene during the infection of Nicotianatabacum by Colletotrichum destructivum[J]. Chen N, Goodwin PH, Hsiang T, JExp Bot, 2003, 54(392): 2449-2456 NbHSR201 、 NbHSR203 and NbHSR515 , and ROS-related genes NbCAT1 and NbGST1 ET synthesis-dependent genes recorded in "Overexpression of the peanut CLAVATA1-like leucine-rich repeat receptor-like kinase AhRLK1 confersincreased resistance to bacterial wilt in tobacco"[J]. Zhang C, Chen H, ZhuangR R, et al. J Exp Bot, 2019, 70(19): 5407-5421 NbEFE26 and NbACO-like , with "Overexpression of a Chinese cabbage BrERF11 transcription factor enhances disease resistance to Ralstonia solanacearum in tobacco" [J]. Lai Y, Dang F, Lin J, et al. Plant Physiol Biochem, 2013, 62: 70-78 and "Asugarcane pathogenesis-related protein, ScPR10, plays a positive role indefense responses under Sporisorium scitamineum, SrMV, SA, and MeJA stresses》[J]. Peng Q, Su Y, Ling H, et al. Plant Cell Rep, 2017, 36(9): 1427-1440 NbEF1-α (GenBank accession number: D63396) is the internal reference gene, and the primer sequences are shown in Table 10.

[0102] Table 10 Primer sequence list

[0103]

[0104] Nine days after inoculation with blue variant of Fusarium solani, the phenotype and DAB staining results showed that transient overexpression ScGSTU9 The leaves of the control group showed obvious lesions, shrinkage and withering, but transient overexpression ScGSTU9 The leaves showed slightly mild symptoms. After DAB staining, the bronze color of the control group 35S::00 was darker, while the color of the overexpression group 35S::ScGSTU9 was lighter. RT-qPCR test results showed that the leaves of the overexpression group 35S::ScGSTU9 were darker at 2d and 9d after inoculation. NbLOX1 、 NbPR1 、 NbHSR201 The expression levels were significantly higher than those in the control, showing an up-regulation trend; NbHSR203 The gene expression levels were not significantly different from those in the control group ( Figure 10 ). In summary, compared with the control group 35S::00, transient overexpression ScGSTU9 Tobacco expressing the gene showed resistance to blue variant of Fusarium solani and may play a positive regulatory role in the defense response to blue variant of Fusarium solani. ScGSTU9 The gene may participate in the response of Nicotiana benthamiana to pathogenic stress by regulating the expression of genes related to JA, SA and HR pathways. ScGSTU9 Genes can improve disease resistance.

[0105] Glutathione S-transferases are a family of multifunctional enzymes widely distributed in plants, playing key catalytic and non-enzymatic roles in plant growth, development, and stress responses. GSTs The expression and function of genes in plant growth and development, the use of related genes to improve plant agronomic traits, and provide a reference for breeding new varieties of stress-resistant crops. In this invention, a gene was cloned from sugarcane. ScGSTU9 The expression of these genes was analyzed and the resistance was evaluated. ScGSTU9 Contains the N-terminal GST_N_Tau and C-terminal GST_C_Tau conserved domains ( Figure 1 Figure D in the figure) is a hydrophobic protein with high stability. ScGSTU9 The gene is constitutively expressed in sugarcane tissues, with the highest expression level in new sugarcane pulp and the lowest expression level in old leaves ( Figure 2 Figure B in the figure). ScGSTU9The tissue expression pattern of the gene reveals its important role in the growth and development of sugarcane, and it plays different functions in different tissues. The study found that most GST proteins are cytoplasmic proteins, and a small number are nuclear proteins. Arabidopsis GSTF11 and GSTU20 were mainly observed in the cytoplasm, and the GFP-GST fusions of all GSTFs and GSTs U2, U7, U9, U11, U19 and U28 were localized in the cytoplasm, while GFP-GSTU12 was completely localized in the nucleus. In onion epidermal cells and Arabidopsis leaf epidermal cells, OsGSTU4 was located in the nucleus and cytoplasm. In the present invention, the localization signal of ScGSTU9::GFP was observed in the cytoplasm and endoplasmic reticulum of the lower epidermal cells of Nicotiana benthamiana leaves ( Figure 4 Figure A in the figure), which may play a role in the cytoplasm and endoplasmic reticulum. The sugarcane ScGSTU9 protein has no autoactivation activity in yeast ( Figure 4 Figure B in the figure).

