Gene TaRBL1 and application
By editing the gene TaRBL1 in wheat and using CRISPR-Cas9 vector to inhibit its expression, the lack of resistance to stripe rust and gibberellosis in wheat varieties was solved, and efficient disease-resistant wheat varieties were created, which enhanced gibberellosis resistance and reduced stripe rust resistance.
Patent Information
- Application Number
- CN202510242781.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-03
- Publication Date
- 2025-07-11
AI Technical Summary
The resistance of existing wheat varieties to wheat stripe rust and wheat gibberellia is easily overcome, and the lack of broad-spectrum, long-lasting disease-resistant materials, and the yield of plaque-like mutant genes is low in crop breeding, which limits their application.
By editing the gene TaRBL1 in wheat, using CRISPR-Cas9 vector to inhibit its expression, reduce resistance to stripe rust and improve resistance to gibberellosis, the gene editing vector was constructed and plant juvenile embryos were transformed using Agrobacterium-mediated genetic transformation method.
Creating wheat varieties with better resistance and no impact on yields provides broad-spectrum, long-lasting disease-resistant materials, enhancing wheat's resistance to gibberellosis and reducing resistance to stripe rust.
Smart Images
Figure CN120290600A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of bio-agriculture, and particularly relates to the gene TaRBL1 and its application. Background Art
[0002] Wheat is the world's largest staple food crop and an important food source on which human life depends. Approximately 35% - 40% of the global population relies on wheat as the main food. Wheat diseases are important factors affecting wheat yield, and the prevention and control of wheat diseases are crucial. Wheat stripe rust and wheat scab are important fungal diseases threatening wheat production, and their prevention and control are particularly important. The virulence of stripe rust fungi frequently mutates, and the resistance of existing wheat varieties is easily overcome, leading to large outbreaks of stripe rust diseases. Therefore, creating broad-spectrum and durable disease-resistant materials is an important way to control wheat stripe rust; Wheat scab is another important fungal disease threatening wheat production. Due to its complex resistance and being controlled by multiple genes, there are currently no highly resistant varieties that can be popularized and applied in production. Therefore, creating broad-spectrum and durable disease-resistant materials is also an important way to control wheat scab.
[0003] Lesion mimic mutant materials can spontaneously produce hypersensitive response necrotic spots without pathogen infection and have broad-spectrum disease resistance, but their low yield limits the application of lesion mimic mutant genes in crop disease-resistant breeding. rbl1 is a lesion mimic mutant line screened from a rice mutant library with whole-genome sequencing, which has good resistance to Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae. Although the rbl1 lesion mimic mutant line has broad-spectrum disease resistance, its yield is extremely low. Its mutant gene RBL1 encodes a cytidine diphosphate-diacylglycerol synthase, and the RBL1 gene regulates programmed cell death and immunity by regulating the biosynthesis of phosphatidylinositol. The RBL1 gene is highly conserved in crops. The present invention obtains the gene TaRBL1 and further studies the role of this gene in the interaction with wheat stripe rust and wheat scab through genetic research means, aiming to provide disease-resistant materials for wheat stripe rust and wheat scab. Summary of the Invention
[0004] To solve the above technical problems, the present invention provides the gene TaRBL1 and its application.
[0005] On the one hand, the present invention provides a gene TaRBL1 (abbreviated as TaRBL1), the nucleotide sequence of the open reading frame of the gene TaRBL1 is shown in SEQ ID NO:1, the protein encoded by the gene TaRBL1 is protein TaRBL1, and the amino acid sequence of the protein TaRBL1 is shown in SEQ ID NO:2.
[0006] On the other hand, the present invention provides the application of the gene TaRBL1 in the cultivation of wheat varieties resistant to stripe rust.
[0007] Furthermore, in the application of the gene TaRBL1 in the cultivation of wheat varieties resistant to stripe rust, suppressing the expression of the gene TaRBL1 reduces the resistance of wheat to stripe rust.
[0008] On the other hand, the present invention provides the application of the gene TaRBL1 in the cultivation of wheat varieties resistant to Fusarium head blight.
[0009] Furthermore, in the application of the gene TaRBL1 in the cultivation of wheat varieties resistant to Fusarium head blight, suppressing the expression of the gene TaRBL1 enhances the resistance of wheat to Fusarium head blight.
