Application of TaRpst9 gene knockout mutant in wheat stripe rust resistance

Abstract

The invention belongs to the technical field of genetic engineering and discloses TaRpst9 Application of gene knockout mutants in wheat stripe rust resistance. The present invention uses a reverse genetics method to construct a gene knockout mutant. TaRpst9 CRISPR / Cas9 gene editing vector of the gene was used to modify the gene in wheat materials through Agrobacterium-mediated method. TaRpst9 The gene was successfully knocked out, and we learned TaRpst9 The gene plays a positive regulatory role in the immune response of wheat to stripe rust. TaRpst9 Genes to improve wheat resistance to stripe rust. TaRpst9 The influence of genes on wheat disease resistance can be used to create wheat germplasm materials resistant to stripe rust.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology and relates to TaRpst9 Application of gene knockout mutants in wheat stripe rust resistance. Background Art

[0002] During the long evolution process, plants have developed a unique immune system that can identify plant pathogens and stimulate plant immune responses, thereby resisting the infection of plant pathogenic fungi. It is currently believed that the plant immune system consists of two layers of immune systems: immunity triggered by pathogen-associated molecular patterns (Pattern-Triggered Immunity, PTI) and immunity triggered by effector proteins (Effector-Triggered Immunity, ETI).

[0003] Among them, PTI is an immune response triggered by pattern recognition receptors (PRRs) located on plant cell membranes that recognize microbial-pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). PRRs-mediated resistance has a broad spectrum and has the potential to promote broad-spectrum disease resistance in plants.

[0004] PRRs include receptor-like kinases (RLKs) and receptor-like proteins (RLPs). RLKs are important participants in the plant PTI immune system and play an important role in the immune regulation process. RLKs can bind to different molecules to identify different external environments. After signal recognition, they cause changes in their own conformation, and ultimately activate downstream signals, causing plants to produce disease-resistant components such as reactive oxygen and callose, and cause the expression of disease-resistant related genes, thereby achieving plant immune defense against external pathogenic microorganisms.

[0005] Wheat stripe rust caused by Puccinia striiformis f.sp.Tritici (Pst) is a major fungal disease in wheat production, which is characterized by wide distribution, rapid spread, and large damage area. The use of traditional chemical pesticides has aggravated environmental pollution and pathogen resistance, making the application of pesticides increasingly restricted. Based on this, the identification of RLKs in wheat materials and the study of the effect of RLKs encoding gene expression levels on wheat disease resistance will have important contributions to the breeding of wheat varieties resistant to stripe rust. Summary of the invention

[0006] On the one hand, the application of traditional pesticides is increasingly restricted; on the other hand, there is still a lack of research on the effect of regulating the expression of wheat receptor kinase genes on wheat disease resistance. Based on this, the present invention studies the effect of wheat receptor kinase gene expression levels on wheat disease resistance from the perspective of genetic engineering, which will have an important contribution value to the cultivation of disease-resistant varieties. Specifically, the present invention adopts the following technical solutions.

[0007] The present invention provides TaRpst9 Application of genes in wheat stripe rust resistance, the TaRpst9 The gene plays a positive regulatory role in wheat's immune response to stripe rust.

[0008] Further, in the above application, the TaRpst9 The gene is located on chromosome 7 of the wheat genome, and there are three copies on chromosome 7, named TaRpst9 -7A, TaRpst9 -7B and TaRpst9 -7D; TaRpst9 -7A nucleotide sequence is shown in SEQ ID NO: 1, TaRpst9 -7B nucleotide sequence is shown in SEQ ID NO: 2, TaRpst9 The nucleotide sequence of -7D is shown in SEQ ID NO:3.

[0009] Further, in the above application, knocking out the TaRpst9 -7A, TaRpst9 -7B and TaRpst9 -7D reduces the resistance of wheat materials to stripe rust.

[0010] The present invention also provides an application of a wheat receptor kinase in wheat stripe rust resistance, wherein the wheat receptor kinase is TaRpst9 Gene encoding; TaRpst9 The gene is located on chromosome 7 of the wheat genome, and there are three copies on chromosome 7, named TaRpst9 -7A, TaRpst9 -7B and TaRpst9 -7D; TaRpst9 -7A nucleotide sequence is shown in SEQ ID NO: 1, TaRpst9 -7B nucleotide sequence is shown in SEQ ID NO: 2, TaRpst9 The nucleotide sequence of -7D is shown in SEQ ID NO:3.

