FtZnBK15 protein and encoding gene and application of the FtZnBK15 protein and encoding gene in enhancing plant disease resistance

Abstract

The application belongs to the technical field of biology, and provides FtZnBK15 protein and application of a coding gene in enhancing plant disease resistance, and the application carries out function research on the disease resistance gene through bioinformatics analysis and overexpression, and determines the physical and chemical properties of the protein and predicts the action mechanism through bioinformatics analysis. Disease resistance analysis is carried out on the FtZnBK15 gene overexpressed Arabidopsis plant, and the results show that FtZnBK15 has resistance to Rhizoctonia solani, and the expression amount is significantly improved under the stress of the pathogenic bacteria, and the enzyme activity of SOD is also correspondingly improved. When buckwheat is infected by Rhizoctonia solani, FtZnBK15 participates in the defense mechanism against Rhizoctonia solani. Through comprehensive evaluation of the pathogenic resistance of the overexpressed Arabidopsis, the function of the gene is determined, and the molecular mechanism of buckwheat in response to Rhizoctonia solani infection is clarified.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of FtZnBK15 protein and its encoding gene in enhancing plant disease resistance. Background Technology

[0002] Plants often face various abiotic stresses during their growth and development, which can be broadly categorized into biotic stresses and abiotic stresses. Biotic stresses mainly include pests and diseases; abiotic stresses mainly include salinity, drought, extreme temperatures, and heavy metals. These stressors affect plant growth, development, and crop yield. Over long-term evolution, plants have gradually developed a series of complex and precise adaptation mechanisms and regulatory networks for abiotic stresses. Enzymes, as one of the main regulatory mechanisms, play a crucial role in plant responses to abiotic stresses. In plants, the alcohol dehydrogenase family is divided into three families based on their substrates: alcohol dehydrogenase (ADH), cinnamyl alcohol dehydrogenase (CAD), and formaldehyde dehydrogenase (FDH). These all belong to the zinc-binding dehydrogenase family and respond to plant abiotic stresses to varying degrees. They are induced not only by stresses such as hypoxia but also regulated by hormones such as ABA, playing a vital role in plant stress resistance.

[0003] Buckwheat (Fagopyrum esculentum), belonging to the Polygonaceae family and the Fagopyrum genus, is a high-quality crop with both medicinal and edible uses, rich in nutrients. Its growth is susceptible to various abiotic stresses. Buckwheat damping-off is a serious soil-borne disease caused by Rhizoctonia solani, which can cause seedlings to wilt and die, affecting yield and quality. Zinc finger proteins are one of the largest families of coenzyme factors in plants and play an important role in plant stress responses. However, current research on the mechanisms of buckwheat disease resistance is limited, especially at the genetic level. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides the application of the FtZnBK15 protein and its encoding gene in enhancing plant disease resistance. By comprehensively evaluating the resistance to pathogens overexpressed in Arabidopsis thaliana, the gene function is clarified, and the molecular mechanism of buckwheat response to Rhizoctonia solani infection is elucidated.

[0005] The technical solution provided by this invention is as follows:

[0006] A buckwheat FtZnBK15 protein, wherein the buckwheat FtZnBK15 protein is a protein as described in either a) or b):

[0007] a) A protein consisting of the amino acid sequence shown in SEQ ID No. 1;

[0008] b) Proteins derived from SEQ ID No. 1 that are derived from the sequence shown in SEQ ID No. 1 by substitution and / or addition and / or deletion of one or more amino acid residues and are related to the grain shape and / or grain weight of rice.

[0009] The present invention also provides a buckwheat FtZnBK15 gene, wherein the buckwheat FtZnBK15 gene encodes the aforementioned buckwheat FtZnBK15 protein.

[0010] Furthermore, the nucleotide sequence of the buckwheat FtZnBK15 gene is shown in SEQ ID NO.2.

[0011] The present invention also provides the expression cassette, recombinant vector, recombinant microorganism or transgenic cell line of the buckwheat FtZnBK15 gene described above.

[0012] The present invention also provides the application of overexpression of the above-mentioned buckwheat FtZnBK15 gene in enhancing plant disease resistance.

[0013] Furthermore, the enhancement of plant disease resistance refers to improving resistance to Rhizoctonia solani.

