Application of wheat taHSF1 gene in improving resistance to scab disease

Abstract

The application discloses a wheat TaHSF1 gene in improving the resistance of wheat to scab, and the wheat TaHSF1 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid is shown as SEQ ID NO. 2. By editing TaHSF1 The gene can significantly improve the disease resistance of the susceptible wheat, provides an effective technical means for preventing and controlling scab in production, further speeds up the wheat breeding process, perfects the prevention and control measures of the disease, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering technology, specifically, relates to wheat TaHSF1 Application of genes in improving wheat fusarium head blight resistance. Background Art

[0002] Wheat is constantly damaged by pests and diseases during its growth. Among them, wheat head blight is caused by Fusarium graminearum complex ( Fusarium graminearum The outbreak of epidemic diseases caused by Fusarium spp. complex species not only seriously threatens wheat yield and quality, but also the toxins such as deoxynivalenol (DON) produced by the pathogen will further contaminate food and feed, threatening the health of humans and livestock.

[0003] Wheat fusarium head blight is common in warm, humid climates, with the wheat-growing areas of my country's middle and lower Yangtze River regions traditionally experiencing it. Due to changes in climatic conditions and tillage systems, improper straw return and handling, and the increasing resistance of the pathogen due to the frequent use of pesticides, wheat fusarium head blight has gradually expanded its reach, moving northward and westward from the wheat-growing areas of the middle and lower Yangtze River regions to become a nationwide epidemic. Climate conditions cannot be altered, and tillage practices have limited effectiveness in preventing and controlling wheat fusarium head blight. While chemical control measures have some effectiveness, they inevitably increase agricultural production costs and pose potential environmental risks. Breeding resistant varieties is a key approach to mitigating the threat posed by the disease.

[0004] Wheat fusarium head blight resistance is a quantitative trait controlled by multiple genes. Although numerous resistance-related quantitative trait loci have been discovered in the wheat genome, the corresponding resistance effects are minimal and cannot be used for breeding. Furthermore, the large size of the wheat genome and the complexity of fusarium head blight resistance mechanisms have hindered the discovery of fusarium head blight resistance genes and the study of their mechanisms. Therefore, the identification of practical resistance genes is a major constraint in wheat fusarium head blight breeding.

[0005] In recent years, using modern molecular biological methods such as gene editing to reduce crop susceptibility has become an increasingly important tool for controlling disease threats. It has been reported that the wheat genome contains numerous scab-susceptibility genes, distributed across multiple chromosomes, but these genes have not been cloned. Cloning and identifying the functions of these genes, and then using gene editing to knock out these genes in susceptible varieties to create scab-resistant wheat germplasm, is one of the key strategies for fundamentally mitigating the damage caused by scab. Summary of the Invention

[0006] In view of the above-mentioned slow progress in the research of wheat scab resistance gene mining and disease resistance mechanism, the present invention provides a wheat TaHSF1 The application of genes in improving wheat scab resistance will TaHSF1Knocking out genes in susceptible wheat varieties can effectively improve wheat resistance to ergot disease, providing genetic resources and theoretical support for breeding wheat varieties resistant to ergot disease.

[0007] In order to achieve the above object, the present invention provides a wheat TaHSF1 Use of genes in any of the following:

[0008] (1) Prevent and control wheat fusarium head blight;

[0009] (2) Preparation of agents for resistance to wheat fusarium head blight;

[0010] (3) Cultivating transgenic plants resistant to wheat fusarium head blight;

[0011] Wherein, 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.

[0012] The second aspect of the present invention provides a method for preventing and treating wheat scab, which comprises the following steps: during the growth of wheat, introducing a gene capable of knocking out the above-mentioned TaHSF1 Gene targets.

[0013] The third aspect of the present invention provides a medicament for resisting wheat scab, which contains TaHSF1 Gene expression substances.

[0014] The fourth aspect of the present invention provides a method for cultivating a transgenic plant resistant to wheat scab, which comprises targeting and knocking out the transgenic plant described in claim 1. TaHSF1 The target site of the gene is transformed into wheat to obtain transgenic plants resistant to wheat fusarium wilt.

[0015] Utilize the technical scheme of the present invention, separate and obtain TaHSF1 The coding region sequence of the gene is designed and connected with the appropriate vector. After being transferred into wheat cells, the TaHSF1 Knockout wheat plants resistant to ergot.