[0011] The present invention further provides a method for cultivating a wheat variety resistant to stripe rust, wherein the method is to overexpress TaRpst9gene; TaRpst9 The gene is located on chromosome 7 of the wheat genome, and there are three copies on chromosome 7, named TaRpst9 -7A, TaRpst9 -7B and TaRpst9 -7D; TaRpst9 -7A nucleotide sequence is shown in SEQ ID NO: 1, TaRpst9 -7B nucleotide sequence is shown in SEQ ID NO: 2, TaRpst9 The nucleotide sequence of -7D is shown in SEQ ID NO:3.

[0012] Compared with the prior art, the present invention TaRpst9 The application of gene knockout mutants in wheat stripe rust resistance has the following beneficial effects:

[0013] The present invention successfully constructed a CRISPR / Cas9 gene editing vector based on CRISPR / Cas9 gene editing technology and achieved TaRpst9 Gene frameshift mutation, to TaRpst9 Gene knockout purpose.

[0014] The present invention has obtained TaRpst9 Gene knockout wheat mutants L91 and L140 strains, wherein the L91 and L140 strains have reduced resistance to stripe rust, TaRpst9 The gene plays a positive regulatory role in wheat's immune response to stripe rust.

[0015] based on TaRpst9 The gene plays a positive regulatory role in wheat's immune response to stripe rust. TaRpst9 Genes are used to create wheat varieties resistant to stripe rust. The specific method is: overexpression in wheat materials TaRpst9 Gene, overexpression TaRpst9 Gene transfer methods are not limited to Agrobacterium-mediated method, gene gun method and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is T1 generation TaRpst9 Sequence alignment of Hi-TOM sequencing targets of gene knockout wheat mutants. “TaRpst9-target” indicates the target sequence, and “TaRpst9-7A”, “TaRpst9-7B”, and “TaRpst9-7D” indicate the wild-type Fielder wheat variety. TaRpst9 The sequences of the genes at the target sites of chromosomes 7A, 7B, and 7D; "TaRpst9-KO-91-7A", "TaRpst9-KO-91-7B", and "TaRpst9-KO-91-7D" represent the sequences of the genes at the target sites of chromosomes 7A, 7B, and 7D, respectively. TaRpst9 Gene knockout mutant strain L91; "TaRpst9-KO-140-7A", "TaRpst9-KO-140-7B", and "TaRpst9-KO-140-7D" represent the 7A, 7B, and 7D chromosomes, respectively. TaRpst9 Knockout mutant line L140.

[0017] Figure 2 It is T1 generation TaRpst9 Phenotypic results of knockout wheat mutants L91 and L140 lines inoculated with the avirulent physiological race CYR23 of stripe rust. "Fielder" means ordinary Fielder wheat plants; "RPst9-KO-91" means TaRpst9 Gene knockout wheat mutant L91 line; "RPst9-KO-140" means TaRpst9 Gene knockout wheat mutant L140 line.

[0018] Figure 3 It is T1 generation TaRpst9 Relative biomass statistics of gene knockout wheat mutants L91 and L140 lines inoculated with the avirulent physiological race CYR23 of stripe rust. "Fielder" means ordinary Fielder wheat plants; "RPst9-KO-91" means TaRpst9 Gene knockout wheat mutant L91 line; "RPst9-KO-140" means TaRpst9 Gene knockout wheat mutant L140 line. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] The primer sequences involved in the embodiments of the present invention are shown in Table 1.

[0021] Table 1, Primer sequences

[0022] .

[0023] Example 1

[0024] This example provides methods for targeted knockout TaRpst9 Construction of recombinant gene vectors.

[0025] The present invention TaRpst9The gene is located on chromosome 7 of the wheat genome. There are three copies of the gene on chromosome 7, named TaRpst9 -7A, TaRpst9 -7B and TaRpst9 -7D, the nucleotide sequences are shown in 1, 2, and 3 respectively. For targeted knockout of the wheat receptor kinase gene TaRpst9 The sequence of the sgRNA is shown in SEQ ID NO: 4, and the last three digits of the sequence are the PAM sequence GGG.