[0014] Furthermore, overexpression of the aforementioned buckwheat FtZnBK15 gene can enhance the plant's antioxidant capacity and reduce oxidative damage to the cell membrane.

[0015] Furthermore, overexpression of the buckwheat FtZnBK15 gene as described in claim 2 is used to upregulate SOD activity in plants infected with Rhizoctonia solani.

[0016] Furthermore, overexpression of the buckwheat FtZnBK15 gene as described in claim 2 is used to inhibit MDA activity in plants infected with Rhizoctonia solani.

[0017] The present invention also provides a method for improving plant disease resistance, the method comprising increasing the content and / or activity of the above-mentioned protein in the target plant to obtain a plant with greater disease resistance than the target plant.

[0018] Beneficial effects

[0019] This invention utilizes bioinformatics analysis and overexpression of disease-resistant genes to conduct functional studies, clarifying the physicochemical properties of proteins and predicting their mechanisms of action. Disease resistance analysis of Arabidopsis plants overexpressing the FtZnBK15 gene showed that FtZnBK15 confers resistance to Rhizoctonia solani, and its expression level significantly increased under pathogen stress, along with a corresponding increase in SOD enzyme activity. When buckwheat is infected with Rhizoctonia solani, FtZnBK15 participates in the defense mechanism against the bacterium. Through a comprehensive evaluation of the pathogen resistance in overexpressed Arabidopsis, gene function was clarified, and the molecular mechanism of buckwheat's response to Rhizoctonia solani infection was elucidated. Attached Figure Description

[0020] Figure 1 Manhattan plot of buckwheat germplasm GWAS;

[0021] Figure 2 The image shows the results of the successful construction of the 293T-hELL2 cell line stably overexpressing human ELL2, as verified by flow cytometry.

[0022] Figure 3 The spectrum of FtZnBK15-pCAMBIA1307;

[0023] Figure 4 Diagram showing the tertiary structure and interactions of proteins;

[0024] Figure 5 Images of DAB staining on leaves of T2 generation Arabidopsis thaliana before and after inoculation;

[0025] Figure 6 The statistical criteria for T2 generation Arabidopsis seedlings and diseased leaves that can be used for infection;

[0026] Figure 7 A statistical chart showing the disease incidence in T2 generation Arabidopsis thaliana;

[0027] Figure 8 A bar chart showing the relative expression level of PR1 after treatment with Rhizoctonia solani. Detailed Implementation

[0028] Example 1

[0029] Disease resistance was evaluated using 20 buckwheat germplasm resources. Genomic DNA was extracted from all germplasm resources using the CTAB method, and the concentration of the extracted products was measured using Nanodrop. After electrophoresis, DNA samples meeting sequencing requirements were selected for GWAS association analysis. Using SNP microarray technology, oligonucleotide probes for a large number of known SNP sites were immobilized on a microarray, and labeled genomic DNA was hybridized to them. The genotype of each SNP site was determined by detecting the intensity of the hybridization signal.

[0030] Based on a genome-wide association analysis (GWAS) of disease resistance in 200 buckwheat accessions, this study investigated five aspects: material selection (200 buckwheat germplasm resources), trait survey and statistics (disease resistance analysis), genotyping, model selection, and association results. Ultimately, the gene FtPinG0302737400.01, associated with disease resistance, was identified and located on chromosome 3 (e.g., Figure 1 ).

[0031] FtbZIPBK15

[0032] Gene ID: FtPinG0302737400.01

[0033] protein sequence

[0034] MPEGSKSDGVITCKAVVCWGPGEALKVEEIQVDPPKSSEVRIKILYSSVCHTDLLLMAGYPSADAFPRAMGHEGVGEVESVGEGVTELKKGDIVIATYVSECKECDNCNFAIRPTNLCCKYPYLMGGVMPDGTSRLSVQGKKLFGFLGSTWSQYVVAHTAYVVKLDPRLPLAHGSFLSCGFSTGLGGSWFDA NVHKGSTVAVLGLGAVGLGAVQGAKIQGASKIIGIDKNPLKREKGEMYGMTDFINPDEASGDKPISQLIMDLTGGQGVDYSFECTGAPALVTQALESTKAGRGLTVVIGAAGKPTMEMHALSLLTGRTIKGSIFGGIKIQSHFPVIVEKCINKEIDLDHLLTHEISFDEMDKALELLRQSDCVKILIKM (SEQ ID No.1).