[0010] Furthermore, suppressing the expression of the gene TaRBL1 is achieved by introducing a gene editing tool into the plant, and the gene editing tool is a CRISPR-Cas9 vector.
[0011] In addition, the present invention also provides a method for cultivating wheat varieties resistant to stripe rust, the method comprising editing the gene TaRBL1 in a plant to obtain a plant variety with edited TaRBL1 gene.
[0012] Furthermore, the method for cultivating wheat varieties resistant to stripe rust comprises constructing a gene editing CRISPR-Cas9 vector for the gene TaRBL1, and transforming the immature embryos of the plant by means of Agrobacterium-mediated genetic transformation using the vector to obtain a plant variety with edited TaRBL1 gene.
[0013] Finally, the present invention also provides a method for cultivating wheat varieties resistant to Fusarium head blight, the method comprising gene editing the gene TaRBL1 in a plant to obtain a plant variety with edited TaRBL1 gene.
[0014] Furthermore, the method for cultivating wheat varieties resistant to Fusarium head blight comprises constructing a gene editing CRISPR-Cas9 vector for the gene TaRBL1, and transforming the immature embryos of the plant by means of Agrobacterium-mediated genetic transformation using the vector to obtain a plant variety with edited TaRBL1 gene.
[0015] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:
[0016] The present invention obtains the gene TaRBL1 and the protein TaRBL1 encoded thereby from the rbl1 lesion mimic mutant line. The amino acid sequence of the protein is shown in SEQ ID NO:2. Through reverse genetics research, the present invention discovers that the gene TaRBL1 plays a positive regulatory role in the interaction with wheat stripe rust and a negative regulatory role in the interaction with wheat head blight. Editing the three alleles of the gene TaRBL1 in wheat cells reduces wheat resistance to stripe rust and increases wheat resistance to head blight. The present invention provides more biological resources for creating wheat varieties resistant to stripe rust and wheat varieties resistant to head blight. Description of the Drawings
[0017] Figure 1 It is the electrophoresis detection result of the positive plants of the T0 generation of the TaRBL1-edited wheat plants. Lane 1 is the 2000bp DNA marker; Lane 1 is the second individual plant of the T0 generation of the TaRBL1-edited wheat plants; Lane 2 is the 19th individual plant of the T0 generation of the TaRBL1-edited wheat plants.
[0018] Figure 2A It is the genotype analysis result of the unedited plants.
[0019] Figure 2B It is the genotype analysis result of the TaRBL1-edited wheat plant TaRBL1-T1-2.
[0020] Figure 2C It is the genotype analysis result of the TaRBL1-edited wheat plant TaRBL1-T1-19.
[0021] Figure 3 It is the test result of the resistance of the TaRBL1-edited wheat plants to stripe rust.
[0022] Figure 4 It is the test result of the resistance of the TaRBL1-edited wheat plants to head blight. Detailed Embodiments
[0023] Next, the technical solutions of the present invention will be described in conjunction with the embodiments. However, the present invention is not limited to the following embodiments.
[0024] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings. However, the embodiments cited are not intended to limit the present invention.
[0025] In the following embodiments, the experimental methods and detection methods are all conventional methods unless otherwise specified; the reagents and materials can all be obtained in the market unless otherwise specified.
[0026] Example 1
[0027] This example is to obtain the gene TaRBL1.
[0028] Given the good stable yield and disease resistance performance of RBL in rice lines, in this example, homologous genes of RBL were searched for in wheat, and the gene TaRBL1 was obtained. The nucleotide sequence of the open reading frame of the gene TaRBL1 is shown in SEQ ID NO:1, and the protein encoded by it is protein TaRBL1. The amino acid sequence of the protein TaRBL1 is shown in SEQ ID NO:2.
[0029] Example 2
[0030] This example is to create gene TaRBL1-edited wheat plants.
