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

Abstract

This invention discloses rice Hd6 Application of genes in enhancing broad-spectrum disease resistance in rice. The rice mentioned... Hd6 The gene has an open reading frame of 1167 bp, encoding an α subunit of casein kinase CK2 composed of 388 amino acids. This invention relates to the rice... Hd6 The open reading frame of the gene was constructed onto the pCXUN plasmid, transformed into Agrobacterium tumefaciens EHA105, and then obtained from the japonica rice variety Zhonghua 11 under the background of Agrobacterium-mediated transformation of mature rice embryos. Hd6 Overexpression plants. Inoculation experiments with *Bacillus oryzae* and *Bacillus oryzae* were conducted on wild-type and overexpression plants, respectively. The results showed that... Hd6 Overexpression significantly enhanced the resistance of plants to infection by rice blast fungus and bacterial blight fungus, indicating that... Hd6 Positive gene regulation of broad-spectrum disease resistance in rice can be applied to the breeding of disease-resistant rice varieties.

Description

TECHNICAL FIELD

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

[0002] Rice (Oryza sativa) is one of the important food crops in China. With the continuous and rapid development of agriculture in China, the rice yield and consumption in China accounts for about 30% of the world. However, there are still many problems in the rice planting areas in China, such as poor rice varieties, poor adaptability to local climate and soil, poor resistance, low yield and the like. In addition, due to the interference of various environmental factors, rice is easily attacked by diseases and pests, and a large amount of economic loss is caused by diseases and pests and adverse environmental factors every year. Rice blast is a fungal disease caused by Magnaporthe oryzae, and bacterial leaf 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 loss every year. Therefore, prevention and control of rice blast and bacterial leaf blight are of great significance to improve the rice yield and ensure food security.

[0003] Protein kinase-mediated phosphorylation of substrates is a key post-translational modification mechanism of proteins, which is involved in the regulation of various biological processes. Casein kinase II (CK2) is a kind of serine / threonine protein kinase tetramer involved in the regulation of protein function, which widely exists in eukaryotes and has a certain conservatism. CK2 is composed of two catalytic alpha subunits and two regulatory beta subunits. Since the first CK alpha subunit was found in maize, the functions of CK2 have been identified in various plants, including Arabidopsis, rice, tobacco and mustard. Plant CK2 plays an important role in regulating light signals, hormone responses, auxin signals, circadian rhythms, DNA double-strand break repair, cell cycle and response to abiotic stress. There are four CK2 alpha proteins (Hd6, OsCK2a3, OspCK2 and OsCK2a2) and two CK2 beta proteins (OsCK2b1 and OsCK2b3) in rice. It is reported that Hd6 gene in indica rice encodes a nuclear-localized CK2 alpha subunit, which can delay the heading time of indica rice under long-day conditions, while the Hd6 gene in japonica rice is lost of function due to single base mutation leading to premature termination.

[0004] At present, there is no report about the function of Hd6 gene in rice immune response. The present application found that the expression level of rice Hd6 gene can be significantly improved by using biotechnology, which can significantly enhance the resistance of rice to Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae (Xoo). SUMMARY

[0005] The application obtains Hd6 overexpression plants through a genetic transformation method, and finds the application of the plants in rice broad-spectrum disease resistance, and the overexpression material of the gene can be used for breeding varieties with broad-spectrum disease resistance.

[0006] To achieve the purpose of the present application, the present application adopts the following technical solutions:

[0007] The rice Hd6 gene, the nucleotide sequence of the open reading frame of which is shown as SEQ ID NO. 1, and the amino acid sequence encoded by the open reading frame of which is shown as SEQ ID NO. 2.

[0008] 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 as SEQ ID NO. 1.

[0009] Further, the broad-spectrum disease resistance of rice is enhanced by overexpressing the Hd6 gene in rice.

[0010] The broad-spectrum disease resistance includes resistance to rice blast and resistance to white blight.

[0011] A method for enhancing the broad-spectrum disease resistance of rice is to increase the expression amount of the Hd6 gene in a receptor rice, so as to obtain transgenic rice with stronger broad-spectrum disease resistance than the receptor rice; the nucleotide sequence of the open reading frame of the Hd6 gene is shown as SEQ ID NO. 1.

[0012] The broad-spectrum disease resistance includes resistance to rice blast and resistance to white blight.