[0035] CDS sequence

[0036]

[0037] Example 2

[0038] Cloning and vector construction of disease resistance genes

[0039] Total RNA was extracted from cultured buckwheat seedlings using a total RNA extraction kit. High-quality RNA samples were obtained by absorbance detection and agarose gel electrophoresis, preparing for subsequent gene cloning. The extracted buckwheat RNA was reverse transcribed using the ReadyScript two-step cDNA synthesis kit (RDRT) to obtain high-quality cDNA samples, which were stored at -20℃ for later use.

[0040] Full-length CDS amplification primers were designed using SnapeGene. The primer sequences are: F:ATGCCTGAGGGAAGCAAATCT (SEQ ID No. 3); R:GTCAAAATCCTCATCAAGATGTGA (SEQ ID No. 4). The full-length CDS of the FtZnBK15 gene was amplified using PCR. The PCR amplification products were separated by agarose gel electrophoresis, and the correct bands were recovered and purified. The target fragment was detected by polyacrylamide gel electrophoresis, and the PCR amplification products were recovered by gel excision. The concentration of the recovered fragment was determined using Nanodrop, and the target band was stored at 4°C for later use.

[0041] The target gene was introduced into heat-shocked *E. coli* DH5α competent cells using a T-vector. Plasmids were extracted, and DNA samples were stored at -20°C to prevent lysis. The extracted DNA was sent to a sequencing facility for sequencing. Universal primers M13F and M13R for T-vectors were selected. The M13F sequence is GTAAAACGACGGCCAGT (SEQ ID No. 5); the M13R sequence is GTCATAGCTGTTTCCTG (SEQ ID No. 6). The DNA containing the sequenced gene fragment completely ligated into the T-vector was used as a template for ligation into the final vector 1307. Primers were designed based on the pCAMBIA1307 map and the restriction enzyme sites SalI and KpnI for PCR amplification. The specificity and concentration of the PCR amplification products were verified by agarose gel electrophoresis. Correct bands were selected and recovered from the gel. Homologous recombination was used for assembly, and the resulting DNA was introduced into *E. coli* DH5α competent cells. Positive clones were selected by agarose gel electrophoresis for restriction enzyme digestion verification and sequencing. The vector containing the correctly sequenced target fragment was stored at -80°C after being added to glycerol for subsequent transfection with Agrobacterium tumefaciens to infect Arabidopsis thaliana.

[0042] Example 3

[0043] Obtaining transgenic plants

[0044] Agrobacterium strains were activated, and the constructed final vector FtZnBK15-pCAMBIA1307 was introduced into Agrobacterium competent cells. FtZnBK15-positive transformed strains were obtained through double-antibody screening and molecular identification to prepare Arabidopsis thaliana infection working solutions. The primer sequences used for molecular identification were: F: atgaaaaagcctgaactca (SEQ ID No. 7); R: gagtggaaaccgacgc (SEQ ID No. 8). Wild-type Arabidopsis thaliana flower buds were infected using the flower-dipping method. After harvesting, FtZnBK15 transgenic Arabidopsis thaliana T0 generation seeds were obtained. Further culture and screening yielded T1 generation transgenic Arabidopsis thaliana. Figure 3 Leaves from T1 generation seedlings were selected as samples for genomic DNA extraction. The obtained genomic DNA was identified, and T1 generation FtZnBK15 transgenic plants were obtained after testing. Seeds were then harvested. The primer sequences used for identification were: F:ATGCCTGAGGGAAGCAAATCT (SEQ ID No. 9); R:GTCAAAATCCTCATCAAGATGTGA (SEQ ID No. 10).

[0045] T1 generation Arabidopsis seeds were disinfected, and disease-resistant lines were screened using MS medium containing hygromycin. DNA was extracted from positive seedlings initially screened in the medium using the CTAB method. Primers were designed for PCR, and the target band was detected by gel electrophoresis. Seedlings that finally detected the target band were identified as transgenic positive lines, and these positive seedlings were transplanted into substrate soil for further cultivation. Simultaneously, *Rhizoctonia solani* AG4-HGI was activated on solid plates for cultivation to obtain highly pathogenic and pure mycelia.