[0016] Through the above technical solution, the present invention achieves the following beneficial effects:

[0017] 1. The present invention cloned the virus from the susceptible wheat variety Fielder using homologous sequence method. TaHSF1 The gene was overexpressed through genetic transformation, its disease susceptibility function was verified, and then it was knocked out from wheat varieties susceptible to ergot disease using gene editing technology. The results showed that knocking out this gene in susceptible varieties can improve wheat resistance to ergot disease.

[0018] 2. By editing TaHSF1The gene can significantly improve the disease resistance of susceptible wheat, provide an effective technical means for the prevention and control of ergot in production, thereby accelerating the process of disease-resistant wheat breeding and improving disease prevention and control measures, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is cloned in the embodiment of the present invention TaHSF1 Genes and verification TaHSF1 Flowchart of gene function;

[0020] Figure 2 It is amplified from Fielder in the embodiment of the present invention TaHSF1 The coding region of the gene was analyzed by gel electrophoresis to detect the amplified product;

[0021] Figure 3 The spikelets of wheat variety Fielder in the embodiment of the present invention are before and after inoculation with Gibberella TaHSF1 Detection of gene expression;

[0022] Figure 4 In the embodiment of the present invention TaHSF1 Detection of gene expression in overexpression wheat plants (T0 generation);

[0023] Figure 5 In the embodiment of the present invention TaHSF1 The diseased spikelet rate of overexpressing wheat plants (T2 generation) 14 days after inoculation with Gibberella fusca. Each box plot is the statistical result of 30 spikelets. Double asterisks (**) indicate that the diseased spikelet rate of transgenic plants is extremely significant compared with the control ( P <0.01) increased;

[0024] Figure 6 In the embodiment of the present invention TaHSF1 The mutation status of the target site in the gene knockout plants (T0 generation), where blue fonts represent the target sequence of gene editing, red fonts represent the mutation site, and “-” represents deletion;

[0025] Figure 7 In the embodiment of the present invention TaHSF1 The diseased spikelet rate of gene knockout plants (T2 generation) 14 days after inoculation with ergot. Each box plot is the statistical result of 30 spikelets. Double asterisks (**) indicate that the diseased spikelet rate of transgenic plants is extremely significant compared with the control ( P <0.01) decreased. DETAILED DESCRIPTION

[0026] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0027] The present invention isolates and clones the wheat scab-susceptible variety Fielder TaHSF1 The coding region of the gene was identified and overexpressed in wheat to verify its susceptibility to the disease. Furthermore, gene editing technology was used to knock out the gene in susceptible wheat varieties to verify disease resistance and create wheat materials resistant to scab, thus laying the foundation for using this gene to improve wheat resistance to scab.

[0028] The technical solution of the present invention is as follows Figure 1 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 technology TaHSF1 The coding region sequence of the gene was linked to an overexpression vector and then transformed into wheat to verify its disease susceptibility. Targets were then designed based on the sequence information and CRISPR / Cas9 gene editing technology combined with Agrobacterium-mediated genetic transformation were used to transform the gene into the wheat cultivar Fielder susceptible to fusarium susceptibility. TaHSF1 Gene knockout, and analysis of scab resistance in genetically transformed plants. Detailed information is provided in the following examples.

[0029] Example 1: Cloning from wheat variety Fielder TaHSF1 Coding region of a gene

[0030] (1) Preparation of total cDNA from Fielder spikelets

[0031] Spikelets of the wheat variety Fielder at the flowering stage were ground into a powder using liquid nitrogen. 100 mg of the powder was quickly weighed and placed in a 1.5 mL centrifuge tube. 1 mL of the plant tissue total RNA extraction reagent TransZol (Beijing Quanshijin Biotechnology Co., Ltd.) was added. Total RNA was extracted from the Fielder spikelets according to the protocol provided in the kit's instructions. 5 μg of total RNA was then removed from the spikelets using DNase I (Invitrogen, USA) to remove genomic DNA. The RNA was then used as a template for the extraction of RNA using oligo(dT)-1-aminobutyric acid (A5). 15 Total cDNA of Fielder spikelets was obtained by reverse transcription using oligo primers and M-MLV reverse transcriptase (Invitrogen, USA).

