Application of rice Hd6 gene in enhancing broad-spectrum disease resistance of rice

Abstract

The invention discloses application of a rice Hd6 gene in enhancing broad-spectrum disease resistance of rice. The length of an open reading frame of the rice Hd6 gene is 1167bp, and the rice Hd6 gene encodes an alpha subunit of casein kinase CK2 composed of 388 amino acids. According to the invention, an open reading frame of the rice Hd6 gene is constructed on a pCXUN plasmid, and after agrobacterium tumefaciens EHA105 is transformed, an Hd6 overexpression plant under the background of a japonica rice variety Zhonghua 11 is obtained by means of an agrobacterium tumefaciens-mediated rice mature embryo transformation technology. According to the present invention, the wild type and the overexpressed plant are subjected to magnaporthe oryzae and Xanthomonas oryzae inoculation experiments respectively, and the results show that the resistance of the Hd6 overexpressed plant to magnaporthe oryzae and Xanthomonas oryzae infection is significantly improved, such that the Hd6 gene can positively regulate the broad-spectrum disease resistance of the rice, and can be used for the breeding of the disease-resistant variety of the rice.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of the rice Hd6 gene in enhancing the broad-spectrum disease resistance of rice. Background Art

[0002] Rice (Oryza sativa) is one of the important food crops in China. With the continuous and rapid development of agriculture in China, the current rice production and consumption in China account for about 30% of the world. However, there are still problems in most rice-growing areas in China, such as poor rice varieties, weak adaptability to local climate and soil, poor resistance, and low yield. In addition, due to the interference of various environmental factors, rice is vulnerable to pests and diseases, resulting in a large amount of economic losses every year due to pests, diseases and adverse environmental factors. Rice blast is a fungal disease caused by Magnaporthe oryzae; bacterial blight is a bacterial disease caused by Xanthomonas oryzae pv. oryzae (Xoo). Both of them seriously affect the rice yield in China and cause huge economic losses every year. Therefore, preventing and controlling rice blast and bacterial blight is of great significance for improving rice yield and ensuring food security.

[0003] Protein kinase-mediated substrate phosphorylation is a key post-translational protein modification mechanism, which is involved in regulating various biological processes. Casein kinase II (CK2) is a serine / threonine protein kinase tetramer involved in protein function regulation, which is widely present in eukaryotes and has a certain degree of conservation. CK2 is composed of two catalytic α subunits and two regulatory β subunits. Since the first plant CKα subunit was discovered in maize, the functions of CK2 have been successively identified in various plants, including Arabidopsis thaliana, rice, tobacco, and mustard, etc. Plant CK2 plays an important role in regulating various physiological processes such as light signal, hormone response, auxin signal, circadian rhythm, DNA double-strand break repair, cell cycle, and response to abiotic stress. There are 4 CK2α proteins (Hd6, OsCK2α3, OspCK2, OsCK2α2) and 2 CK2β proteins (OsCK2β1 and OsCK2β3) in rice. It is reported that the Hd6 gene in indica rice encodes a nuclear-localized CK2α subunit, which can delay the heading time of indica rice under long-day conditions, while the Hd6 gene in japonica rice has a premature termination due to a single-base mutation, resulting in the loss of function.

[0004] At present, there is no report on the function of the Hd6 gene in the rice immune response. The present invention finds through research that by using biotechnological means to increase the expression level of the rice Hd6 gene, the resistance of rice to Magnaporthe oryzae and Xanthomonas oryzae can be significantly enhanced. Summary of the Invention

[0005] The present invention obtained Hd6 overexpression plants through genetic transformation and discovered its application in the broad-spectrum disease resistance of rice. The overexpression materials of this gene can be used to breed varieties with broad-spectrum disease resistance.

[0006] To achieve the objectives of the present invention, the following technical solutions are adopted: The rice Hd6 gene, the nucleotide sequence of its open reading frame is shown in SEQ ID NO.1, and the amino acid sequence encoded by its open reading frame is shown in SEQ ID NO.2.

[0007] The application of the rice Hd6 gene in enhancing the broad-spectrum disease resistance of rice; the nucleotide sequence of the open reading frame of the Hd6 gene is shown in SEQ ID NO.1.

[0008] Furthermore, the broad-spectrum disease resistance of rice is enhanced by overexpressing the Hd6 gene in rice; The broad-spectrum disease resistance includes resistance to rice blast and bacterial blight.

[0009] A method for enhancing the broad-spectrum disease resistance of rice, which is to increase the expression level of the Hd6 gene in recipient rice to obtain transgenic rice with stronger broad-spectrum disease resistance than the recipient rice; the nucleotide sequence of the open reading frame of the Hd6 gene is shown in SEQ ID NO.1; The broad-spectrum disease resistance includes resistance to rice blast and bacterial blight.