[0046] Leaves of T2 generation transgenic Arabidopsis thaliana were collected after approximately 30 days of growth and inoculated with fungi to verify whether the overexpressed gene exhibited resistance to Rhizoctonia solani AG-4 HGI 3, and to comprehensively assess the extent to which the FtZnBK15 gene participates in disease resistance. The incidence rate and lesion area percentage of the transgenic lines were statistically analyzed. Detached leaves of the treated FtZnBK15 transgenic lines were stained with DAB staining solution to further observe the lesion condition. The expression pattern of the FtZnBK15 gene in the FtZnBK15 transgenic lines under different treatments was analyzed, and the activities of enzymes reflecting disease resistance were measured.

[0047] Overexpressing Arabidopsis thaliana T2 strains were plated on MS plates and transplanted into nutrient soil after 10 days of growth. Leaves were collected before bolting during vigorous growth for inoculation. Leaves of uniform growth were placed in square Petri dishes lined with sterile filter paper. Rhizoctonia solani was inoculated with mycelium cakes, ensuring the mycelium was in contact with the upper surface of the leaf. Fifteen leaves were collected from each strain, with three strains used as replicates for each transgenic material. Blank medium served as a control. Disease incidence and disease index were recorded 16 hours after inoculation. Disease incidence on buckwheat leaves was investigated according to the following grading criteria:

[0048] Table 1. Disease severity grading criteria for Arabidopsis leaves

[0049]

[0050] Incidence rate = Disease Index (DI) =

[0051] The disease incidence statistics show that the disease index of FtZnBK15 OE was 23.49, indicating resistance compared to the control. The disease index of wild-type Arabidopsis thaliana col-0 was 48.69, while the disease index of Arabidopsis thaliana AtOE (control) transformed with the overexpression vector pCAMBIA1307 was 49.51.

[0052] Example 4

[0053] Gene function research

[0054] Bioinformatics analysis of FtZnBK15 using online platforms such as Protparam, Interpro, SOPMA, and SWISS-MODEL revealed that FtZnBK15 is a stable hydrophobic protein (e.g., ...). Figure 4 Furthermore, the proportion of α-helices in the secondary structure is not high. Interaction analysis shows that this protein is mainly present in the cytosol and participates in the formaldehyde decomposition and metabolism process in the organism. It exerts its function by controlling the activity of pyruvate decarboxylase, the binding of thiamine pyrophosphate and the binding of magnesium ions. It can be further inferred that FtZnBK15 has certain disease resistance in buckwheat.

[0055] Diaminobenzidine (DAB) produces a brown precipitate under the catalysis of peroxidase, which usually indicates the generation of reactive oxygen species. DAB staining results ( Figure 5The results showed that transgenic Arabidopsis carrying the FtZnBK15 gene exhibited significantly higher resistance to Rhizoctonia solani than wild-type Arabidopsis. Twenty-four hours after Rhizoctonia solani infection, detached leaves of wild-type Arabidopsis (WT) showed more pronounced infection marks, while detached leaves of the transgenic line showed less infection. This result further confirms that transgenic Arabidopsis has stronger resistance to Rhizoctonia solani.

[0056] Superoxide dismutase (SOD) is an antioxidant enzyme associated with the scavenging of reactive oxygen species (ROS). Increased SOD activity can reduce free radical damage to cell membranes, thereby mitigating oxidative damage to plants. Furthermore, the measurement of malondialdehyde (MDA) activity can indirectly reflect the degree of damage to plant cell membranes and the plant's ability to cope with stress under corresponding environments. Experimental results showed that 24 hours after Rhizoctonia solani infection, SOD activity was significantly upregulated in Arabidopsis thaliana lines transgenic with the FtZnBK15 gene, while SOD activity in wild-type Arabidopsis thaliana (WT) showed no significant change. This indicates that transgenic Arabidopsis thaliana, under Rhizoctonia solani stress, can enhance its antioxidant capacity by upregulating SOD activity, thereby reducing oxidative damage to cell membranes.