[0032] (2) TaHSF1 Obtaining the coding region of a gene

[0033] The whole genome of the wheat variety Chinese Spring has been sequenced. TraesCS5D02G553300 Based on the sequence information of the locus, a pair of PCR primers TaHSF1-1F (5′- GGATCC ATGGACGGCGGGGTAGCTGCTG-3′) (underlined BamHI restriction site), TaHSF1-1R (5′- GGATCC TCACCCTCTATGGTTGGATGAC-3′) (underlined Bam HI restriction site), using the total cDNA of Fielder spikelets obtained above as a template, amplified TaHSF1 The coding regions were separated by 1% agarose gel electrophoresis ( Figure 2 The amplified product was recovered using an agarose gel DNA recovery kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), ligated with the TA cloning vector pCE2-TA / Blunt-Zero (Nanjing Novozymes Biotechnology Co., Ltd.), and the positive clones were sequenced to obtain TaHSF1 Sequence information of gene coding regions, TaHSF1 The coding region of the gene consists of 1569 nucleotides (SEQ ID NO.1) and encodes 522 amino acids (SEQ ID NO.2).

[0034] SEQ ID NO.1:

[0035]

[0036] SEQ ID NO.2:

[0037] MDGGVAAVAAAAAAASTVTTAVAPPGAAGAGAPPPFLMKTYDMVDDPATDAVVSWGPANNSFIVWNTPEFARDLLPKYFKHNNFSSFVRQLNTYGFRKVDPDKWEFANEGFLRGQKHLLKTITRRKPSHANNQVQVPQQQPQQQHQQQPQLQNAPMPSCVEVGKFGMEEEIEMLKRDKNVLMQELVRLRQQQQTTDHQLQTLGKRLHGMEQRQQQMMSFLAKAMQSPGFLAQFVQQNENSKRRIVAANKKRRLPKQDDGLNSESALLDGQIIKYQPMINEAAKAMLRKILQQDTSPHRFESMGNSDNLLLENCMPSAQTFDSSSSTRNSAVTLAEVPGNSGMPYMPTSSGLSAICSSSTPPEMQCPVLDSNSSTQLPTQLPNMSAASSIPKAMTPGLSDISIPGFPDLHDLITEDAINIPVENYAMPGPECIFPLPDEGSDDSVPMDPIDTDETDDTQKLPGIIDSFWEQFLCASPLSIDNDEVDSGLLDTREAQEENGWTRTENLANLTEQMGLLSSNHRG

[0038] Example 2: Verification TaHSF1 The gene is a wheat scab resistance gene

[0039] (1) TaHSF1 Expression analysis of the gene after inoculation with scab fungus

[0040] The wheat spikes before and after inoculation with scab fungus were taken, total RNA was extracted and reverse transcribed into cDNA. The expression of the gene before and after inoculation was detected by qPCR technology. The detection primer sequence was TaHSF1-RTF (5'-AAGAGGAGAATGGATGGA-3') and TaHSF1-RTR (5'-TCTATGGTTGGATGACAG-3'). The results showed that in the scab-susceptible wheat variety Fielder, TaHSF1 The expression of the gene 24 h and 48 h after inoculation with scab fungus was significantly higher than that before inoculation TaHSF1 P <0.01), indicating Figure 3 TaHSF1 ​​The gene is induced to express by Fusarium.

[0041] (2) TaHSF1 Creation of wheat plants overexpressing the gene

[0042] The TaHSF1 coding region of the gene is ligated to the wheat overexpression vector pUbiGW (wheat expression vector pUbiGW is donated by Professor He Yi of Jiangsu Academy of Agricultural Sciences, which is 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, and the resulting above biological material is only used for repeating the experiments of the present application and cannot be used for other purposes). The vector carries a maize ubiquitin promoter with constitutive and overexpression characteristics. The successfully constructed vector is transformed into the callus of wheat variety Fielder by Agrobacterium-mediated genetic transformation method (Zhou Z, Yang Y, Ai G, et al. Overcoming genotypic dependency and bypassing immature embryos in wheat transformation by using morphogenic regulators. Sci China Life Sci. 2024, 67:1535-1538.), and plants are regenerated, and a total of 12 independent transformed plants are obtained.

