Application of wheat TaHSF1 gene in improvement of wheat scab resistance

Abstract

The invention discloses an application of a wheat TaHSF1 gene in improvement of wheat scab resistance. The nucleotide sequence of the wheat TaHSF1 gene is shown as SEQ ID NO.1, and the wheat TaHSF1 gene encodes amino acid shown as SEQ ID NO.2. The invention also discloses an application of the wheat TaHSF1 gene in improvement of wheat scab resistance. By editing the TaHSF1 gene, the disease resistance of susceptible wheat can be remarkably improved, an effective technical means is provided for prevention and control of gibberellic disease in production, then the wheat disease resistance breeding process is accelerated, disease prevention and control measures are perfected, and the TaHSF1 gene has wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to wheat. TaHSF1 Application of genes in improving wheat resistance to Fusarium head blight. Background Technology

[0002] Wheat is constantly susceptible to diseases and pests during its growth process. Among them, wheat scab is caused by the Fusarium complex (Fusarium graminearum). Fusarium graminearum Outbreaks of infectious diseases caused by complex species not only seriously threaten wheat yield and quality, but toxins such as deoxynivalenol (DON) produced by the pathogens can further contaminate food and feed, threatening the health of humans and livestock.

[0003] Fusarium head blight (FHB) is prevalent in warm and humid regions, with the Yangtze River mid-lower reaches wheat-growing areas in my country being a traditional outbreak zone. However, due to changes in climate and farming practices, improper straw return to the field, and the increased resistance of the pathogen caused by frequent pesticide use, the affected area of ​​FHB has gradually expanded, moving north and west from the Yangtze River mid-lower reaches wheat-growing areas to become a nationwide epidemic. While climate conditions cannot be changed, farming practices have limited effectiveness in controlling FHB. Although chemical control has some effect, it inevitably increases agricultural production costs and may cause potential environmental pollution. Developing disease-resistant varieties is a crucial means of mitigating the threat of this disease.

[0004] Wheat scab resistance is a quantitative trait controlled by multiple genes. Although numerous resistance trait loci have been identified in the wheat genome, the resistance effects corresponding to these loci are minimal and cannot be used for breeding. Furthermore, the large size of the wheat genome and the complexity of the scab resistance mechanism have slowed progress in the discovery of scab resistance genes and the study of resistance mechanisms. Therefore, the availability of practical resistance genes is a major obstacle to scab breeding in wheat.

[0005] In recent years, using modern molecular biology techniques such as gene editing to reduce crop susceptibility to diseases has gradually become an important means of controlling disease threats. It has been reported that the wheat genome contains a large number of genes susceptible to Fusarium head blight, distributed across multiple chromosomes, but these genes have not been cloned. Cloning and identifying the functions of these genes, and then using gene editing technology to knock out these genes in susceptible varieties, creating Fusarium head blight-resistant wheat germplasm, is one of the important strategies for fundamentally mitigating the damage caused by Fusarium head blight. Summary of the Invention

[0006] To address the slow progress in the discovery of wheat scab resistance genes and the study of resistance mechanisms, this invention provides a wheat... TaHSF1 The application of genes in improving wheat resistance to Fusarium head blight will TaHSF1Knocking out genes in susceptible wheat varieties can effectively improve wheat resistance to Fusarium head blight, providing genetic resources and theoretical support for breeding wheat varieties resistant to Fusarium head blight.

[0007] To achieve the above objectives, the present invention provides, in one aspect, a wheat TaHSF1 The application of genes in any of the following: (1) Control of wheat scab; (2) Preparation of agents to resist wheat scab; (3) Cultivating transgenic plants resistant to wheat scab; Among them, the wheat TaHSF1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, which encodes the amino acids shown in SEQ ID NO.2.

[0008] A second aspect of the present invention provides a method for preventing and controlling wheat scab, comprising the following steps: during the wheat growth process, introducing an agent capable of knocking out the aforementioned... TaHSF1 Gene targets.

[0009] A third aspect of the present invention provides a medicament for resisting wheat scab, said medicament containing an inhibitor of the above-mentioned... TaHSF1 Substances involved in gene expression.

[0010] A fourth aspect of the present invention provides a transgenic plant for breeding resistance to wheat scab, comprising targeting and knocking out the pathogen described in claim 1. TaHSF1 The steps involved in converting a gene target into wheat to obtain transgenic plants resistant to wheat scab.

[0011] Using the technical solution in this invention, the following can be separated: TaHSF1 The coding region sequence of the gene is determined, and a target site is designed and linked to a suitable vector. After being transformed into wheat cells, it can yield... TaHSF1 Knockout wheat plants that are resistant to Fusarium head blight.

