Hongyoumai TaRGA4 gene as well as encoding protein and application thereof

Abstract

The invention belongs to the technical field of biological agriculture, and particularly relates to Hongyoumai TaRGA4 gene as well as a coding protein and application thereof. According to the Hongyoumai TaRGA4 gene and the application thereof, the full length of a Hongyoumai TaRGA4 gene coding region is obtained from a local powdery mildew high-resistance variety Hongyoumai for the first time, the nucleotide sequence of the TaRGA4 gene coding region is disclosed, the sequence is shown as SEQ ID NO: 2, the sequence of Hongyoumai TaRGA4 protein is shown as SEQ ID NO: 3, the gene and protein functions are further researched through a gene silencing technology, and the application of the gene and the protein is developed. The gene and the protein have the function of regulating and controlling the wheat powdery mildew resistance and can be applied to improvement of the wheat powdery mildew resistance, and a new biological resource is provided for prevention and treatment of plant powdery mildew.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological agriculture, and in particular relates to a TaRGA4 gene of Triticum aestivum and its encoded protein and application. Background Art

[0002] Wheat powdery mildew (Blumeria graminis f.sp. tritici) is a major disease in wheat production. Currently, chemical control remains the primary method for its prevention and treatment. However, long-term use of chemical pesticides has led to increased resistance in the pathogen, and the presence of pesticide residues poses environmental and food safety concerns. Therefore, identifying and utilizing wheat's own disease-resistance genes and cultivating disease-resistant varieties are key strategies for sustainable agricultural development.

[0003] Several wheat powdery mildew resistance genes (such as Pm3, Pm8, and Pm21) have been identified, but some of these genes gradually lose resistance due to pathogen race variation. Furthermore, some resistance genes may be associated with adverse agronomic traits such as yield reduction during breeding. Therefore, the discovery of new broad-spectrum, durable resistance genes, especially superior resistance genes from local varieties, is crucial for wheat disease resistance breeding.

[0004] Hongqie wheat is a local wheat variety in Henan Province. Long-term indoor resistance identification shows that it is highly resistant to powdery mildew and has a stable disease-resistant phenotype. However, there have been no reports on disease-resistant genes in Hongqie wheat varieties. Current research mainly focuses on Hongqie wheat molecular markers. For example, Chinese patent CN105112416A discloses a molecular marker that is closely linked to the powdery mildew resistance gene of Hongqie wheat. The disease-resistant wheat Hongqie wheat and the susceptible wheat Huixianhong were hybridized to construct F1, F2 and F3 family groups, and the disease-resistant gene was located on chromosome 7BL through resistance identification, molecular marker technology and physical positioning. Chinese patent CN117844967A discloses a KASP marker that is co-segregated with the wheat powdery mildew resistance gene pmHYM. The disease-resistant wheat Hongqie wheat and the susceptible wheat Ningmaizi 119 were hybridized to construct F1, F2 and F3 family groups. 2:3 Through chromosome sorting and sequencing of a family population, candidate genes for pmHYM were identified, and the functional SNP was used to develop the KASP marker Xmp1394. While molecular markers have accelerated breeding compared to traditional breeding, the process is complex, time-consuming, and labor-intensive, requiring significant human and financial investment. Therefore, exploring wheat disease-resistant proteins and genes and developing new disease-resistant materials using genetic engineering techniques is the most direct, effective, and economical approach to controlling wheat powdery mildew. Summary of the Invention

[0005] Based on the above technical problems, the present invention cloned the full-length coding region of the TaRGA4 gene of red wheat for the first time from the local variety red wheat with high resistance to powdery mildew, obtained the red wheat TaRGA4 protein, and used genetic technology to conduct in-depth research on the protein and gene function. It was found that the gene and protein are key biological molecules for regulating wheat powdery mildew resistance, which can be used to regulate wheat powdery mildew resistance and to improve wheat varieties resistant to powdery mildew.

[0006] On the one hand, the present invention provides a TaRGA4 gene of the red wheat, and the nucleotide sequence of the coding region of the TaRGA4 gene of the red wheat is shown in SEQ ID NO: 2.

[0007] In a second aspect, the present invention provides a TaRGA4 protein from Rhizoctonia ovata, wherein the TaRGA4 protein is encoded by the TaRGA4 gene from Rhizoctonia ovata, and the amino acid sequence of the TaRGA4 protein from Rhizoctonia ovata is shown in SEQ ID NO:3.

