Application of the heat shock transcription factor TaHsfA1 gene in improving resistance to wheat stem base rot

Abstract

This invention belongs to the field of genetic engineering technology, specifically involving TaHsfA1 Application of genes in improving resistance to wheat stem base rot. This invention discovers... TaHsfA1 Genes play a significant role in improving wheat resistance to stem rot. Knocking out wheat genes using CRISPR / Cas9 gene editing technology... TaHsfA1 And obtained a homozygous mutant with TaHsfA1 knocked out; 28 days after inoculation with the pathogen, TaHsfA1 The gene knockout mutant exhibits superior phenotype and physiological indicators compared to the wild-type wheat variety Cangmai 6005, specifically as follows: TaHsfA1 The mutant exhibits less browning symptoms at the stem base compared to the wild-type Cangmai 6005, a lower disease index, less H2O2 accumulation in leaves, higher chlorophyll content, and better growth. The mutant also shows significantly enhanced resistance to stem base rot compared to the wild-type wheat. This invention provides a novel method for rapidly developing new wheat varieties resistant to stem base rot.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically involving TaHsfA1 Application of gene knockout in improving resistance to wheat stem base rot. Background Technology

[0002] Wheat stem base rot (Fusarium crown rot, FCR) is a serious soil-borne disease worldwide. Infection of the wheat stem base and roots leads to tissue necrosis, impairing the plant's ability to transport water and nutrients upwards, resulting in reduced grain number per ear, grain weight, plant height, tiller number, and yield. Furthermore, the fungal toxins secreted by the pathogen can contaminate the grains, causing food safety issues. However, currently promoted wheat varieties do not exhibit the expected resistance to wheat stem base rot, and resistant germplasm resources are scarce, with the vast majority of germplasm being highly susceptible. Therefore, breeding wheat varieties with high resistance to stem base rot is crucial for stable wheat yield and quality. Targeted improvement of disease resistance in major wheat varieties through gene editing technology will significantly shorten the breeding cycle and accelerate the development of disease-resistant wheat varieties. Summary of the Invention

[0003] The purpose of this invention is to provide TaHsfA1 Application of genes in improving resistance to wheat stem rot TaHsfA1 This invention provides a novel genetic resource for disease-resistant wheat breeding using genetic engineering techniques by negatively regulating stem base rot resistance in wheat. The technical solution employed in this invention is as follows: This invention provides TaHsfA1 The application of genes in improving wheat stem base rot resistance, the aforementioned TaHsfA1 The accession numbers for the three homologous genes are: MW756130, MW756131, and MW756132. This invention inhibits the TaHsfA1 Gene expression or knockout TaHsfA1 Genes that enhance wheat's resistance to stem base rot.

[0004] As a preferred embodiment, the application utilizes construction and suppression TaHsfA1 Gene-related biological materials were used to obtain wheat varieties with enhanced resistance to stem rot.

[0005] In some embodiments of the present invention, the biological material does not include reproductive material. In some embodiments of the present invention, the biomaterial includes nucleic acid molecules, carriers, and cells. In some embodiments of the present invention, the nucleic acid molecule includes an inhibitor. TaHsfA1 Functional microRNAs, siRNAs, shRNAs, dsRNAs, sgRNAs, and / or antisense oligonucleotides. In some embodiments of the present invention, the sequence of the sgRNA is shown in SEQ ID NO.1. In some embodiments of the present invention, the sgRNA is used in conjunction with a CRISPR / Cas9 vector to achieve gene knockout. In some embodiments of the present invention, the CRISPR / Cas9 vector further includes an expression vector containing the sgRNA, and may also include Cas9 protein or an expression vector for expressing Cas9 protein. In some embodiments of the present invention, the carrier includes, but is not limited to, other carriers commonly used in the art such as Cas9. In some embodiments of the present invention, the cells include at least one of *Escherichia coli* and *Agrobacterium*. *Escherichia coli* is a commonly used host cell in the art for constructing vectors and plasmids, while *Agrobacterium* is a common tool in the art for delivering DNA molecules to plants.

[0006] This invention also provides a method for breeding wheat varieties highly resistant to stem rot, including reducing the amount of wheat... TaHsfA1 Steps for determining gene expression levels and / or activity.

[0007] In some embodiments of the present invention, the reduction of wheat TaHsfA1 The steps for regulating gene expression and / or activity are as described in this invention, involving the inhibition of... TaHsfA1 Introducing gene-related biological materials into wheat tissues or wheat cells; In some embodiments of the present invention, the introduction method includes using at least one of Ti plasmid, Ri plasmid, plant virus vector, microinjection, and electroporation.