[0012] Through the above technical solution, the present invention achieves the following beneficial effects: 1. This invention utilizes the homologous sequence method to clone wheat from the disease-susceptible variety Fielder. TaHSF1 The gene was overexpressed through genetic transformation to verify its disease-susceptibility function. Then, gene editing technology was used to knock it out of wheat varieties susceptible to Fusarium head blight. The results showed that knocking out the gene in susceptible varieties could improve wheat resistance to Fusarium head blight.

[0013] 2. Through editing TaHSF1 Genes can significantly improve the disease resistance of susceptible wheat, providing an effective technical means for the prevention and control of Fusarium head blight in production, thereby accelerating the process of wheat disease resistance breeding, improving disease control measures, and having broad application prospects. Attached Figure Description

[0014] Figure 1 This is a clone in the embodiments of the present invention. TaHSF1 Genes and verification TaHSF1 Flowchart of gene function; Figure 2 This is an amplification from Fielder in the embodiments of the present invention. TaHSF1 The coding region of the gene is analyzed by gel electrophoresis to detect the amplification products. Figure 3 This refers to the inoculation of the spikelets of the wheat variety Fielder with Fusarium head blight before and after inoculation in this embodiment of the invention. TaHSF1 Gene expression level detection; Figure 4 This is an embodiment of the present invention. TaHSF1 Detection of gene expression levels in overexpressed wheat plants (T0 generation); Figure 5 This is an embodiment of the present invention. TaHSF1 The incidence of diseased spikelets in overexpressing wheat plants (T2 generation) 14 days after inoculation with Fusarium graminearum. Each box plot represents the statistical results of 30 spikelets. Double asterisks (**) indicate that the incidence of diseased spikelets in transgenic plants was extremely significant compared with the control. P <0.01) increase; Figure 6 This is an embodiment of the present invention. TaHSF1 Mutations at the target site in gene knockout plants (T0 generation), where blue text indicates the gene editing target sequence, red text represents the mutation site, and "-" represents deletion; Figure 7 This is an embodiment of the present invention. TaHSF1 The percentage of diseased spikelets in gene knockout plants (T2 generation) 14 days after inoculation with Fusarium head blight is shown in each box plot, representing the statistical results of 30 spikelets. Double asterisks (**) indicate that the percentage of diseased spikelets in transgenic plants was extremely significant compared to the control. P <0.01) decrease. Detailed Implementation

[0015] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0016] This invention isolates and clones wheat varieties susceptible to Fusarium head blight, specifically the Fielder variety. TaHSF1 The coding region of the gene was identified, and its susceptibility was verified by overexpressing it in wheat. Subsequently, gene editing technology was used to knock out the gene in susceptible wheat varieties to verify resistance, creating wheat materials resistant to Fusarium head blight, thus laying the foundation for improving wheat resistance to Fusarium head blight using this gene.

[0017] The technical solution of the present invention is as followsFigure 1 As shown: Based on the sequenced wheat variety Chinese Spring TraesCS5D02G553300 The sequence information of the locus was obtained from the wheat variety Fielder using RT-PCR. TaHSF1 The coding region sequence of the gene was extracted, ligated into an overexpression vector, and transformed into wheat to verify its susceptibility. Subsequently, target sites were designed based on the sequence information, and CRISPR / Cas9 gene editing technology combined with Agrobacterium-mediated genetic transformation was used to induce Fusarium head blight in the wheat variety Fielder. TaHSF1 Gene knockout was used to analyze the resistance of genetically transformed plants to Fusarium head blight. See the following examples for details.

[0018] Example 1: Cloning from the wheat variety Fielder TaHSF1 coding region of a gene (1) Preparation of total cDNA from Fielder spikelets Spikes of the wheat variety Fielder, harvested at flowering stage, were ground into powder using liquid nitrogen. 100 mg of the powder was quickly weighed and placed in a 1.5 mL centrifuge tube. 1 mL of TransZol (Beijing TransGen Biotech Co., Ltd.) plant tissue total RNA extraction reagent was added. Total RNA was extracted from the Fielder spikelets according to the instructions provided with the kit. Then, 5 μg of total RNA was taken, and DNase I (Invitrogen, USA) was added to remove genomic DNA. This RNA was then used as a template for oligo(dT) extraction. 15 Total cDNA from Fielder spikelets was obtained by reverse transcription using oligomeric primers and M-MLV reverse transcriptase (Invitrogen, USA).