[0043] The leaves of the genetically transformed plants are taken, total RNA is extracted and total cDNA is obtained by reverse transcription, and the expression amount of the gene in the genetically transformed plants is detected by qPCR technology. The sequences of the detection primers TaHSF1-RTF and TaHSF1-RTR are as shown above. The results show that compared with the wild type Fielder, the expression amount of the gene in the genetically transformed plants is extremely significantly (P<0.01) increased. TaHSF1 TaHSF1 P <0.01). Figure 4

[0044] (3) TaHSF1 Analysis of Fusarium head blight resistance of wheat plants overexpressing the gene

[0045] Two TaHSF1 ​​​The T0 generation genetically transformed plants with increased gene expression were propagated, and at the T2 generation flowering stage, the single flower drip method (Jiang P, Zhang X, Wu L, et al. A novel QTL on chromosome 5AL ofYangmai 158 increases resistance to Fusarium head blight in wheat. PlantPathol. 2020, 69: 249–258) was used to inoculate Fusarium head blight. The pathogen spores were injected into the wheat ears. Two weeks after inoculation, the number of diseased spikelets and the total number of spikelets were counted, and the proportion of the number of diseased spikelets to the total number of spikelets was used to measure the disease of the plant. The results showed that compared with the control Fielder, TaHSF1 Transformed plants with increased gene expression were significantly more susceptible to disease ( P <0.01) enhanced (see Figure 5 These results indicate that TaHSF1 The gene is a fusarium susceptibility gene.

[0046] Example 3: TaHSF1 Application of Genes in Improving Wheat Fusarium Head Blight Resistance

[0047] (1) TaHSF1 Gene editing target design and vector construction

[0048] The three subgenomes (A, B, and D) of the wheat variety Fielder were obtained using the WheatOmics (http: / / wheatomics.sdau.edu.cn / ) database. TaHSF1 The sequences of some homologous genes were selected in the conserved region of the first exon, which could simultaneously target three homologous genes (see 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 document “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 to repeat the experiments of the present invention and cannot be used for other purposes).

[0049] (2) TaHSF1 Obtaining and identifying knockout plants

[0050] The constructed vector was introduced into the wheat variety Fielder by using the Agrobacterium-mediated genetic transformation method (same as in Example 2 (2)). A total of 7 independent transformed plants were obtained in the present invention. DNA of the transformed plants was extracted at the seedling stage and the primers TaHSF-NGS-F (5′- ggagtgagtacggtgtgc GGGCGCGCCGCCGCCGTTCC-3′) (underlined is the sequencing adapter sequence) and TaHSF-NGS-R (5′- gagttggatgctggatgg GGAGGAGGTCCCGGGCGAACTC-3′) (underlined for the sequencing adapter sequence) amplified a 142 bp product including the editing target site for next-generation sequencing analysis. The results showed that TaHSF1 Transformed plants with all three homologous genes knocked out (see Figure 6 ).

[0051] (3) TaHSF1 Disease resistance analysis of knockout plants

[0052] In the T2 generation TaHSF1 Knockout plants were inoculated for analysis of fusarium head blight. The pathogen spores were injected into the spikelets during the flowering period of wheat. The results of the investigation two weeks after inoculation showed that compared with the control Fielder, TaHSF1 The disease resistance of gene knockout plants was significantly enhanced ( P <0.01) ( Figure 7 TaHSF1 ).

[0053] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within 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 scope of protection of the present invention.

[0054] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0055] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. Knockout wheat TaHSF1 Use of genes in any of the following: (1) Prevent and control wheat fusarium head blight; (2) Cultivating wheat resistant to ergot; in, The wheat TaHSF1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and it encodes the protein shown in SEQ ID NO.

2.

2. A method for preventing and treating wheat scab, characterized in that: The following steps are involved: Knockout wheat TaHSF1 Gene, the wheat TaHSF1 The nucleotide sequence of the gene is shown as SEQ ID NO.1, and it encodes the protein shown as SEQ ID NO.2; wheat with enhanced resistance to ergot disease is obtained.

3. A method for cultivating wheat resistant to scab, characterized in that: The following steps are involved: Knockout wheat TaHSF1 Gene, the wheat TaHSF1 The nucleotide sequence of the gene is shown as SEQ ID NO.1, and it encodes the protein shown as SEQ ID NO.2; wheat with enhanced resistance to ergot disease is obtained.

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