[0008] In a third aspect, the present invention also provides a gene silencing fragment of the TaRGA4 gene of red wheat, wherein the gene silencing fragment comprises a gene silencing fragment 1 and a gene silencing fragment 2, the nucleotide sequence of the gene silencing fragment 1 is shown in SEQ ID NO: 5, and the nucleotide sequence of the gene silencing fragment 2 is shown in SEQ ID NO: 6.

[0009] In a fourth aspect, the present invention further provides a gene silencing vector comprising the nucleotide sequence shown in SEQ ID NO: 5 or the nucleotide sequence shown in SEQ ID NO: 6.

[0010] In a fifth aspect, the present invention also provides the use of the TaRGA4 gene of R. truncatum or the TaRGA4 protein of R. truncatum or the gene silencing fragment or the gene silencing vector in regulating the resistance of R. truncatum to wheat powdery mildew.

[0011] In a sixth aspect, the present invention also provides a method for cultivating wheat varieties susceptible to powdery mildew, silencing the TaRGA4 gene of red wheat to reduce the resistance of wheat to powdery mildew.

[0012] In a seventh aspect, the present invention also provides a method for cultivating wheat varieties susceptible to powdery mildew, reducing the expression or biological activity of TaRGA4 protein in red wheat, and reducing the resistance of wheat to powdery mildew.

[0013] In an eighth aspect, the present invention provides a method for cultivating wheat varieties susceptible to powdery mildew, wherein the gene silencing vector of the present invention is introduced into wheat to reduce the wheat's resistance to powdery mildew. Furthermore, in the cultivation method, the gene silencing vector is transformed into wheat cells using BSMV.

[0014] Finally, the present invention also provides a method for improving wheat powdery mildew resistance, comprising introducing the TaRGA4 protein of the present invention or the TaRGA4 gene of the present invention into wheat.

[0015] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0016] (1) Compared with traditional disease-resistant breeding technology, plant disease-resistant genetic engineering technology can break through the reproductive isolation and incompatibility of distant hybridization between species, achieve targeted improvement of target traits in a shorter period of time, and provide more comprehensive, continuous, and broad-spectrum protection for crops. The present invention discovered a new wheat disease-resistant gene, the TaRGA4 gene, through gene screening research on a wheat variety highly resistant to powdery mildew, the TaRGA4 gene. Further functional research on the gene revealed that the TaRGA4 gene plays a positive regulatory role in the defense response of wheat to powdery mildew invasion, that is, silencing the TaRGA4 gene reduces the ability of wheat to resist powdery mildew. Reducing the expression of the TaRGA4 gene in plants significantly reduces wheat's resistance to powdery mildew.

[0017] (2) The present invention provides a method for cultivating wheat varieties susceptible to powdery mildew. This method uses RNA interference technology to transiently silence the TaRGA4 gene in wheat plants, significantly reducing wheat resistance to powdery mildew. The present invention provides new genetic resources and technical ideas for regulating wheat resistance to powdery mildew from a molecular biological perspective. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the electrophoresis result of the TaRGA4 gene of the red scorpion. Lane M is the marker DL6000; lane 1 is the TaRGA4 gene fragment of the red scorpion.

[0019] Figure 2 The amino acid sequence alignment results for the TaRGA4 protein from red scorpion wheat and the TaRGA4 protein from Huixian red wheat are shown in Figure 1. HYM-RGA4.seq is the amino acid sequence of the TaRGA4 protein from red scorpion wheat; HXH-RGA4.seq is the amino acid sequence of the TaRGA4 protein from Huixian red wheat; consensus is the alignment result.

[0020] Figure 3 The expression levels of the TaRGA4 gene in TaRGA4-silenced plants at different time points are shown in Figure 2. BSMV-γ is the control; BSMV:TaRGA4-1as is the BSMV:TaRGA4-1as-silenced plant; and BSMV:TaRGA4-2as is the BSMV:TaRGA4-2as-silenced plant.

[0021] Figure 4 This is a diagram of the agronomic traits of red wheat TaRGA4 gene silenced plants. Figure 4 Middle A is the agronomic traits of TaRGA4 gene-silenced plants before inoculation with powdery mildew; MOCK (blank control); BSMV-TaPDS is the plant transformed with BSMV: α+β+PDS; BSMV-γ is the plant transformed with BSMV: α+β+γ; TaRGA4-1as is the plant transformed with BSMV: α+β+TaXXX-1as, and TaRGA4-1as is the plant transformed with BSMV: α+β+TaXXX-2as. Figure 4 Figure B shows the agronomic characteristics of powdery mildew resistance in TaRGA4 gene-silenced plants. Mock is the red wheat control inoculated with powdery mildew; BSMV-γ is the empty transgenic control inoculated with powdery mildew; TaRGA4-1as is the gene-silenced plant Ta-GRA4-1as inoculated with powdery mildew; and TaRGA4-2as is the gene-silenced plant Ta-GRA4-2as inoculated with powdery mildew. DETAILED DESCRIPTION

[0022] The technical solutions of the present invention are described below with reference to the following embodiments; however, the present invention is not limited to the following embodiments.