[0026] synthesis TaRpst9 The sgRNA sequence of the gene (Shaanxi Zhongke Chuang Biotechnology Co., Ltd.), in which the oligonucleotide pair of sgRNA directly anneals to form a double strand, the system is:

[0027] Rpst9 -gR1-F (10 μM): 1 μL, Rpst9 -gR1-R (10μM): 1μL, 1×TE: 5μL, ddH2O to make up to 10μL. Boil in boiling water for 5min, then cool naturally to room temperature.

[0028] The annealed product was connected to the intermediate vector after BtgZ1 digestion. The connection reaction system was:

[0029] 5×ligation Buffer: 2μL, sgRNA oligonucleotide pair: 4μL, intermediate vector after enzyme digestion: 2μL, T4ligase: 1μL, ddH2O to 10μL, carry out ligation reaction at 16℃ overnight.

[0030] The ligation product was transformed into Escherichia coli, and the recombinant plasmid was transformed into DH5α competent cells. After that, it was cultured on LB plates containing kanamycin until a monoclonal strain grew out. The strain was picked for bacterial liquid PCR identification and sequencing. After the sequencing results were correctly aligned, the plasmid was extracted from the bacteria.

[0031] The intermediate vector was connected to the final pCas9 using the LR reaction. The system is:

[0032] Intermediate vector: 2 μL, pCas9: 0.5 μL, LR enzyme: 0.5 μL, 1×TE to make up to 5 μL.

[0033] Then transform DH5α competent cells and culture on LB plates containing kanamycin until clones grow out. Pick a single clone for bacterial liquid PCR identification and sequencing. After the sequencing results are correct, shake the bacteria to extract the plasmid. TaRpst9 The CRISPR / Cas9 gene editing vector was successfully constructed.

[0034] Example 2

[0035] This embodiment provides TaRpst9 Breeding and identification of gene knockout wheat mutants.

[0036] The CRISPR / Cas9 gene editing vector was transferred into the Agrobacterium EHA105 strain, and the callus tissue of the Fielder wheat material was used as the transformation receptor. The CRISPR / Cas9 gene editing vector was introduced into the Fielder recipient wheat material by the Agrobacterium-mediated method, and T0 generation transgenic plants were produced through callus kanamycin resistance screening, pre-regeneration, regeneration, rooting and other steps. After the seedlings grew well, leaves were collected and DNA was extracted using the CTAB method, and then the extracted DNA was used as a template, and Cas9-F and Cas9-R were used as primers to detect whether the CRISPR / Cas9 gene editing vector was successfully transformed, and the plants that were successfully transformed were treated for additional generations.

[0037] TaRpst9 Whether the gene knockout is successful requires deep sequencing to determine. The detection fragment is amplified using the above-extracted DNA as a template and specific primers on both sides of the target sequence (Rpst9-gR1-F, Rpst9-gR1-R). The amplified product is sequenced by Hi-TOM (sequencing group of Hangzhou Rice Research Institute, Zhejiang Province). The results are as follows: Figure 1 shown. Figure 1 In the text, “TaRpst9-target” indicates the target sequence, and “TaRpst9-7A”, “TaRpst9-7B”, and “TaRpst9-7D” indicate the wild-type Fielder wheat variety. TaRpst9 The sequences of the genes at the target sites of chromosomes 7A, 7B, and 7D; "TaRpst9-KO-91-7A", "TaRpst9-KO-91-7B", and "TaRpst9-KO-91-7D" represent the sequences of the genes at the target sites of chromosomes 7A, 7B, and 7D, respectively. TaRpst9 Gene knockout mutant strain L91; "TaRpst9-KO-140-7A", "TaRpst9-KO-140-7B", and "TaRpst9-KO-140-7D" represent the 7A, 7B, and 7D chromosomes, respectively. TaRpst9 Gene knockout mutant line L140. Figure 1 The results showed that frameshift mutations occurred in the three copies of chromosomes 7A, 7B, and 7D in the two strains L91 and L140, proving that TaRpst9 The gene knockout was successful.

[0038] Example 3

[0039] This embodiment provides TaRpst9 Disease resistance experiment of gene knockout mutant wheat plants.