[0008] In some embodiments of the present invention, the content of wheat is reduced. TaHsfA1 The specific steps for determining protein expression levels and / or activity are as follows: (1) Design TaHsfA1 The target sequence sgRNA of the gene was used to construct wheat TaHsfA1 Gene-editing CRISPR / Cas9 vectors; (2) Transform the CRISPR / Cas9 vector described in step (1) into Agrobacterium competent cells to obtain Agrobacterium containing the CRISPR / Cas9 vector; (3) Infect the embryos of Cangmai 6005 with the Agrobacterium infection solution obtained in step (2), and re-obtain seedlings through tissue culture. Screen wheat. TaHsfA1 Stable genetic mutant lines that have gene mutations, do not contain exogenous Cas9 protein, and have variations in the target sequence.

[0009] In some embodiments of the present invention, the carrier is pWMB110-Cas9; In some embodiments of the present invention, the host cell is Agrobacterium C58C1; In some embodiments of the present invention, the wheat variety is Cangmai 6005.

[0010] The beneficial effects of this invention are: this invention is the first to discover... TaHsfA1 Application of genes in regulating resistance to wheat stem base rot. This was achieved by [implanting gene regulation in wheat]. TaHsfA1 Repression and knockout of gene expression TaHsfA1 Genes can significantly improve wheat resistance to stem base rot, providing a simple and effective technical means for rapidly creating new wheat lines resistant to stem base rot. Attached Figure Description

[0011] Figure 1 for TaHsfA1 Schematic diagram of gene mutation methods Figure 2 Wild type and TaHsfA1 Phenotypic diagrams of mutant wheat (A) and wheat infected with stem base rot (B). Figure 3 Wild type and TaHsfA1 Image of the stem base of a mutant infected with stem base rot Figure 4 Wild type and TaHsfA1 Disease index diagram of mutant wheat infected with stem base rot Figure 5 Wild type and TaHsfA1 DAB staining image of leaves of mutant wheat infected with stem base rot Figure 6 wild type and TaHsfA1 Image of chlorophyll content in leaves of mutant wheat infected with stem base rot Detailed Implementation The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0012] Retrieve using the Ensembl plants database TaHsfA1 homologous sequences TaHsfA1-A , TaHsfA1-B , TaHsfA1-D ,right TaHsfA1Homologous sequence alignment analysis was performed, and sequences containing the PAM domain of NGG were selected from their conserved regions. One of these sequences was chosen as the target site, with a sequence length of 23 bp, and named sgRNA. The sgRNA sequence is shown in SEQ ID NO.1.

[0013] Example 2: Construction and identification of the CRISPR / Cas9-TaHsfA1 vector 1) Construction TaHsfA1 The target primers for the gene editing vector, the sgRNA target primer TaHsfA1-gR1-F sequence is shown in SEQ ID NO.2, and the TaHsfA1-gR1-R sequence is shown in SEQ ID NO.3; 2) Using TaU3 as a template, four primers—UF, gR-R, TaHsfA1-gR1-F, and TaHsfA1-gR1-R—were added to the first reaction for PCR amplification of the sgRNA target. The PCR program was set as follows: Stage 1: 95℃, 5 min; Stage 2 (35 cycles): 95℃, 30 sec; 60℃, 30 sec; 72℃, 1 min; Stage 3: Store at 4℃. The UF sequence is shown in SEQ ID NO. 4, and the gR-R sequence is shown in SEQ ID NO. 5. 3) Using the PCR product from the above steps as a template, a second round of PCR amplification was performed using primers ZWHind-UF and ZWHindR containing restriction enzyme sites. The PCR amplification products were detected by agarose gel electrophoresis, and the gel was excised and purified. The ZWHind-UF sequence is shown in SEQ ID NO.6, and the ZWHindR sequence is shown in SEQ ID NO.7. 4) The restriction endonuclease HindIII was used to digest the vector backbone pWMB110-Cas9 to linearize the vector, and the vector was detected by agarose gel electrophoresis and purified using a DNA purification and concentration kit. 5) The second-round PCR product and the digested vector DNA were ligated using In-Fusion ligase and transformed into competent E. coli cells. 6) Colony PCR identification: Select colonies with band size consistent with the target product, shake the colony and send it for sequencing. For clones with correct sequencing, reproduce them again, extract plasmids, and transform them into Agrobacterium C58C1 competent cells. Select positive clones and send them to the Chinese Academy of Agricultural Sciences for genetic transformation. The recipient is the wheat variety Cangmai 6005.