[0019] (2) TaHSF1 Acquisition of the coding region of a gene The whole genome of the wheat variety Chinese Spring has been completed, based on the characteristics of this variety. TraesCS5D02G553300 Based on the sequence information of the site, a pair of PCR primers TaHSF1-1F (5′- GGATCC ATGGACGGCGGGGTAGCTGCTG-3′(underlined) Bam HI restriction site), TaHSF1-1R (5′- GGATCC TCACCCTCTATGGTTGGATGAC-3′(underlined) Bam Using the total cDNA from Fielder spikelets obtained above as a template, HI restriction sites were used to amplify the resulting... TaHSF1 The coding region was separated by 1% agarose gel electrophoresis. Figure 2The amplification products were recovered using an agarose gel DNA recovery kit (Tiangen Biotech (Beijing) Co., Ltd.), ligated into the TA cloning vector pCE2-TA / Blunt-Zero (Nanjing Novizan Biotechnology Co., Ltd.), and positive clones were sequenced to obtain... TaHSF1 Sequence information of the gene coding region, TaHSF1 The coding region of the gene consists of 1569 nucleotides (SEQ ID NO.1) and encodes 522 amino acids (SEQ ID NO.2).

[0020] SEQ ID NO.1: SEQ ID NO.2: MDGGVAAVAAAAAAASTVTTAVAPPGAAGAGAPPPFLMKTYDMVDDPATDAVVSWGPANNSFIVWNTPEFARDLLPKYFKHNNFSSFVRQLNTYGFRKVDPDKWEFANEGFLRGQKHLLKTITRRKPSHA NNQVQVPQQQPQQQHQQQPQLQNAPMPSCVEVGKFGMEEEIEMLKRDKNVLMQELVRLRQQQQTTDHQLQTLGKRLHGMEQRQQQMMSFLAKAMQSPGFLAQFVQQNENSKRRIVAANKKRRLPKQDDGLN SESALLDGQIIKYQPMINEAAKAMLRKILQQDTSPHRFESMGNSDNLLLENCMPSAQTFDSSSSTRNSAVTLAEPGNSGMPYMPTSSGLSAICSSSTPPEMQCPVLDSNSSTQLPTQLPNMSAASSIPK AMTPGLSDISIPGFPDLHDLITEDAINIPVENYAMPGPECIFPLPDEGSDDSVPMDPIDTDETDDTQKLPGIIDSFWEQFLCASPLSIDNDEVDSGLLDTREAQEENGWTRTENLANLTEQMGLLSSNHRG Example 2: Verification TaHSF1 The gene is a wheat scab susceptibility gene. (1) TaHSF1 Gene expression analysis after inoculation with Fusarium graminearum Wheat ears were collected before and after inoculation with Fusarium head blight pathogens. Total RNA was extracted and reverse transcribed into cDNA. qPCR was used for detection. TaHSF1 The changes in gene expression levels before and after inoculation were studied. The primer sequences used for detection were TaHSF1-RTF (5′-AAGAGGAGAATGGATGGA-3′) and TaHSF1-RTR (5′-TCTATGGTTGGATGACAG-3′). Results showed that in the susceptible wheat variety Fielder, [the gene expression level was observed]. TaHSF1 The expression levels of the gene were extremely significant 24 h and 48 h after inoculation with Fusarium graminearum. P <0.01) is higher than the expression level before inoculation ( Figure 3 ),show TaHSF1 The gene was induced to express by Fusarium graminearum.

[0021] (2) TaHSF1 Creation of wheat plants with overexpressed genes Will TaHSF1 The gene coding region is linked to the wheat overexpression vector pUbiGW (the wheat expression vector pUbiGW was donated by Researcher He Yi of Jiangsu Academy of Agricultural Sciences and disclosed in the literature "He Y, Yang X, Xia X, et al. A phase-separated protein hub modulates resistance to Fusarium head blight in wheat. Cell Host Microbe. 2024, 32:710-726". The public can obtain the above biological material from the applicant. The obtained biological material is only used to repeat the experiments of this invention and cannot be used for other purposes). This vector carries a maize ubiquitin promoter with constitutive and overexpression characteristics. Using Agrobacterium-mediated genetic transformation (Zhou Z, Yang Y, Ai G, et al. Overcoming genotypic dependency and bypassing immatureembryos in wheat transformation by using morphogenic regulators. Sci ChinaLife Sci. 2024, 67:1535-1538.), the successfully constructed vector was transferred into the callus tissue of the wheat variety Fielder and regenerated to obtain plants. A total of 12 independently transformed plants were obtained.