[0023] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below with reference to specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0024] The experimental methods and detection methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0025] Example 1

[0026] This example is to obtain the TaRGA4 gene and protein of Rhizoma rubripes.

[0027] Based on the sequence of the disease-resistant transcript Wheat_Chr_Trans_newGene_16173 (SEQ ID NO: 17) obtained earlier in the inventors' laboratory, specific primers W33-F and W33-R:5 were designed and synthesized. Using red sedge wheat cDNA as a template, the target gene was cloned by PCR. The reaction system and procedure were as follows: 20 μL of 2×P515 enzyme, 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), 3 μL of template, and sterile ddH2O was added to the reaction system to a total of 40 μL. The reaction procedure was as follows: 95°C for 10 min; 30 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 30 s / kb; and 72°C for 10 min. The target gene obtained by PCR was detected by 1% agarose gel electrophoresis. The target gene fragments with the expected size were recovered according to the instructions of the Magen gel recovery kit (HiPure Gel Pure DNA Mini Kit). The recovered target fragments were stored at -20°C for future use.

[0028] The target fragment recovered above was connected to the T vector. The connection reaction system is as follows: 1 μL of 5×TA / Blunt-ZeroCloning Mix, 1-4 μL of PCR purified product, and 5 μL of ddH2O. Flick the bottom of the tube to mix, centrifuge at low speed to collect all the liquid at the bottom of the centrifuge tube, and react at room temperature (20℃~37℃) for 5 minutes. After the reaction is completed, place the centrifuge tube on ice to obtain the target fragment T vector connection product, and transform the target fragment T vector connection product into DH5α competent cells. Colony PCR was used to identify the positive strain carrying the target fragment, and the results were as follows: Figure 1 The identification primer sequences for the PCR identification are W33-M13F and W33-M13R. The W33-M13F and W33-M13R primer sequences and the primer sequence information involved below are shown in Table 1.

[0029] Table 1 Primer sequence information

[0030]

[0031] The identification results showed that the size of the PCR product was between 6000bp and 4000bp, which was consistent with the expected size, and was determined to be a positive strain.

[0032] After extracting the plasmid from the above positive strains, the plasmids were sent to a biological company for sequencing to obtain the TaRGA4 gene sequence of the red wheat. The full length of the TaRGA4 gene of the red wheat is 4999 bp, and its nucleotide sequence is shown in SEQ ID NO: 1.

[0033] The nucleotide sequence shown in SEQ ID NO: 1 was analyzed using ORF Finder software to obtain the coding region nucleotide sequence of the TaRGA4 gene of the red sedge wheat. The coding region nucleotide sequence of the TaRGA4 gene of the red sedge wheat is shown in SEQ ID NO: 2. The amino acid sequence of the TaRGA4 protein of the red sedge wheat is shown in SEQ ID NO: 3.

[0034] Those skilled in the art can also prepare the TaRGA4 gene of the red sedge wheat as shown in SEQ ID NO: 2 and the TaRGA4 protein of the red sedge wheat as shown in SEQ ID NO: 3 through conventional technical means such as whole gene synthesis and amino acid sequence synthesis.

[0035] Example 2

[0036] Red sedge wheat exhibits high resistance to wheat powdery mildew, suggesting that it carries a powdery mildew-resistance gene located on wheat chromosome 7BL. However, detailed information on this gene has not been found. Example 1 of the present invention provides the full-length gene and the amino acid sequence of its encoded protein. To further characterize its function, the amino acid sequence of this protein was compared with the amino acid sequence of the TaRGA4 protein from Huixianhong, a wheat variety highly susceptible to powdery mildew. The amino acid sequence of the TaRGA4 protein from Huixianhong is shown in SEQ ID NO:4.

[0037] The comparison results are as follows Figure 2 As shown, the TaRGA4 protein sequence of the wheat powdery mildew-resistant variety Hongqimai and the wheat powdery mildew-susceptible variety Huixianhong has a homology of 93.3%, suggesting that the TaRGA4 protein of Hongqimai may be related to its high resistance to wheat powdery mildew.