[0057] Regarding MDA activity, the WT line showed significantly higher MDA activity than the control group under normal conditions 24 hours after Rhizoctonia solani infection; while the FtZnBK15 gene-transgenic line also showed an increase in MDA activity after Rhizoctonia solani treatment, but the increase was significantly lower than that of the WT line. This further demonstrates that transgenic Arabidopsis thaliana can effectively inhibit the increase in MDA activity by upregulating SOD activity, thereby mitigating cell membrane damage to some extent. Therefore, compared with WT, the FtZnBK15 gene-transgenic line exhibits stronger disease resistance (e.g., ...). Figure 6 and 7 ).

[0058] Following infection with Rhizoctonia solani, the PR1 gene in the SA signaling pathway was the first to be upregulated and showed the greatest upregulation in Arabidopsis thaliana lines transgenic with the FtZnBK15 gene (e.g., ...). Figure 8 The NPR1 gene was upregulated slightly later, while the TGA1 gene was upregulated the latest and with the smallest magnitude. In the JA signaling pathway, the PDF1.2 gene was upregulated earliest and with the largest magnitude, while the VSP2 and LOX2 genes were upregulated slightly later and with smaller magnitudes. The expression patterns of these genes reflect the active response of different signaling pathways to the FtZnBK15 gene under pathogenic stress.

[0059] This invention, using a Arabidopsis thaliana model with heterologous overexpression of the FtZnBK15 gene, revealed that this gene significantly upregulated superoxide dismutase (SOD) activity and reduced malondialdehyde (MDA) activity under Rhizoctonia solani stress, thereby alleviating cell membrane damage and enhancing plant resistance. It is speculated that FtZnBK15 may maintain cellular protein homeostasis and improve buckwheat resistance to Rhizoctonia solani by participating in the jasmonic acid (JA) and salicylic acid (SA) signaling pathways. In summary, this invention has explored and studied the gene function of FtZnBK15 through genome-wide association analysis, bioinformatics analysis, gene cloning, and overexpression vector construction, preliminarily verifying its resistance effect against Rhizoctonia solani. This provides a theoretical basis for constructing high-quality buckwheat germplasm resources and also serves as a reference for other crop breeding projects.

Claims

1. A buckwheat FtZnBK15 protein, characterized in that, The buckwheat FtZnBK15 protein is either a) or b) of the following: a) A protein consisting of the amino acid sequence shown in SEQ ID No. 1; b) Proteins derived from SEQ ID No. 1 that are derived from the sequence shown in SEQ ID No. 1 by substitution and / or addition and / or deletion of one or more amino acid residues and are related to the grain shape and / or grain weight of rice.

2. A buckwheat FtZnBK15 gene, characterized in that, The buckwheat FtZnBK15 gene encodes the buckwheat FtZnBK15 protein as described in claim 1.

3. The buckwheat FtZnBK15 gene according to claim 2, characterized in that, The nucleotide sequence of the buckwheat FtZnBK15 gene is shown in SEQ ID NO.

2.

4. The expression cassette, recombinant vector, recombinant microorganism, or transgenic cell line of the buckwheat FtZnBK15 gene as described in claim 2.

5. The application of overexpression of the buckwheat FtZnBK15 gene as described in claim 2 in enhancing plant disease resistance.

6. The application according to claim 5, characterized in that, The enhancement of plant disease resistance refers to improving resistance to Rhizoctonia solani.

7. The application according to claim 5, characterized in that, Overexpression of the buckwheat FtZnBK15 gene as described in claim 2 is used to enhance the antioxidant capacity of plants and reduce oxidative damage to cell membranes.

8. The application according to claim 5, characterized in that, Overexpression of the buckwheat FtZnBK15 gene as described in claim 2 is used to upregulate SOD activity in plants infected with Rhizoctonia solani.

9. The application according to claim 5, characterized in that, Overexpression of the buckwheat FtZnBK15 gene as described in claim 2 is used to inhibit MDA activity in plants infected with Rhizoctonia solani.

10. A method for improving plant disease resistance, characterized in that, The method includes increasing the content and / or activity of the protein of claim 1 in the target plant to obtain a plant with greater disease resistance than the target plant.

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