Application of gene osprx30 related to bacterial blight resistance

Active Publication Date: 2020-07-31
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the functions of C III PRXs genes are not very clear, but the expression of PRXs family genes will change significantly after plants are stimulated by stress

Method used

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  • Application of gene osprx30 related to bacterial blight resistance
  • Application of gene osprx30 related to bacterial blight resistance
  • Application of gene osprx30 related to bacterial blight resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Cloning and sequence analysis of rice OsPRX30 gene

[0030] 1) Extraction of rice total RNA

[0031] Get rice H4 (in literature " rice blast resistance protein Pik 2Cloning of -H4 gene and screening of interacting proteins[J].Guangdong Agricultural Sciences, 2014, (04):156-160.") rice seedlings at the 3-4 leaf stage were frozen in liquid nitrogen and ground into powder, Transfer to a 1.5mL centrifuge tube, and add Trizol reagent (Invitrogen Company) according to the ratio of 1mL extraction reagent per 100mg of material, and mix well; Discard the middle and lower organic phases, collect the upper aqueous phase and transfer to a new centrifuge tube; add 600 μL of isopropanol, mix well, let stand at room temperature for 20 minutes, centrifuge at 12,000 rpm, 4°C for 15 minutes, collect the precipitate, and wait for the isopropanol to volatilize Dissolve in RNase-free ultrapure water and freeze at -80°C for later use.

[0032] 2) Cloning of OsPRX30 gene

[0033...

Embodiment 2

[0039] Example 2 OPRX30 expression trend analysis of different rice after bacterial blight inoculation

[0040] The expression pattern of OsPRX30 gene after inoculation with bacterial blight was analyzed by quantitative RT-PCR. Inoculation of bacterial blight type IV bacteria in H4 (in the literature "Study on the resistance of rice bacterial blight-resistant near-isogenic lines to South China strains [J]. Phytopathological Journal, 2006,36(2):177-180 Disclosed in . ") after different time points (1d, 2d, 3d, 4d, 5d) leaves were collected and total RNA was extracted, and the reverse transcription kit ReverTraAce was used to synthesize the first strand of reverse transcription cDNA. Then, according to the instructions of the SYBR Premix ExTaq kit, the expression level of OsPRX30 was analyzed using the AB StepOne Plus fluorescent quantitative PCR detector, and Actin was used as an internal reference gene.

[0041] The primers and sequences used are:

[0042] OsPRX30-RT-F: 5'-C...

Embodiment 3

[0047] Example 3 Construction of rice OsPRX30 gene editing vector and acquisition of osprx30-ko

[0048] Using CRISPR / Cas9 technology to construct osprx30-ko plants, the specific operation method refers to the method of Ma et al. (2015). The 20bp DNA fragment in the first exon of OsPRX30 was designed to contain the PAM (protospacer-adjacent motif) sequence, and the primers and sequences used were:

[0049] OsPRX30-U3-F: 5'-ggcAACGTCGAGCTCCTCTGCCC-3' (SEQ ID NO: 10);

[0050] OsPRX30-U3-R: 5'-aaacGGGCAGAGGAGCTCGACGT-3' (SEQ ID NO: 11).

[0051] The sequence was fused with the U6a-gRNA expression cassette, and the fused fragment was digested with BsaI and inserted into pYLCRISPR / Cas9P ubi -H carrier ( Figure 4 ) (disclosed in the document "A robust CRISPR / Cas9 System for Convenient, High-efficiency Multiplex Genome Editing in Monocot and Dicot plants. Mol Plant, 2015, 8(8): 1274-84"), construct the transformation vector pYLCRISPR / Cas9P ubi -OsPRX30. This plasmid was transf...

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Abstract

The invention discloses an application of a bacterial blight resistance related gene OsPRX30, belonging to the technical field of plant genetic engineering. The expression of OsPRX30 gene is increasedafter induction by Xanthomonas oryzae pv. Oryzae pv. Oryzae. The gene is up-regulated after induction by Xanthomonas oryzae pv. Oryzae and the resistance to Xanthomonas oryzae pv. Oryzae could be enhanced by knockout. OsPRX30 edit site sequence is ligated with U3 promoter and cloned into gene editing vector pYLCRISPR / Cas9Pubi-H, and that gene can negatively regulate the resistance of rice to bacterial blight. The gene editing vector constructed by the invention can be used in rice breeding for resistance to bacterial blight. The present invention is helpful to better understand the mechanismof action of OsPRX30, and the cloning of OsPRX30 is helpful to further understand rice. Bacterial blight pathogen interaction, disease resistance signal transduction pathway laid the foundation, has great application value in breeding.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering and relates to the application of a rice disease resistance-related gene, in particular to the application of a bacterial blight resistance-related gene OsPRX30. Background technique [0002] Rice bacterial blight disease is caused by bacteria (Xanthomonas oryzae pv.oryzae), accumulating genetic and molecular data show that the molecular mechanism of rice resistance to bacterial blight and the major resistance gene (Major Resistance gene, MR) patterns are quite different. Most of the main disease resistance genes in rice are recessive regulation of bacterial blight resistance. Among the 37 reported R genes, 14 have resistance to bacterial blight after recessive mutation in natural populations, but the main Most of the fungi, nematodes, and viruses triggered by the effector genes are dominantly regulated. At present, only seven major MR genes of bacterial blight have been cloned...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415A01H5/00A01H6/46
CPCC07K14/415C12N15/8281
Inventor 王加峰郭涛刘浩肖武名陈志强王慧
Owner SOUTH CHINA AGRI UNIV
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