[0038] Example 3

[0039] This example is to create TaRGA4 gene silenced plants of R. rubrum and determine their resistance to wheat powdery mildew.

[0040] 3.1 Construction of gene silencing vector

[0041] Two specific fragments were selected from the TaRGA4 gene of the red wheat as shown in SEQ ID NO:1, and were named TaRGA4-1as and TaRGA4-2as. The nucleotide sequence of TaRGA4-1as is shown in SEQ ID NO:5, and the nucleotide sequence of TaRGA4-2as is shown in SEQ ID NO:6. Primers TaRGA4-1as-F, TaRGA4-1as-R and TaRGA4-2as-F, TaRGA4-2as-R with BSMV vector homology arms were designed. The two specific fragments were amplified using a high-fidelity enzyme, and the above two specific fragments were ligated to the BSMV:γ vector using homologous recombination technology to obtain BSMV:TaXXX-1as and BSMV:TaXXX-2as recombinant plasmids.

[0042] 3.2 BSMV vector linearization

[0043] The recombinant viral vectors BSMV:α, BSMV:β, BSMV:γ, BSMV:TaXXX-1as, BSMV:TaXXX-2as, and BSMV:PDS were subjected to enzyme digestion and linearization treatment. After the enzyme digestion reaction, the undigested vector was used as a control and agarose electrophoresis was performed to detect whether the vector was linear. The linearized vectors were stored at -20°C for future use.

[0044] 3.3 In vitro transcription of BSMV vector linearization product

[0045] Use an in vitro transcription kit to in vitro transcribe the linearized plasmid prepared in 3.2.

[0046] Add the following reagents to an RNAase-free PCR tube in the following order: 2 μL of T7 Transcription 5× Buffer, 0.5 μL of RNAase Inhibitor, 0.5 μL of rATP, 0.5 μL of rCTP, 0.5 μL of rGTP, 0.5 μL of rUTP, 0.5 μL of Ribo m7GCap Analog, 1 μL of Enzyme Mix (T7), and 4 μL of linearized plasmid. Gently mix and centrifuge briefly. Incubate the PCR reaction at 37°C for 1 hour. After the reaction, aspirate 1 μL of the PCR product and add 5 μL of RNAase-free water. Detect the in vitro transcription product by agarose gel electrophoresis. The presence of a bright band indicates successful in vitro transcription. Store the α, β, γ, PDS, TaXXX-1as, and TaXXX-2as transcripts at -80°C until ready for use.

[0047] 3.4 Creation of TaRGA4 gene-silenced plants by inoculation with BSMV virus

[0048] Barley streak mosaic virus (BSMV) was inoculated into plants using the friction inoculation method, and PDS was used as an indicator control.

[0049] (1) Select wheat seeds with full grains and plant 15 seeds in each flower pot. Inoculate the seeds when the wheat has two leaves and one heart.

[0050] (2) Combine the in vitro transcription products of α, β, γ, PDS, TaXXX-1as, and TaXXX-2as in 3.3 to form different types of treatments. Control group: MOCK (blank control), BSMV: α+β+PDS (BSMV-TaPDS), BSMV: α+β+γ (BSMV-γ); Experimental group: BSMV: α+β+TaXXX-1as (TaRGA4-1as), BSMV: α+β+TaXXX-2as (TaRGA4-1as); Mix each combination product with 800 μL of FES buffer, gently pipette to mix, and use for inoculation.

[0051] (3) Inoculate 5 wheat pots for each combination. Wear rubber gloves during inoculation and rub the mixture on the second leaf of the wheat. After inoculation, place the wheat in a 25°C artificial climate chamber, protect from light and keep moist overnight, and then culture under normal light / dark alternation.

[0052] (4) After about 10 to 13 days of exposure, observe closely whether the poison has developed. Figure 4 As shown in middle A, the leaves of the blank control MOCK showed no abnormalities, while the leaves of the indicator control BSMV-TaPDS showed photobleaching, indicating that the poisoning occurred and the gene silencing was successful. The inoculation with BSMV:α+β+TaXXX-1as and BSMV:α+β+TaXXX-2as was successful, and the TaRGA4 gene-silenced plants BSMV:TaRGA4-1as and BSMV:TaRGA4-2as of red wheat were successfully constructed.

[0053] 3.5 Testing of resistance of TaRGA4 gene-silenced plants to wheat powdery mildew

[0054] Ten days after inoculation with BSMV, the gene-silenced plants (BSMV:TaRGA4-1as, BSMV:TaRGA4-2as, and control plants) obtained in 3.4 were inoculated with E. graminis E09. Disease development was assessed 10-15 days after inoculation. Leaves free of E. graminis E09 were removed prior to inoculation, and leaves showing E. graminis E09 were inoculated.

