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Application of OsHDA710 epigenetic regulatory factor gene in rice development and stress resistance

A technology of regulatory factors and epigenetic regulation, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems that the mechanism of stress resistance has not been elucidated

Inactive Publication Date: 2021-02-05
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In summary, plants are affected by different hormones and various environmental stimuli during their growth and development, but there are few studies on the ability of HDACs epigenetic regulators to enhance rice's resistance to salt stress. mechanism has not been elucidated

Method used

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  • Application of OsHDA710 epigenetic regulatory factor gene in rice development and stress resistance
  • Application of OsHDA710 epigenetic regulatory factor gene in rice development and stress resistance
  • Application of OsHDA710 epigenetic regulatory factor gene in rice development and stress resistance

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1: Cloning of the OsHDA710 gene

[0039] The clone (LOC_Os02g12380) of the gene OsHDA710 of the present invention is mainly obtained by the method of RT-PCR (method reference: J. Sam Brook, EF Fritsch, T Mani Atis, translated by Huang Peitang, Wang Jiaxi, etc., Molecular Cloning Experiment Guide (Third Edition), Science Press, Beijing, 2002 edition). The specific operation steps are as follows:

[0040] (1) Extract RNA from seedling leaves of rice variety "Zhonghua 11" (from Institute of Crop Science, Chinese Academy of Agricultural Sciences), and use Invitrogen's Trizol extraction kit for RNA extraction (see the kit's instruction manual for specific steps);

[0041] (2) RT-PCR reverse transcription to synthesize the first strand of cDNA: ①Preparation of mixed solution 1: total RNA 4μg, DNaseI 2U, 10xDNaseI buffer 1μl, add DEPC (diethyl pyrocarbonate, a strong inhibitor of RNase) treated water (0.01% DEPC) to 10 μl, after mixing, place the mixture 1 at 37°C fo...

Embodiment 2

[0048] Embodiment 2: Construction of OsHDA710 overexpression vector

[0049] (1) Design primers suitable for constructing overexpression vectors and add enzyme-cut linkers of Kpn I and BamH I at both ends of the primers, use the full-length cDNA obtained in Example 1 as a template, and amplify by PCR The CDs sequence of OsHDA710 was obtained.

[0050] (2) digest the PCR product and the overexpression vector plasmid pU1301 with Kpn I and BamH I respectively (see figure 1 ), the target fragment is recovered and purified and connected with ligase, and the connected product is introduced into Escherichia coli DH10B (purchased from Promega (Beijing) Biotechnology Co., Ltd.), in LA containing 250ppm kanamycin (purchased from Roche Biological Company products) (LA formulation is referred to J. Sambrook, EF Fritsch, T Mani Artis, Huang Peitang , translated by Wang Jiaxi, etc., Molecular Cloning Experiment Guide (Third Edition), Science Press, Beijing, 2002 edition) cultured on resi...

Embodiment 3

[0054] Embodiment 3: Construction of CRISPR / CAS9 vector

[0055] According to the full-length sequence of the OsHDA710 gene obtained in Example 1, the gRNA sequence was designed, and the U3-promoter-gRNA sequence was constructed by fusion PCR, and then the cloned plasmid was digested with KpnI, and finally connected to the pCXUN- On the Cas9 vector, positive clones containing foreign fragments were screened.

[0056] Specific steps: Find the target site for knockout, using the website CRISPR-P developed by the Bioinformatics Center of the State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University (Lei et al 2014; Liu et al 2017). Copy the genome sequence or coding region sequence of the target gene to the homepage of the website, execute the search function, and select the point with the fewest possible off-target sites and the highest Score as the target site at the 5′ end close to the gene coding region sequence, and design primers.

[0057] The CRI...

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Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to application of an OsHDA710 epigenetic regulatory factor gene in rice development and stress resistance. TheOsHDA710 epigenetic regulatory factor gene is an HDAC family histone deacetylase gene related to stress resistance, and the nucleotide sequence of the OsHDA710 epigenetic regulatory factor gene is asshown in SEQ ID NO: 1. The gene is induced by adversity such as drought simulation, cold, salt and heat shock and plant hormones such as ABA, NAA, 6-BA, JA and IAA. The setting percentage of a gene mutant is remarkably reduced under natural conditions. NaCl and ABA are used for treating overexpression, mutants and wild type plant seedlings, it is found that the mutants of the gene are resistant to NaCl and ABA, and meanwhile growth and the seed germination rate of overexpression plants of the gene are both affected by exogenous ABA. After ABA treatment, the expression level detection of stress resistance related genes of transgenic family plants of different mutants shows that the gene has an important regulation and control function in the rice resistance process.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. It specifically relates to the functional verification of a rice HDAC family histone deacetylase gene OsHDA710 and its application in rice development and stress resistance. Background technique [0002] Due to its fixed growth characteristics, plants will experience various abiotic environmental stresses during their growth and development, such as drought, flood, high salinity, high temperature, mineral deficiency and so on. These abiotic stresses have important effects on the yield of plants, especially crops. (Boyer 1982. Plant productivity and environment. Science 218:443-448). At present, about 50% of the food crops in the world are reduced each year due to abiotic stress (Mantri et al. 2012. Abiotic stress responses in plants: present and future. Abiotic stress responses in plants: Springer). Among them, the salt stress caused by soil salinization has been the main env...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N15/82A01H5/10A01H6/46
CPCC12N9/80C12N15/8261C12N15/8273C12Y305/01098
Inventor 赵毓徐秋涛周道绣
Owner HUAZHONG AGRI UNIV
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