[0014] Example 3 TaHsfA1 Identification of knockout wheat lines Genomic DNA was extracted from leaves of T0 generation transgenic plants using a DNA extraction kit (Kangwei Century Biotechnology Co., Ltd.). Primer pairs were designed for PCR-RE detection of gene sequences at each target site. The detection primer sequences are as follows: Chromosome A F: AGC ACC ACC AGC AGC AGC ATC AG R:TCA GGA CCA GGC ATT GCG AAA Chromosome B F: CCC AGC AGC AGC ATC AGC AGC AGC A R:GCA TAG TTC TCT ACA GGA ATA T C chromosome F:CCC AGC AGC AGC AGC AGC AGC AGC AGC R:TGG GAA GTT GTG TCG ATG AGT TGG PCR amplification products corresponding to the edited plants identified by PCR-RE were selected and sent to a biotechnology company for sequencing. The editing type was analyzed based on the sequencing results, and different types of edited plants were statistically analyzed. Homozygous mutant materials were retained, and finally, two homozygous mutants were obtained, denoted as M-1 and M-2. TaHsfA1 Gene mutation types such as Figure 1 As shown.

[0015] Example 4 TaHsfA1 Analysis of stem base rot resistance in gene knockout lines

[0016] The cultivation and inoculation method for wheat seedlings involves selecting plump, uniformly sized wheat seeds and germinating them at a constant temperature for 36 hours. Twelve seeds with consistent growth are then sown at a depth of 7 cm. 3 After culturing in square flowerpots for 24 hours, diseased rice grains were inoculated. Specifically, 50 g of 0.5% bacterial soil (containing 0.25 g of diseased rice grains) was added to the square flowerpot, and the pot was covered with a transparent lid to maintain humidity for 4 days. After uncovering, the seedlings were cultured under the following conditions: temperature 25 ℃, alternating 14 h light (15000 lx light intensity) / 10 h dark, and relative humidity 60%. Disease incidence was recorded 28 days after inoculation, the disease index (DI) was calculated, and photographs were taken.

[0017] wild type and TaHsfA1 Phenotypic differences in mutant wheat: Under normal conditions, both wild-type and mutants M-1 and M-2 showed good growth and were free from pathogen infection. Figure 2 A); 28 days after inoculation, wild-type Cangmai 6005 and mutant wheat showed different degrees of disease ( Figure 2 B), the browning and withering of the stem base of Cangmai 6005 was significantly more severe than that of mutants M-1 and M-2.Figure 3 The disease index was significantly higher than that of the two mutant wheat varieties. Figure 4 ).

[0018] wild type and TaHsfA1 DAB staining analysis of mutant wheat leaves: DAB staining is a classic histochemical staining method in plant pathology for in situ localization of hydrogen peroxide (H2O2), specifically used to visually display the location and content of reactive oxygen species accumulation in plant tissues. 28 days after inoculation, DAB staining revealed differences between wild-type Cangmai 6005 and... TaHsfA1 Both mutants M-1 and M-2 showed a brownish color, but the leaves of the wild-type wheat were darker, indicating that the wild-type wheat leaves accumulated more H2O2 and suffered more severe damage to the leaf tissue. TaHsfA1 mutant ( Figure 5 ).

[0019] wild type and TaHsfA1 Chlorophyll content determination of mutant wheat leaves under normal culture conditions, wild-type Cangmai 6005 and TaHsfA1 The chlorophyll content in the leaves of the mutant systems was not different; 28 days after inoculation, TaHsfA1 The chlorophyll content in the leaves of mutant M-2 was significantly higher than that of wild-type wheat, indicating that: TaHsfA1 The mutant system showed improved resistance to stem rot and better growth compared to wild-type wheat. Figure 6 ).

Claims

1. A wheat heat shock transcription factor TaHsfA1 The application of gene knockout in improving resistance to wheat stem rot; TaHsfA1 The three homologous gene nucleotide sequence registration numbers are MW756130, MW756131, and MW756132.

2. With knockout TaHsfA1 Application of gene-related biomaterials in wheat breeding; The TaHsfA1 The nucleotide sequence accession numbers of the gene are: MW756130, MW756131, MW756132; The application is for building and knocking out. TaHsfA1 Gene-related biological materials were used to obtain wheat varieties with improved resistance to stem base rot; the biological materials do not include propagation materials. The biomaterials include nucleic acid molecules, carriers, and cells; The nucleic acid molecules include inhibitors TaHsfA1 A functional sgRNA; the sequence of the sgRNA is shown in SEQ ID NO: 1; The vector includes a CRISPR / Cas9 vector; The cells include at least one of Escherichia coli and Agrobacterium.

3. A method for breeding wheat varieties resistant to stem base rot, comprising reducing the amount of wheat... TaHsfA1 Steps for determining gene expression levels and / or activity; The wheat variety possesses the following characteristics: increased resistance to stem rot compared to the recipient wild-type wheat; The reduction of wheat TaHsfA1 The step of adjusting gene expression levels and / or activity is to combine the gene expression level and / or activity described in claim 2 with the knockout step. TaHsfA1 Gene-related biological materials are introduced into wheat tissues or wheat cells.

4. The method according to claim 3, characterized in that: The importation method includes at least one of Ti plasmid, Ri plasmid, plant virus vector, microinjection, and electroporation.

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