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mir395 gene and its regulatory sites and applications

A gene and rice technology, applied to the miR395 gene and its regulatory sites and application fields, can solve the problems of rice disease resistance phenotype changes, unclear and other problems, and achieve the effect of enhancing accumulation and enhancing broad-spectrum disease resistance.

Active Publication Date: 2021-07-30
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many disease resistance-related genes have been identified in rice, and the mechanism of some rice resistance-related genes has been studied in depth, the mechanism of action of most disease-resistance-related genes in rice disease resistance response, as well as individual disease resistance Whether related genes cause changes in rice resistance phenotypes is unclear

Method used

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  • mir395 gene and its regulatory sites and applications
  • mir395 gene and its regulatory sites and applications
  • mir395 gene and its regulatory sites and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Acquisition of miR395 target genes and overexpression materials

[0043] (1) Acquisition of miR395 gene recognition site

[0044] Using the principle of sequence complementarity between plant miRNA and target mRNA, the applicant obtained the recognition site of the miR395 gene in rice through genome-wide scanning of rice (Dai et al., 2018; Warthmann et al., 2008). There are three target gene mRNAs of miR395 gene in rice, which are transcribed from Os03g53230, Os03g09930, and Os03g09940 genes, respectively. The recognition site of the miR395 gene is a nucleotide sequence with a length of 21nt. There is a good sequence match between the miR395 gene and the target gene mRNA at the corresponding recognition site. The results are shown in figure 2 .

[0045] (2) Construction of miR395 gene overexpression vector

[0046] This example is about the construction method of pU1301-pUBIQUITIN-miR395 vector.

[0047] First, using the rice variety Zhonghua 11 rice DNA...

Embodiment 2

[0055] Example 2: Correlation analysis and functional verification of rice lines with overexpression of miR395 gene

[0056] (1) Phenotype analysis of resistance to bacterial blight of rice lines with overexpression of miR395 gene at booting stage

[0057] In summer fields in Hubei, Wuhan, China, rice lines overexpressing miR395 gene and wild-type controls were inoculated with bacterial blight. The results showed that rice lines with overexpression of miR395 gene invented by this department were inoculated with pathogenic race PXO347 of bacterial blight at the booting stage. The disease length of the positive individual plants was significantly shorter than that of the negative individual plants and the wild-type control (p Figure 7 . The above results indicated that the overexpression of miR395 gene can enhance the resistance of rice to the pathogenic race PXO347 of bacterial blight.

[0058] (2) Analysis of broad-spectrum resistance of rice lines with overexpression of miR...

Embodiment 3

[0066] Example 3: Analysis of sulfate ion content in rice lines with overexpression of miR395 gene

[0067] (1) Analysis of sulfate ion content in leaves of rice lines with overexpression of miR395 gene

[0068] Rice lines with overexpression of miR395 gene were planted in Daejeon of Hubei and Wuhan in China in summer, and the content of sulfate ion in flag leaves was determined at the booting stage. The main analysis steps are as follows: crush the leaves with liquid nitrogen, dissolve them in deionized water, incubate in a water bath at 80°C for 3 hours, filter the supernatant after centrifugation with a 0.22 micron filter membrane, and measure sulfate ions in the filtrate on an ion chromatograph content. Compared with the wild type, the content of sulfate ion in the flag leaves of rice lines overexpressing miR395 gene was higher (see Figure 12 ). This result indicated that the miR395 gene had a positive regulatory effect on sulfate ion accumulation in rice leaves.

[0...

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Abstract

The invention belongs to the field of plant genetic engineering, and specifically relates to miR395 gene and its regulatory site and application. The miR395 gene is cloned from rice, and its nucleotide sequence is shown in SEQ ID NO:1. The regulatory sites of miR395 gene are Os03g53230, Os03g09930 and Os03g09940. The miR395 gene and its regulatory sites can be used in rice varieties with enhanced resistance to bacterial blight and bacterial leaf spot. Through the overexpression of miR395, it was found that miR395 can affect the ability of rice to resist bacterial blight and bacterial streak disease. That is, overexpression of miR395 can improve the resistance of rice to bacterial blight and bacterial streak, and overexpression of miR395 can increase the endogenous sulfate concentration of rice. The gene and its regulating site of the invention can be used for rice disease resistance breeding and sulfate-mediated resistance research and development.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to miR395 gene and its regulation site and application. The gene is rice miR395 gene, and the control sites of the miR395 gene are Os03g53230, Os03g09930 and Os03g09940. The gene and its regulatory sites can be used in rice varieties with enhanced resistance to bacterial blight and bacterial leaf spot. Background technique [0002] Plants are infected by a variety of pathogenic bacteria during their growth. There are many kinds of plant pathogens, mainly bacteria, fungi, oomycetes, viruses and nematodes. In the long-term evolution process, plants have evolved some disease-resistant genes to deal with the infection of various pathogens. Discovering and using these genes to improve the disease resistance of plants is the fundamental way to prevent and control diseases under the premise of protecting the environment. [0003] Many genes are involved in t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/29C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/113C12N15/8218C12N15/8243C12N15/8281C12N2310/141
Inventor 王石平袁猛杨泽宇惠述刚
Owner HUAZHONG AGRI UNIV