[0031] (1) Construction of CRISPR-Cas9 vector
[0032] The three alleles of the gene TaRBL1 in wheat, TaRBL-3A, TaRBL-3B, and TaRBL-3D, all have complete open reading frames. In order to knockout TaRBL-3A, TaRBL-3B, and TaRBL-3D, the target sites of sgRNA were designed in the conserved regions of TaRBL-3A, TaRBL-3B, and TaRBL-3D. A total of two sgRNAs were designed, and the sequence information of the sgRNAs is as follows:
[0033] sgRNA1: 5’-TGGGAATAGAGCCAATGGGACGG-3’,
[0034] sgRNA2: 5’-CCTACTCAGAAGATCCAGCGAAG-3’.
[0035] sgRNA1 and sgRNA2 were tandemly connected to the vector VK005 to construct the TaRBL1-VK005-gRNA1-sgRNA2 vector. The specific process of constructing the TaRBL1-VK005-gRNA1-sgRNA2 vector was carried out according to the instructions of the plant Cas9 / gRNA plasmid construction kit (Beijing Weishang Lide Biotechnology). The recombinant plasmid was transformed into Escherichia coli, and then cultured on an LB(kan) plate until colonies grew. Single colonies were picked for sequencing. The sequencing results detected the target sequence gRNA of sgRNA1-sgRNA2, and at the same time, the TaU3 promoter sequence was detected upstream of the target sequence. The sequencing results indicated that the TaRBL1-VK005-gRNA1-sgRNA2 expression cassette was successfully constructed and successfully assembled into the VK005 expression vector, proving that the CRISPR / Cas9 gene editing vector of the gene TaRBL1 was successfully constructed.
[0036] (2) Creation of gene TaRBL1-edited wheat varieties
[0037] The TaRBL1-VK005-gRNA1-sgRNA2 vector was transferred into Agrobacterium tumefaciens EHA05, and immature embryos of Fielder wheat were transformed by Agrobacterium-mediated transformation. When leaves and roots differentiated from the callus, the seedlings were transplanted from the MS medium to the flower substrate, and acclimated in an incubator at 16°C with 16 h of light / 14°C with 8 h of darkness and a relative humidity of 70% for two weeks, and then transplanted to the greenhouse for cultivation. Thus, the TaRBL1-edited wheat variety, designated as the T0 generation of TaRBL1-edited wheat, was obtained.
[0038] (3) Identification of positive plants of TaRBL1-edited wheat plants
[0039] When the TaRBL1-edited wheat plants of the T0 generation grew to the one-leaf and one-heart stage, their leaf tissues were collected, genomic DNA was extracted, and primers were designed to perform PCR amplification on the target fragments of TaRBL-3A, TaRBL-3B, and TaRBL-3D, respectively.
[0040] The genomic DNA of TaRBL1-edited wheat plants was obtained by extraction using the CTAB method. The specific steps are as follows:
[0041] (a) The CTAB extraction solution was preheated in an oven at 65°C for standby, and isopropanol was pre-cooled in a -20°C refrigerator for standby. Approximately 2 cm of wheat leaf segments were taken from each individual TaRBL1-edited wheat plant and placed in a 2 mL centrifuge tube containing 2 steel beads. After quick-freezing in liquid nitrogen, it was broken by a shaking crusher for 2 min.
[0042] (b) After adding 850 μL of the preheated CTAB extraction solution, it was gently shaken to prevent DNA breakage, and incubated in a water bath at 65°C for 45 min (inverted and mixed up and down every 15 min).
[0043] (c) After the wheat cells were fully lysed, the sample was cooled to room temperature, an equal volume of phenol-chloroform was added, mixed for 2 min, and centrifuged at 12000 rpm at room temperature for 10 min.
[0044] (d) 650 μL of the supernatant was taken into a 1.5 mL centrifuge tube, 650 μL of chloroform was added, and centrifuged at 12000 rpm at room temperature for 1 min.
[0045] (e) 500 μL of the supernatant was taken into a 1.5 mL centrifuge tube, 500 μL of isopropanol and 50 μL of 3M sodium acetate were added, mixed well, and left standing in a -20°C refrigerator for more than 3 h. The precipitated white flocculent substance was DNA.
[0046] (f) Centrifuge at 12,000 rpm for 10 min at room temperature and discard the supernatant. Wash the DNA with 600 μL of 75% ethanol and absolute ethanol respectively, discard the supernatant, dry the DNA overnight at room temperature, add 50 μL of sterile water to dissolve the DNA and detect its concentration, and store it at -20 °C in the refrigerator for later use.