[0013] 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 as SEQ ID NO. 1.

[0014] Further, the expression amount of the Hd6 gene in a receptor rice is increased, so as to obtain transgenic rice with stronger broad-spectrum disease resistance than the receptor rice.

[0015] The broad-spectrum disease resistance includes resistance to rice blast and resistance to white blight.

[0016] The present application has the following beneficial effects:

[0017] In the present application, after overexpression of the rice Hd6 gene, the ability of the Hd6 overexpression plant to resist rice blast and white blight is significantly improved compared with Zhonghua 11 (ZH11) wild type rice, and the Hd6 gene can be used for breeding or cultivating rice varieties with broad-spectrum disease resistance. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1The results of the identification of the expression level of Hd6 in the plants overexpressing the Hd6 gene. ZH11: wild-type rice material Zhonghua 11; OEHd6-1, OEHd6-2 and OEHd6-3 are rice lines overexpressing the Hd6 gene.

[0019] Figure 2 The results of the investigation of the plants overexpressing the Hd6 gene 7 days after being punched and inoculated with Magnaporthe grisea. ZH11: wild-type rice material Zhonghua 11; OEHd6-1, OEHd6-2 and OEHd6-3 are rice lines overexpressing the Hd6 gene.

[0020] Figure 3 The results of the investigation of the plants overexpressing the Hd6 gene 15 days after being inoculated with Xanthomonas oryzae. ZH11: wild-type rice material Zhonghua 11; OEHd6-1, OEHd6-2 and OEHd6-3 are rice lines overexpressing the Hd6 gene. DETAILED DESCRIPTION

[0021] The application will be described in further detail below with specific embodiments.

[0022] Example 1: Obtaining of the Hd6 gene

[0023] 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, with a full length of 1167 bp (SEQ ID NO. 1). Sequencing found that there was one base difference between the predicted cDNA sequence of the Hd6 gene in japonica rice Nipponbare, proving that there was a single base mutation in the Hd6 gene in japonica rice Nipponbare. The nucleotide sequence of the primers used to clone the open reading frame of the Hd6 gene in indica rice Kasalath is as follows:

[0024] Hd6ORF-F (SEQ ID NO. 3): 5'-ATGACCGATGCGCCTCCGC-3',

[0025] Hd6ORF-R (SEQ ID NO. 4): 5'-TCATTGTGGTCGTGCTCTGC-3'

[0026] Example 2: Construction of the Hd6 overexpression vector

[0027] The full-length open reading frame (SEQ ID NO. 1) of the Hd6 gene in indica rice Kasalath was constructed into the pCXUN plasmid using single enzyme digestion, obtaining the pCXUN-Hd6 overexpression vector; the enzyme digestion site of the vector is XcmI.

[0028] (1) Primer design

[0029] 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 by infusion system):

[0030] OEHd6-F (SEQ ID NO. 5):

[0031] 5'-ATTACGCTCCAATACTTATGACCGATGCGCCTCCGC-3',

[0032] OEHd6-R (SEQ ID NO. 6):

[0033] 5'-TTCGGATCCCCAATACCTATTGTGGTCGTGCTCTGC-3'

[0034] (2) High-fidelity enzyme amplification of target fragments

[0035] The PCR amplification system is as follows:

[0036]

[0037] The PCR amplification procedure is as follows:

[0038]

[0039] (3) Enzymatic digestion of pCXUN vector and recovery

[0040] The pCXUN plasmid is subjected to XcmI single enzyme digestion, and the enzyme digestion reaction system is as follows:

[0041]

[0042] The nucleic acid electrophoresis detection band is recovered by agarose gel.

[0043] (4) Homologous recombination of target fragments and vectors by infusion system

[0044]

[0045] The 10 μL reaction product is transformed into E. coli competent cells. Single colonies are shaken, sequenced, and subjected to sequence alignment. If the sequence is consistent with the Hd6 full-length open reading frame nucleotide sequence, the construction is successful.