[0106] GSTs actively participate in the plant abiotic stress response process and can improve the plant's ability to adapt to adversity. In the present invention, the results of prokaryotic expression and plate stress experiments showed that under the treatment of D-mannitol, NaCl and 4°C low temperature, ScGSTU9 The gene enhances the tolerance of E. coli cells ( Figure 8 ). This indicates ScGSTU9 Genes can respond to drought, salt and low temperature stress, reflecting the diversity of gene functions.

[0107] In the present invention, ScGSTU9 The gene also showed a response to sugarcane smut fungus stress, and the response in the resistant variety YT93-159 was significantly higher than that in the susceptible variety ROC22, which may be due to ScGSTU9 The increase in gene expression enhanced the defense mechanism of sugarcane against smut fungus, thus ScGSTU9 The gene plays a positive regulatory role in sugarcane defense against smut ( Figure 2 SA and JA are two important defense signaling molecules in plants. They can activate defense responses and enhance plant resistance to pathogens. ScGSTU9 The gene responded positively to SA but had no significant response to MeJA stress ( Figure 2 Figures C and D in Figure 2 show that ScGSTU9 It may play a key role in the SA signal transduction pathway, affecting plant defense responses by regulating the expression of related genes. ScGSTU9 Gene, detection of immune-related gene expression also confirmed ScGSTU9 It may activate immune response by mediating SA signaling pathway ( Figure 10 Figure B in the figure). ScGSTU9The gene can also positively regulate tobacco's defense response to the blue variant of Fusarium solani by regulating the transcription levels of genes related to the JA and HR pathways.

[0108] In summary, GST Genes play an important role in plant response to pathogenic bacteria stress. ScGSTU9 The preliminary study on the disease resistance function of sugarcane has laid a solid foundation for in-depth study of the function and mechanism of action of this gene in the process of sugarcane responding to pathogen stress.

[0109] All other parts not described in detail are prior art. Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Those skilled in the art can also derive other embodiments based on this embodiment without inventiveness, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A sugarcane with stress resistance ScGSTU9 A gene characterized by described ScGSTU9 The coding sequence of the gene is SEQ ID NO:

1.

2. A sugarcane ScGSTU9 protein with stress resistance, characterized in that: The ScGSTU9 protein is the protein with stress resistance function as claimed in claim 1 ScGSTU9 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

3. A ScGSTU9 A plasmid containing a gene, characterized in that Said inclusion ScGSTU9 The plasmid of the gene is a plasmid having stress resistance function as claimed in claim 1 ScGSTU9 The gene was connected to a cloning vector.

4. A ScGSTU9 A recombinant gene vector, characterized in that Said inclusion ScGSTU9 The recombinant vector of the gene is a ScGSTU9 The PCR amplification product of the gene or ScGSTU9 The plasmid of the gene is connected to the vector to obtain; described ScGSTU9 The coding sequence of the gene is shown in SEQ ID NO:

1.

5. A ScGSTU9 A strain of genes, characterized in that Said inclusion ScGSTU9 The strain containing the gene ScGSTU9 Agrobacterium containing genes ScGSTU9 Yeast containing the gene or the ScGSTU9 Gene of Escherichia coli; Said inclusion ScGSTU9 The Agrobacterium containing the gene is the one described in claim 4 ScGSTU9 The recombinant vector of the gene is transformed into Agrobacterium; Said inclusion ScGSTU9 The yeast containing the gene is used as claimed in claim 4 ScGSTU9 The recombinant vector of the gene is transformed into yeast; Said inclusion ScGSTU9 The Escherichia coli containing the gene is used as claimed in claim 4 ScGSTU9 The recombinant vector of the gene or the above-mentioned ScGSTU9 The plasmid containing the gene was transformed into Escherichia coli.

6. A method according to claim 1 ScGSTU9 The application of genes in improving disease resistance is characterized by: The application is overexpressed in tobacco ScGSTU9 Gene to improve the defense ability against infection by blue variant of Fusarium solani.

7. A method for breeding plants with disease resistance, characterized in that: The breeding method is to overexpress ScGSTU9 Genes to obtain tobacco with defense capabilities against infection by the blue variant of Fusarium solani; described ScGSTU9 The coding sequence of the gene is shown in SEQ ID NO: 1.

Images