[0047] Using the genomic DNA of the T0 generation of the TaRBL1-edited wheat plants obtained above as a template, amplify the TaRBL-3A, TaRBL-3B, and TaRBL-3D genes with the designed detection primers. The sequences of the detection primers are as follows:
[0048] TaRBL1-ORF-F1: 5’-AAGGATACAGGCTCTGGTGA-3’,
[0049] TaRBL1-ORF-R1: 5’-ACGAACACAAAGCCT-3’.
[0050] TaRBL1-ORF-F2: 5’-AGGCTTTGTGTTCGT-3’,
[0051] TaRBL1-ORF-R2: 5’-GTGAAGAAGAAGTG-3’.
[0052] The electrophoresis pattern of the amplification products of the positive plants of the T0 generation of the TaRBL1-edited plants is as Figure 1 shown, Figure 1 In the figure, lane M is a 2000 bp DNA marker; lane 1 is the second individual plant of the T0 generation of the TaRBL1-edited wheat plants; lane 2 is the 19th individual plant of the T0 generation of the TaRBL1-edited wheat plants. The TaRBL1 gene fragment was detected in both lane 1 and lane 2, indicating that both the second individual plant (T0-2) and the 19th individual plant of the T0 generation of the TaRBL1-edited wheat plants are positive plants (T0-19) edited by the TaRBL1 gene.
[0053] Recover the amplification products, send them to Beijing Tsingke Biotechnology Co., Ltd. for sequencing, analyze the sequenced gene sequences and peak maps with DNAMAN and SnapGene, obtain two genotypes, both genotypes correspond to insertion mutants, and the plants corresponding to the above two genotypes are all positive plants of the T0 generation of the TaRBL1-edited wheat plants.
[0054] Example 3
[0055] This example is to prepare and identify the T1 generation of TaRBL1-edited plants.
[0056] Select the seeds of the T0-generation gene TaRBL1-edited wheat positive plants T0-2 and T0-19 obtained in Example 2 to prepare the T1-generation gene TaRBL1-edited plants. For the gene TaRBL1-edited wheat T1-generation plants TaRBL1-T1-2 (T1-2) and TaRBL1-T1-19 (T1-19) obtained by planting, use the positive plant identification method for the gene TaRBL1-edited wheat plants in Example 2 to identify positive plants. The identification results are as Figure 2A and Figure 2B 、 Figure 2C shown. The results show that there are differences in genotypes between TaRBL1-T1-2 and the unedited plants. There is an insertion of 1 bp of base T before the 6th base on sgRNA2 in TaRBL-T1-2, and the base insertion causes a frameshift mutation; there is an insertion of 1 bp of base T before the 7th base on sgRNA2 in TaRBL-T1-19, and the base insertion causes a frameshift mutation.
[0057] Example 4
[0058] This example is for the analysis of the resistance of gene TaRBL1-edited plants to wheat stripe rust.
[0059] Preparation of wheat stripe rust bacterial liquid: Put wheat stripe rust into the electronic fluorinated liquid to prepare a bacterial liquid with a certain concentration (1 spoonful of stripe rust bacteria added to 2 mL of electronic fluorinated liquid), and the wheat stripe rust bacterial liquid is obtained.
[0060] Analysis of the resistance of gene TaRBL1-edited plants to wheat stripe rust: Select the T1-generation gene TaRBL1-edited wheat positive plants T1-2 and T1-19 prepared in Example 3 and the control Fielder. When the above-mentioned wheat grows to the two-leaf and one-heart stage, draw a line mark on the second leaf of the wheat, inoculate the wheat stripe rust bacterial liquid on the second leaf of the wheat, spray water to keep it moist, and culture it in the dark for 24 h. During this period, spray water multiple times until the phenotype is collected 14 days after inoculation, and observe the disease incidence.
[0061] The results are as Figure 3 shown, indicating that gene TaRBL1 plays a role in the regulation of wheat against stripe rust, gene TaRBL1 plays a positive regulatory role in the interaction between wheat and stripe rust, and the resistance of gene TaRBL1-edited plants to wheat stripe rust is weakened.
[0062] Example 5
[0063] This example is for the analysis of the resistance of gene TaRBL1-edited plants to Fusarium head blight.