[0046] Example 3: Identification of Hd6 overexpression plants

[0047] The constructed pCXUN-Hd6 overexpression vector was used to infect and transform ZH11 callus by the method of genetic transformation of callus induced by mature embryo mediated by Agrobacterium tumefaciens EHA105 to obtain Hd6 overexpression plants. In order 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'-CAAGAAAATCTACAGCCTCA-3'. The genomic DNA of the Hd6 overexpression plant was extracted as a template, and the genomic DNA of the wild type ZH11 was used as a control for PCR amplification. After amplification, the overexpression positive plants were identified by agarose gel electrophoresis (1090bp band was amplified in the overexpression positive plants, and no band was amplified in the wild type rice). The total RNA of the wild type and the positive overexpression plants was extracted, and the cDNA was obtained by reverse transcription. The cDNA was used as a template to carry out qRT-PCR experiment to identify the expression level of Hd6 gene in the positive plants. The primer sequences for identifying the expression level of Hd6 gene were SEQ ID NO. 9: 5'-AAGCTCTTGTTGGGAGGCATAG-3' and SEQ ID NO. 10: 5'-TTGTGGTCGTGCTCTGCTATT-3'. Three strains with significantly higher expression level of Hd6 gene than the wild type were obtained, and were named as OEHd6-1, OEHd6-2 and OEHd6-3, respectively. The results are shown in Figure 1

[0048] Example 4: Resistance identification of Hd6 overexpression plants to Magnaporthe oryzae by punching inoculation

[0049] The ZH11 rice and the Hd6 overexpression plants were planted in an artificial intelligence incubator, and punching inoculation experiment of Magnaporthe oryzae was carried out after 4 weeks of culture. A small puncher was used to punch at a position 1 / 3 away from the leaf tip, and a wound was left on the upper surface of the leaf (attention should be paid to not break the leaf tissue), so as to facilitate the infection of Magnaporthe oryzae. 10 μL of Magnaporthe oryzae Guy11 spore suspension (concentration about 2 x 10 5 The inoculated rice was transferred to normal light conditions for further culture, and the disease incidence was investigated after 7-10 days. The results are shown in Figure 2 Compared with the wild type ZH11 rice, the leaf lesion area of the Hd6 overexpression plants was significantly smaller and the relative biomass of the fungus carried was less, indicating that the resistance of rice to Magnaporthe oryzae was significantly enhanced after overexpression of Hd6 gene.

[0050] Example 5: Resistance identification of Hd6 overexpression plants to Xanthomonas oryzae pv. oryzae by inoculation

[0051] ​The rice Zhonghua 11 and the Hd6 overexpression plant are planted in the experimental rice field, and when the rice is naturally grown for 60 days, the rice bacterial blight is transplanted and cultivated, the concentration is adjusted to OD 600 =1.0, the scissors are immersed in the bacterial solution, and the part of the leaf is cut horizontally at 1 / 3 of the leaf by the scissors. After 15 days, the disease incidence is counted, and by counting the bacterial relative biomass ( Figure 3 ), it is found that compared with the wild type rice Zhonghua 11, the leaf spot expansion speed of the Hd6 overexpression plant is slower, the length of the leaf spot is shorter, and the relative biomass of the bacteria carried is less, and a more resistant phenotype is shown.

[0052] The above is only the preferred embodiment of the present application, and any equivalent changes and modifications made within the scope of the patent application of the present application shall be included in the scope of the present application.​

Claims

1. Hd6 The use of a gene in enhancing broad spectrum disease resistance in rice, characterized in that: By overexpressing in rice Hd6 Genes are used to enhance the broad-spectrum disease resistance of rice; Hd6 The nucleotide sequence of the open reading frame of the gene is shown in SEQ ID NO.1; the broad-spectrum disease resistance is selected from resistance to rice blast and resistance to bacterial blight.

2. A method of enhancing broad spectrum disease resistance in rice, characterized by: Increase receptor rice Hd6 The gene expression level was adjusted to obtain transgenic rice with broad-spectrum disease resistance stronger than the recipient rice; Hd6 The nucleotide sequence of the open reading frame of the gene is shown in SEQ ID NO.1; the broad-spectrum disease resistance is selected from resistance to rice blast and resistance to bacterial blight.

3. Hd6 The use of the genes in the breeding of transgenic rice with enhanced broad-spectrum disease resistance, characterized in that: The expression of the gene in the receptor rice is increased Hd6 The transgenic rice has stronger broad-spectrum disease resistance than the receptor rice; the Hd6 The nucleotide sequence of the open reading frame of the gene is shown in SEQ ID NO. 1; the broad-spectrum disease resistance is selected from resistance to rice blast and resistance to bacterial leaf blight.

Images