[0055] (1) Use a marker pen to mark the third and fourth leaves of the poisonous plants to ensure that the inoculation position can be clearly identified to facilitate subsequent sampling and phenotypic observation.

[0056] (2) Real-time fluorescence quantitative and histological samples were collected at different time points (0 h, 24 h, 48 h, 72 h, and 96 h) after inoculation. The real-time fluorescence quantitative samples were quickly frozen in liquid nitrogen. Total RNA was extracted from the real-time fluorescence quantitative samples and reverse transcribed. The primer pairs TaRGA4-quantitative-F and TaRGA4-quantitative-R were used to detect the content of the TaRGA4 gene. Real-time fluorescence quantitative PCR was performed. All experiments were carried out according to the corresponding kit instructions.

[0057] (3) Observe the control group for full disease development 7 days after inoculation. After full disease development, observe the phenotype of the silent plants and take photos.

[0058] Table 2 4-grade grading standard for wheat powdery mildew

[0059]

[0060] The results of fluorescence quantitative PCR detection of TaRGA4 gene in BSMV:TaRGA4-1as and BSMV:TaRGA4-2as silenced plants at different time points after inoculation with wheat powdery mildew are shown in the figure. Figure 3 As shown, the results showed that compared with the control BSMV:γ, the expression of the TaRGA4 gene in the silenced plants BSMV:TaRGA4-1as and BSMV:TaRGA4-2as was significantly reduced at 0h-96h.

[0061] Agronomic traits of resistance of silenced plants BSMV:TaRGA4-1as and BSMV:TaRGA4-2as to wheat powdery mildew Figure 4 The four-grade wheat powdery mildew grading standard is shown in Table 2, as shown in Figure B. The results show that after silencing the TaRGA4 gene in red wheat, the resistance phenotype of red wheat to wheat powdery mildew changed from highly resistant to highly susceptible. This indicates that the TaRGA4 gene plays an important role in red wheat's resistance to wheat powdery mildew.

[0062] As described above, the basic principles, main features and advantages of the present invention are well described. The above embodiments and descriptions are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the scope of protection determined by the present invention.

Claims

1. The TaRGA4 gene of red wheat is characterized by: The nucleotide sequence of the TaRGA4 gene coding region of the red wheat is shown in SEQ ID NO:

2.

2. TaRGA4 protein from red wheat, characterized in that The red sedge TaRGA4 protein is encoded by the red sedge TaRGA4 gene according to claim 1, and the amino acid sequence of the red sedge TaRGA4 protein is shown in SEQ ID NO:

3.

3. A gene silencing fragment of the TaRGA4 gene of red wheat, characterized in that: The gene silencing fragment comprises a gene silencing fragment 1 and a gene silencing fragment 2. The nucleotide sequence of the gene silencing fragment 1 is shown in SEQ ID NO: 5, and the nucleotide sequence of the gene silencing fragment 2 is shown in SEQ ID NO:

6.

4. A gene silencing vector, characterized in that: The gene silencing vector contains the nucleotide sequence shown in SEQ ID NO: 5 or the nucleotide sequence shown in SEQ ID NO: 6 according to claim 3.

5. Use of the TaRGA4 gene of red sedge wheat according to claim 1, the TaRGA4 protein of red sedge wheat according to claim 2, the gene silencing fragment of claim 3, or the gene silencing vector of claim 4 in regulating resistance of red sedge wheat to wheat powdery mildew.

6. A method for cultivating wheat varieties susceptible to powdery mildew, characterized in that: The TaRGA4 gene of red wheat is silenced in the wheat, thereby reducing the resistance of wheat to powdery mildew.

7. A method for cultivating wheat varieties susceptible to powdery mildew, characterized in that: Reducing the expression or biological activity of TaRGA4 protein in red wheat reduces wheat resistance to powdery mildew.

8. A method for cultivating wheat varieties susceptible to powdery mildew, characterized in that: The gene silencing vector according to claim 4 is introduced into the wheat to reduce the resistance of the wheat to powdery mildew.

9. The cultivation method according to claim 8, characterized in that The gene silencing vector was transformed into wheat cells via BSMV.

10. A method for improving wheat powdery mildew resistance, characterized in that: The method comprises transferring the TaRGA4 gene of claim 1 or the TaRGA4 protein of claim 2 into wheat.

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