[0064] Preparation of sodium carboxymethyl cellulose medium (CMC medium): Weigh 15 g of carboxymethyl cellulose, 1 g of NH4NO3, 1 g of KH2PO4, 0.5 g of MgSO4·7H2O, and 1 g of yeast extract, dissolve them with distilled water, and make up the volume to 1 L with distilled water. Sterilize it by moist heat at 121 °C for 25 min.
[0065] Preparation of Gibberella fujikuroi spore suspension: Scratch the Gibberella fujikuroi mycelial cake with a pipette tip and put it into the CMC medium. Incubate at 25 °C and 170 rpm. After 3 days of culture, filter the spores with a filter cloth, discard the supernatant impurities. After centrifuging the lower layer at 3500 rpm for 8 min, discard the supernatant to obtain the spores. Resuspend the spores with clear water and count them with a hemocytometer. Dilute the spores to 2×10 5 cells / mL to obtain the Gibberella fujikuroi spore suspension.
[0066] Analysis of Fusarium head blight resistance of TaRBL1 gene-edited plants: Select the positive plants T1-2 and T1-19 identified in Example 3 and the control Fielder. When the above-mentioned wheat grows to the early flowering stage, select the wheat in the middle of the ear and inoculate 10 μL of Gibberella fujikuroi spore suspension. After inoculation, cover it with a bag and keep it moist for three days. Collect the phenotypes 14 days after inoculation and observe the disease incidence.
[0067] The results are as Figure 4 shown. Figure 4 On the left is the statistical result of the Fusarium head blight disease index of wheat. Figure 4 On the right are the agronomic traits of the positive plants T1-2, T1-19 and the control Fielder against Fusarium head blight of wheat. Among them, the agronomic traits of the Fielder group on the left and right are 2 replicates of the Fielder group, and both T1-2 and T1-19 are set with 2 replicates. The results show that the TaRBL1 gene is involved in the interaction between wheat and Fusarium graminearum, plays a negative regulatory role in the interaction between wheat and Fusarium head blight, and the TaRBL1 gene-edited plants have enhanced resistance to Fusarium head blight of wheat.
[0068] As described above, the present invention can be preferably implemented. The above embodiments are only used to describe the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the present invention.
Claims
1. A gene TaRBL1, characterized in that, The nucleotide sequence of the open reading frame of the gene TaRBL1 is shown in SEQ ID NO: 1; The protein encoded by the gene TaRBL1 is protein TaRBL1, and the amino acid sequence of the protein TaRBL1 is shown in SEQ ID NO:
2.
2. Use of the gene TaRBL1 described in claim 1 in the cultivation of wheat varieties resistant to stripe rust.
3. The application according to claim 2, characterized in that Inhibiting the expression of the gene TaRBL1 reduces the resistance of wheat to stripe rust.
4. Use of the gene TaRBL1 described in claim 1 in the cultivation of wheat varieties resistant to Fusarium head blight.
5. The application according to claim 4, wherein Inhibiting the expression of the gene TaRBL1 enhances the resistance of wheat to Fusarium head blight.
6. The application according to claim 3 or 5, characterized in that, Inhibiting the expression of the gene TaRBL1 is achieved by introducing a gene editing tool into the plant, and the gene editing tool is a CRISPR-Cas9 vector.
7. A method for cultivating wheat varieties resistant to stripe rust, characterized in that, Editing the gene TaRBL1 in the plant to obtain a plant variety with edited TaRBL1 gene.
8. The method for cultivating wheat varieties resistant to stripe rust according to claim 7, including constructing a gene editing CRISPR-Cas9 vector for the gene TaRBL1, and transforming the immature embryos of the plant by the method of Agrobacterium-mediated genetic transformation to obtain a plant variety with edited TaRBL1 gene.
9. A method for cultivating wheat varieties resistant to Fusarium head blight, characterized in that, Editing the gene TaRBL1 in the plant to obtain a plant variety with edited TaRBL1 gene.
10. The method for cultivating wheat varieties resistant to Fusarium head blight according to claim 9, including constructing a gene editing CRISPR-Cas9 vector for the gene TaRBL1, and transforming the immature embryos of the plant by the method of Agrobacterium-mediated genetic transformation to obtain a plant variety with edited TaRBL1 gene.