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Application of gene OsERF65 related to plant disease resistance

A disease resistance, gene technology, applied in the field of plant genetic engineering

Pending Publication Date: 2022-05-13
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report about the relationship between OsERF65 gene and resistance to rice sheath blight, especially the research on improving rice resistance to sheath blight by changing OsERF65 gene through genetic engineering technology

Method used

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  • Application of gene OsERF65 related to plant disease resistance
  • Application of gene OsERF65 related to plant disease resistance
  • Application of gene OsERF65 related to plant disease resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Analysis of the expression characteristics of the OsERF65 gene

[0045] Samples of rice variety Xudao 3 at different growth stages were taken and stored in liquid nitrogen. Cultivate Xudao 3 under normal conditions until the end of tillering stage to inoculate with sheath blight bacteria, cut off the rice leaf sheaths 1cm above and below the inoculum before inoculation (0h) and 12h, 24h and 48h after inoculation, and store them in liquid nitrogen . According to the steps of the manual (Invitrogen Company), the total RNA of each rice sample was extracted with Trizol, and then the total RNA was digested with DNaseI (RNase free, Promega Company) to remove genomic DNA contamination (see the DNaseI manual for the method), and then reverse transcriptase (TaKaRa Company) reverse-transcribed the total RNA to synthesize the first strand of cDNA (for the method, refer to the manual of reverse transcriptase), the reaction conditions were: 37°C, 30min; 85°C, 5min; 4°C, ...

Embodiment 2

[0047] Example 2 Vector Construction, Genetic Transformation and Detection

[0048] The main steps of constructing the knockout vector are as follows: (1) Design the gene knockout target; (2) Amplify the sgRNA cassette. Using the pYLgRNA-OsU6a / LacZ plasmid as a template, the OsU6a-target fragment and the gRNA-target fragment were amplified in two reaction systems, respectively. (3) Construct the p-cas9 / gRNA knockout vector; (4) Select positive clones and confirm by sequencing; positive clone plasmids and final vector plasmids are mixed for recombination reaction; (5) PCR and plasmid digestion verify positive clones; (6) The recombinant positive plasmid is transferred into Agrobacterium; (7) Verification and preservation of positive Agrobacterium single clone. Two transgenic lines were obtained in the experiment, named OsERF65-KO3 and OsERF65-KO4 respectively.

[0049] The main steps of the construction of the overexpression vector are as follows: high-fidelity DNA polymerase...

Embodiment 3

[0050] Example 3 Identification of resistance to sheath blight of transgenic rice

[0051] The identification of sheath blight resistance was inoculated by the toothpick embedding method, and the strain was the medium-strong pathogenic strain YN-7 provided by the Plant Protection Department of Yangzhou University. For the identification of resistance at the adult plant stage in the greenhouse, the present invention uses 3 transgenic lines and 3 plants of the control wild type to be cultivated outdoors until the end of tillering stage, and then inoculated under high temperature and high humidity conditions in the greenhouse, and investigated 5 days, 10 days and 15 days after inoculation. Lesion length in days.

[0052] The results showed that the length of sheath blight lesions in the transgenic overexpression line was significantly higher than that of the wild type ( figure 2 A, B, C), the length of the sheath blight lesion of the knockout line was significantly lower than t...

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Abstract

The invention discloses an application of an OsERF65 gene in regulating and controlling the resistance of rice sheath blight disease. The invention also discloses a method for cultivating the transgenic rice with improved disease resistance, which comprises the step of reducing the expression quantity and / or activity of the OsERF65 gene in the receptor rice to obtain the transgenic rice. The disease resistance of the transgenic rice is higher than that of the receptor rice. According to the invention, sheath blight resistance identification and expression pattern analysis are carried out on the OsERFs family, and the over-expression of one gene OsERF65 can reduce the resistance of the rice sheath blight disease, and the inhibition of the expression quantity of the gene can enhance the resistance of the rice sheath blight disease. The main agronomic traits between an OsERF65 overexpression line and a knockout line and a control variety are further compared, it is found that the OsERF65 does not affect the main agronomic traits of rice, and it is indicated that the OsERF65 has important application value in the aspect of sheath blight resistance breeding.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to the application of a gene OsERF65 related to plant disease resistance. Background technique [0002] Sheath blight is one of the three major diseases of rice. Due to the lack of sheath blight resistance gene resources and the lag in research on disease resistance mechanisms, the breeding of sheath blight resistance has been slow. Discovery of sheath blight resistance genes can provide genetic resources and theoretical basis for molecular breeding of sheath blight resistance. [0003] ERF is a plant-specific transcription factor located downstream of the ethylene signaling pathway. It contains a relatively conserved DNA-binding domain consisting of about 60 amino acids, namely the AP2 domain. In addition to the DNA binding domain, each member also contains a nuclear localization signal domain (NLS) and a transcriptional regulatory domain, and some me...

Claims

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Application Information

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IPC IPC(8): C12N15/82C07K14/415C12N15/29A01H5/00A01H6/46
CPCC12N15/8282C12N15/8218C07K14/415Y02A40/146
Inventor 左示敏史小品曹文磊胡珂鸣冯志明陈宗祥谢文亚卢帅兵
Owner YANGZHOU UNIV
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