[0010] The application of the Hd6 gene in cultivating transgenic rice with enhanced broad-spectrum disease resistance, the nucleotide sequence of the open reading frame of the Hd6 gene is shown in SEQ ID NO.1; Furthermore, increase the expression level of the Hd6 gene in recipient rice to obtain transgenic rice with stronger broad-spectrum disease resistance than the recipient rice; The broad-spectrum disease resistance includes resistance to rice blast and bacterial blight.

[0011] The present invention has the following beneficial effects: In the present invention, after the rice Hd6 gene is overexpressed, compared with the wild-type rice Zhonghua 11 (ZH11), the ability of Hd6 overexpression plants to resist rice blast and bacterial blight is significantly improved. The Hd6 gene can be used to breed or cultivate rice varieties with broad-spectrum disease resistance. Brief Description of the Drawings

[0012] Figure 1 It is the identification result of the Hd6 expression level in positive plants overexpressing the Hd6 gene. ZH11: Zhonghua 11, the wild-type rice material; OEHd6-1, OEHd6-2, and OEHd6-3 are rice lines overexpressing the Hd6 gene.

[0013] Figure 2Investigation results 7 days after inoculating Magnaporthe oryzae into holes of plants overexpressing the Hd6 gene. ZH11: Zhonghua 11, a wild-type rice material; OEHd6-1, OEHd6-2, and OEHd6-3 are rice lines overexpressing the Hd6 gene.

[0014] Figure 3 Investigation results 15 days after inoculating Xanthomonas oryzae pv. oryzae into plants overexpressing the Hd6 gene. ZH11: Zhonghua 11, a wild-type rice material; OEHd6-1, OEHd6-2, and OEHd6-3 are rice lines overexpressing the Hd6 gene. Specific implementation manners The present invention will be further described in detail below in conjunction with specific implementation manners.

[0016] Example 1: Obtaining of the Hd6 gene According to the predicted cDNA sequence (LOC_Os03g55389) of the Hd6 gene in the genome of japonica rice Nipponbare in the RGAP database, we successfully cloned the open reading frame of the Hd6 gene from the leaf cDNA of indica rice Kasalath using primers Hd6ORF-F and Hd6ORF-R. The full length is 1167 bp (SEQ ID NO.1). Sequencing found that there is a base difference between it and the predicted cDNA sequence of the Hd6 gene in japonica rice Nipponbare, proving that there is a single-base mutation in the Hd6 gene in japonica rice Nipponbare. The nucleotide sequences of the primers used to clone the open reading frame of the Hd6 gene of indica rice Kasalath are as follows: Hd6ORF-F (SEQ ID NO.3): 5’-ATGACCGATGCGCCTCCGC-3’, Hd6ORF-R (SEQ ID NO.4): 5’-TCATTGTGGTCGTGCTCTGC-3’ Example 2 Construction of the Hd6 overexpression vector The full-length open reading frame (SEQ ID NO.1) of the Hd6 gene of indica rice Kasalath was constructed onto the pCXUN plasmid by single enzyme digestion to obtain the pCXUN-Hd6 overexpression vector; the enzyme digestion site of the vector is XcmI.

[0017] (1) Primer design The nucleotide sequences of the primers for constructing the pCXUN-Hd6 overexpression vector are as follows (the primers are added with vector adapters and enzyme digestion sites for vector construction using the infusion system): OEHd6-F (SEQ ID NO.5): 5’-ATTACGCTCCAATACTTATGACCGATGCGCCTCCGC-3’, OEHd6-R (SEQ ID NO.6): 5’-TTCGGATCCCCAATACCTATTGTGGTCGTGCTCTGC-3’ (2) Amplify the target fragment with high-fidelity enzyme The PCR amplification system is as follows: The PCR amplification program is as follows: (3) Enzyme digestion and recovery of pCXUN vector Perform single enzyme digestion of pCXUN plasmid with XcmI. The enzyme digestion reaction system is as follows: Detect the bands by nucleic acid electrophoresis, and recover the enzyme digestion fragment with agarose gel.

[0018] (4) Perform homologous recombination of the target fragment and the vector in the infusion system Transform the 10 μL reaction product into Escherichia coli competent cells. Pick monoclonal colonies, shake the bacteria, sequence, and perform sequence alignment. If it is consistent with the nucleotide sequence of the full-length open reading frame of Hd6, the construction is successful.