[0040] Will TaRpst9The knockout mutant wheat lines L91 and L140 were soaked and germinated and then sown in 15×15×12 cm pots. Wild-type Fielder wheat materials were sown at the same time as a control and inoculated with the non-toxic physiological race CYR23 of stripe rust. The seeds were kept in a 16℃ moisturizing box in the dark for 24 h and then transferred to a growth incubator with a photoperiod of 16℃ light for 16 h and 14℃ darkness for 8 h. The phenotypic changes of the experimental and control groups were observed after 14 days.

[0041] The experimental results are shown in Figure 2 "Fielder" refers to ordinary Fielder wheat plants; "RPst9-KO-91" refers to TaRpst9 Gene knockout wheat mutant L91 line; "RPst9-KO-140" means TaRpst9 Gene knockout wheat mutant L140 line. Figure 2 As shown in the figure, there are no obvious stripe rust spores on the leaves of wild-type Fielder wheat materials, while obvious spore piles appear on the leaves of L91 and L140 strains, indicating that the knockout TaRpst9 Gene, wheat resistance to stripe rust reduced, thus knowing TaRpst9 The gene plays a positive regulatory role in wheat's immune response to stripe rust.

[0042] Example 4

[0043] This example provides biomass detection of wheat materials inoculated with stripe rust.

[0044] Fielder wheat leaves and stripe rust summer spores not inoculated with stripe rust were collected, and DNA was extracted according to the method in Example 2. The extracted DNA concentration was determined, and the extracted DNA was diluted to the same concentration with sterilized ddH2O. Then, wheat-stripe rust interaction cDNA with different dilution gradients was used as a template, and the DNA was extracted using TaEF1α ( TaEF1α -F. TaEF1α -R), and PstEF ( PstEF -F. PstEF -R) were used as primers for qRT-PCR, and a standard curve of the biomass ratio of wheat and stripe rust was drawn.

[0045] The DNA of diseased leaves 14 days after inoculation was extracted according to the method in Example 2, and the DNA of diseased leaves was extracted using TaEF1α ,and PstEF qRT-PCR was performed with primers, and the biomass was calculated based on the standard curve to obtain the relative biomass of stripe rust relative to wheat. Figure 3 "Fielder" indicates ordinary Fielder wheat plants; "RPst9-KO-91" indicates TaRpst9Gene knockout wheat mutant L91 line; "RPst9-KO-140" means TaRpst9 Gene knockout wheat mutant L140 line. Figure 3 It can be seen that the biomass of L91 and L140 strains inoculated with the avirulent physiological race CYR23 of stripe rust was significantly higher than that of the wild-type Fielder wheat material, indicating that the knockout TaRpst9 The results show that the resistance of wheat to stripe rust is reduced, which further verifies the conclusion in Example 3.

[0046] The embodiments described above are only some embodiments of the present invention, not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. Application of knocking out TaRpst9 gene in reducing wheat resistance to stripe rust, characterized in that: The TaRpst9 gene is located on chromosome 7 of the wheat genome, and there are three copies on chromosome 7, namely TaRpst9-7A, TaRpst9-7B and TaRpst9-7D; The nucleotide sequence of TaRpst9-7A is shown in SEQ ID NO: 1; The nucleotide sequence of TaRpst9-7B is shown in SEQ ID NO: 2; The nucleotide sequence of TaRpst9-7D is shown in SEQ ID NO: 3; Knocking out TaRpst9-7A, TaRpst9-7B and TaRpst9-7D reduces wheat resistance to stripe rust.

2. The use of knocking out wheat receptor kinase in reducing wheat resistance to stripe rust, characterized in that: The wheat receptor kinase is encoded by the TaRpst9 gene, which is located on chromosome 7 of the wheat genome. There are three copies of the TaRpst9 gene on chromosome 7, namely TaRpst9-7A, TaRpst9-7B and TaRpst9-7D. The nucleotide sequence of TaRpst9-7A is shown in SEQ ID NO: 1; The nucleotide sequence of TaRpst9-7B is shown in SEQ ID NO: 2; The nucleotide sequence of TaRpst9-7D is shown in SEQ ID NO: 3; Knocking out TaRpst9-7A, TaRpst9-7B and TaRpst9-7D reduces wheat resistance to stripe rust.

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