Rice OsNBARC1 protein and application of coding gene of rice OsNBARC1 protein in regulation and control of resistance of rice to bacterial leaf blight

A technology of leaf blight resistance and coding gene, applied in the biological field, can solve problems such as loss of variety resistance, and achieve the effect of improving resistance and improving resistance

Active Publication Date: 2020-06-05
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the high degree of variability of rice bacterial blight, after large-scale promotion and planting of disease-resistant varieties carrying a single major gene, potential toxic races will become dominant races or new toxic races will appear due to mutations, which can easily lead to Variety resistance loss

Method used

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  • Rice OsNBARC1 protein and application of coding gene of rice OsNBARC1 protein in regulation and control of resistance of rice to bacterial leaf blight
  • Rice OsNBARC1 protein and application of coding gene of rice OsNBARC1 protein in regulation and control of resistance of rice to bacterial leaf blight
  • Rice OsNBARC1 protein and application of coding gene of rice OsNBARC1 protein in regulation and control of resistance of rice to bacterial leaf blight

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1, the cloning of rice OsNBARC1 gene

[0041] 1. Obtaining the genome sequence of the rice OsNBARC1 gene

[0042] Extract leaf DNA of rice variety FF329, use this DNA as a template, and use:

[0043] Forward primer OsNBARC1-F: 5'-ACTCACTCCAAAATCCTGAGTAGT-3',

[0044] Reverse primer OsNBARC1-R: 5'-AGCGGCCAATATGCTCGGAAT-3';

[0045] PrimeSTAR GXL DNA Polymerase (Code: R050A, Takara) was used for PCR amplification to obtain the amplified product, namely SEQ ID NO.2 in the sequence listing, OsNBARC1 gene sequence.

[0046] 2. Obtaining the sequence of the coding region of the rice OsNBARC1 gene

[0047]Take the total RNA of leaves of rice variety FF329, and use FastKing gDNA Dispelling RT SuperMix (Code: KR118, TIANGEN) to synthesize cDNA, use this cDNA as a template, and use primers:

[0048] OsNBARC1-CDS-F:5'-ATGGAGTTCGCCACAGGGGC-3',

[0049] OsNBARC1-CDS-R:5'-TTACCTCCTATTGTCAAGGTATTCTC-3';

[0050] PrimeSTAR GXL DNA Polymerase (Code: R050A, Takara) was u...

Embodiment 2

[0051] Example 2, Construction of Rice OsNBARC1 Complementary Vector and Gene Editing Vector

[0052] 1. Construction of rice OsNBARC1 complementary vector

[0053] The OsNBARC1 gene complementary vector pCAMBIA1300-OsNBARC1 driven by the OsNBARC1 gene's own promoter to drive the full-length genome sequence was constructed. The operation steps are as follows:

[0054] (1) Extract the leaf DNA of the rice variety FF329, use the DNA as a template, and use:

[0055] Forward primer OsNBARC1-CP-F: 5'-TTGGAAGAGCGAGAGACTTCG-3',

[0056] Reverse primer OsNBARC1-CP-R:5'-AAAAGCAGCCACTCTTATCTCG-3';

[0057] Perform PCR amplification with PrimeSTAR GXL DNA Polymerase (Code: R050A, Takara) to obtain the amplified product (ie, SEQ ID NO.4 in the sequence listing, the full-length sequence of the OsNBAR C1 gene's own promoter-driven genome), and perform gel cutting Recycle.

[0058] (2) Add the carrier homologous sequence to the amplification product obtained in step (1), the specific st...

Embodiment 3

[0096] Embodiment 3, the acquisition of transgenic plants

[0097] Transgenic rice was prepared using pCAMBIA1300-OsNBARC1 and CRISPR / Cas9-OsNBARC1 in Example 2, respectively, and blank vectors pCAMBIA1300 and pYLCRISPR / Cas9Pubi-H were used as controls. Huanghuazhan (HHZ) and FF329 were used as recipient plants for the preparation of transgenic rice, among which rice variety Huanghuazhan (HHZ) showed high sensitivity to bacterial blight strain PXO99A, and rice variety FF329 showed high resistance to bacterial blight strain PXO99A. Specific steps are as follows:

[0098] (1) Take out the mature seeds of the plant, remove the shells, and pick the plump, smooth and spot-free seeds for disinfection.

[0099] (2) Inoculate the sterilized seeds on the induction medium, culture in the dark at 28° C. for about 14 days, and select calli with good appearance and good growth ability.

[0100] (3) Take the recombinant vector pCAMBIA1300-OsNBARC1 constructed in Example 2 and transfer it ...

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Abstract

The invention relates to rice OsNBARC1 protein and application of a coding gene of the rice OsNBARC1 protein in regulation and control of resistance of rice to bacterial leaf blight. An amino acid sequence of the rice OsNBARC1 protein, a sequence of the coding gene of the rice OsNBARC1 protein, and a sequence of a coding region of the coding gene of the rice OsNBARC1 protein are disclosed, and functional verification is conducted by means of gene complementation and gene editing technologies. Experimental results prove that the resistance of OsNBARC1-complementary transgenic plants to bacterial leaf blight is significantly improved, the lesion length is reduced by about 61.9% compared with those of wild-type plants, the resistance of plants with OsNBARC1 genes knocked out to bacterial leafblight is significantly reduced, and the lesion length is improved by about 2.8 times compared with those of the wild-type plants. The experimental results show that the rice OsNBARC1 protein has a function of positively regulating and controlling the disease resistance of rice, can be used for improving the resistance of rice to bacterial leaf blight, and has important significance for cultivation of a novel rice variety with resistance to bacterial leaf blight.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the function and application of a gene related to rice disease resistance, in particular to the application of rice OsNBARC1 protein and its coding gene in regulating rice resistance to bacterial blight. Background technique [0002] Rice bacterial blight is an important bacterial disease caused by Xanthomonas oryz ae pv. oryzae (Xoo), which has a wide range of diseases. In normal years, rice production can be reduced by about 10%, and in severe cases, it can be reduced by 50%-60%. Breeding disease-resistant varieties using resistance genes is currently the most economical and effective measure to control rice bacterial blight. [0003] So far, 45 rice bacterial blight resistance genes have been reported in the prior art (http: / / www.shigen.nig.ac.jp / rice / oryzabase / gene / list). However, the resistance genes derived from wild rice are difficult to use, some resistance genes only have re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/8281
Inventor 周永力卢家玲张帆黎志康
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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