[0019] Example 3: Identification of Hd6 overexpression plants The constructed pCXUN-Hd6 overexpression vector was used to infect and transform the callus of Zhonghua 11 (ZH11) through the genetic transformation method of callus induction mediated by Agrobacterium tumefaciens EHA105, and Hd6 overexpression plants were obtained. To identify positive plants, a pair of identification primers were designed on the overexpression vector and the target gene fragment, and the primer sequences were SEQ ID NO.7: 5’-TACCCATACGATGTTCCAGATT-3’ and SEQ ID NO.8: 5’-CAAGAAAATCTACAGCC TCA-3’, respectively. The genomic DNA of Hd6 overexpression plants was extracted as a template, and the genomic DNA of wild-type ZH11 was used as a control for PCR amplification. After amplification, the overexpression positive plants were identified by agarose gel electrophoresis (a 1090bp band was amplified for the overexpression positive plants, and no band was amplified for wild-type rice). The total RNA of wild-type and positive overexpression plants was extracted and reverse transcribed into cDNA, and then qRT-PCR experiments were carried out with cDNA as a template to identify the expression level of the Hd6 gene in positive plants. The primer sequences for identifying the expression level of the Hd6 gene were SEQ ID NO.9: 5’-AAGCTCTTGTTG GGAGGCATAG-3’ and SEQ ID NO.10: 5’-TTGTGGTCGTGCTCTGCTATT-3’, respectively. Three lines with significantly higher Hd6 gene expression levels than the wild-type were obtained and named OEHd6-1, OEHd6-2, and OEHd6-3, and the results are as Figure 1 shown.

[0020] Example 4: Identification of the resistance of Hd6 overexpression plants to Magnaporthe oryzae by punching inoculation Zhonghua 11 rice and Hd6 overexpression plants were planted in an artificial intelligence incubator. After 4 weeks of cultivation, an experiment of punching and inoculating Magnaporthe oryzae was carried out. Punch holes at a position 1 / 3 from the leaf tip with a small puncher, just leave wounds on the leaf surface (note not to break the leaf tissue), which is conducive to the infection of Magnaporthe oryzae. Take 10 μL of the spore suspension of Magnaporthe oryzae Guy11 (concentration about 2×10 5 cells / mL) and drop it on the wound, and then wrap the leaf at this place with transparent tape to form a small chamber. The inoculated rice was transferred to normal light conditions for continued cultivation, and the disease incidence was investigated 7-10 days later. The results are shown in Figure 2 . Compared with wild-type rice Zhonghua 11, the leaf lesion area of Hd6 overexpression plants was significantly smaller and the relative fungal biomass carried was less, indicating that the resistance of rice to Magnaporthe oryzae was significantly enhanced after the overexpression of the Hd6 gene.

[0021] Example 5: Identification of the resistance of Hd6 overexpression plants to Xanthomonas oryzae pv. oryzae Plant Zhonghua 11 rice and Hd6 overexpressing plants in the experimental rice field. When the rice grows naturally for 60 days, transfer and cultivate Xanthomonas oryzae pv. oryzae, adjust the concentration to OD 600 = 1.0. Immerse the scissors in the bacterial solution and horizontally cut some leaves at the 1 / 3 position of the leaf with the scissors. After 15 days, count the disease incidence, and count the lesion expansion length and the relative bacterial biomass ( Figure 3 ). We found that compared with the wild-type rice Zhonghua 11, the leaves of Hd6 overexpressing plants had a slower lesion expansion rate, shorter lesion length and less relative bacterial biomass, showing a more disease-resistant phenotype.

[0022] The above are only the preferred embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims

1. Hd6 The application of the gene in enhancing the broad-spectrum disease resistance of rice is characterized by: Said Hd6 The nucleotide sequence of the open reading frame of the gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: By overexpression in rice Hd6 Genes to enhance broad-spectrum disease resistance in rice.

3. The use according to claim 1, characterized in that: The broad-spectrum disease resistance includes resistance to rice blast and resistance to bacterial blight.

4. A method for enhancing broad-spectrum disease resistance of rice, characterized in that: Increase the recipient rice Hd6 The expression level of the gene is increased to obtain transgenic rice with a broad-spectrum disease resistance stronger than that of the recipient rice; Hd6 The nucleotide sequence of the open reading frame of the gene is shown in SEQ ID NO.

1.

5. The method according to claim 4, characterized in that: The broad-spectrum disease resistance includes resistance to rice blast and resistance to bacterial blight.

6. Hd6 The application of the gene in breeding transgenic rice with enhanced broad-spectrum disease resistance is characterized by: Said Hd6 The nucleotide sequence of the open reading frame of the gene is shown in SEQ ID NO.

1.

7. The use according to claim 6, characterized in that: Increase the recipient rice Hd6 The expression level of the gene is increased to obtain transgenic rice with a broad-spectrum disease resistance that is stronger than that of the recipient rice.

8. The use according to claim 6, characterized in that: The broad-spectrum disease resistance includes resistance to rice blast and resistance to bacterial blight.

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