[0022] Leaves were taken from genetically transformed plants, total RNA was extracted and reverse transcribed to obtain total cDNA, and the cDNA was detected by qPCR. TaHSF1 Gene expression levels were detected using the sequences of primers TaHSF1-RTF and TaHSF1-RTR, as shown above. The results indicated that, compared to wild-type Fielder, the genetically transformed plants showed significantly higher expression levels. TaHSF1 Gene expression levels were extremely high ( P <0.01) increased ( Figure 4 ).

[0023] (3) TaHSF1 Analysis of Fusarium head blight resistance in wheat plants with overexpressed gene Select 2 plants TaHSF1Transformed plants of the T0 generation with elevated gene expression were propagated. At the flowering stage of the T2 generation, they were inoculated with Fusarium head blight using a single-flower drip method (Jiang P, Zhang X, Wu L, et al. A novel QTL on chromosome 5AL of Yangmai 158 increases resistance to Fusarium head blight in wheat. PlantPathol. 2020, 69: 249–258). The fungal spores were injected into the wheat ears. Two weeks after inoculation, the number of diseased spikelets and the total number of spikelets were counted. The disease severity was assessed by the proportion of diseased spikelets to the total number of spikelets. Results showed that, compared to the control Fielder, TaHSF1 Genetically transformed plants with elevated gene expression levels are highly susceptible to disease. P <0.01) Enhancement (see) Figure 5 These results indicate that... TaHSF1 The gene is a gene that makes the plant susceptible to Fusarium wilt.

[0024] Example 3: TaHSF1 Application of genes in improving wheat scab resistance (1) TaHSF1 Gene editing target design and vector construction Using the WheatOmics database (http: / / wheatomics.sdau.edu.cn / ), we obtained the three subgenomes (A, B, and D) of the wheat variety Fielder. TaHSF1 Select editing sites in the conserved region of the first exon of some homologous gene sequences that can simultaneously target three homologous genes (see [link to source code]). Figure 6 The DNA element containing the editing site was ligated into the wheat gene editing vector pBUE411 (the wheat gene editing vector pBUE411 was donated by Researcher He Yi of Jiangsu Academy of Agricultural Sciences and disclosed in the literature "He Y, Yang X, Xia X, et al. A phase-separated protein hub modulates resistance to Fusarium head blight in wheat. Cell Host Microbe. 2024, 32:710-726". The public can obtain the above-mentioned biological material from the applicant. The obtained biological material is only used for repeating the experiments of this invention and cannot be used for other purposes).

[0025] (2) TaHSF1 Obtaining and identifying knockout plants The successfully constructed vector was introduced into the wheat variety Fielder using Agrobacterium-mediated genetic transformation (same as in Example 2 (2)). A total of 7 independently transformed plants were obtained in this invention. DNA was extracted from the transformed plants at the seedling stage and transformed using primers TaHSF-NGS-F (5′- ggagtgagtacggtgtgc GGGCGCGCCGCCGCCGTTCC-3′ (underlined is the sequencing adapter sequence) and TaHSF-NGS-R (5′- gagttggatgctggatgg The 142 bp product containing the editing target site, amplified from GGAGGAGGTCCCGGGCGAACTC-3′ (underlined adapter sequence), was analyzed by next-generation sequencing. The results showed that... TaHSF1 Genetically transformed plants with all three homologous genes knocked out (see...) Figure 6 ).

[0026] (3) TaHSF1 Disease resistance analysis of knockout plants In the T2 generation TaHSF1 Plants were knocked out for inoculation analysis with Fusarium head blight. During the wheat flowering stage, fungal spores were injected into the spikelets. Results two weeks after inoculation showed that, compared to the control Fielder, [the following was observed]... TaHSF1 Gene knockout plants exhibited significantly enhanced disease resistance. P <0.01)( Figure 7 ).

[0027] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0028] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0029] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A kind of wheat TaHSF1 Use of genes in any of the following: (1) Prevent and control wheat fusarium rust; (2) Preparation of agents for resisting wheat fusarium head blight; (3) Cultivating transgenic plants resistant to wheat fusarium rust; in, The wheat TaHSF1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and it encodes the amino acid shown in SEQ ID NO.

2.

2. A method for preventing and treating wheat scab, characterized in that: The following steps are involved: During the growth of wheat, the introduction of TaHSF1 Gene targets.

3. A medicament for resisting wheat scab, characterized in that: The drug contains the substance that inhibits the TaHSF1 Gene expression substances.

4. A method for cultivating a transgenic plant resistant to wheat scab, characterized in that: Including targeting and knocking out the TaHSF1 The target site of the gene is transformed into wheat to obtain transgenic plants resistant to wheat ergot.

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