Application of micromolecular heat shock protein gene improving stress resistance of oryza sativa

A heat shock protein, rice stress resistance technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problem of short gene sequence, and achieve the effect of short coding sequence, high safety performance and easy access

Inactive Publication Date: 2012-10-17
HEFEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These small-molecule heat-shock proteins are rice's own genetic resources. The gene sequence is short and easy to clone and genetically manipulate. However, which small heat-shock protein genes have the ability to improve rice heat resistance has not yet been identified at the molecular level. Whether they also have drought, salt, etc. Stress functions, whether their expression affects the phenotype of other major agronomic traits of rice, and how to apply them to the improvement of stress resistance traits of important food crops such as rice are yet to be studied

Method used

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  • Application of micromolecular heat shock protein gene improving stress resistance of oryza sativa
  • Application of micromolecular heat shock protein gene improving stress resistance of oryza sativa
  • Application of micromolecular heat shock protein gene improving stress resistance of oryza sativa

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: Cloning of rice OsHSP18 gene

[0026] 1. Reagents

[0027] RNA extraction reagent Trizol was purchased from Invitrogen; reverse transcriptase ReverTraAce was purchased from Toyobo; high-fidelity DNA polymerase PrimeStar was purchased from TaKaRa; cloning vector pEASY TM -Blunt Simple Cloning Vector was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; the primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd., and the rest of the reagents were imported or domestic analytically pure products.

[0028] 2. E. coli strains and plant material

[0029] Escherichia coli (Escherichia coli) strain DH5α was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; rice japonica variety Nipponbare (Oryza sative L.ssp.japonica cv.Nipponbare) seeds were provided by Sichuan Academy of Agricultural Sciences.

[0030] 3. Medium and solution

[0031] LB medium: tryptone 10g / L, yeast powder 5g / L, NaCl 10g / L. Adjust the pH to 7.0 with NaOH and aut...

Embodiment 2

[0083] Example 2: Analysis of rice OsHSP18 gene expression pattern

[0084] 1. Reagents

[0085] RNA extraction reagent Trizol was purchased from Invitrogen; reverse transcriptase ReverTraAce was purchased from Toyobo; real-time quantitative PCR reagent TransStart TM Green qPCR SuperMix was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

[0086] 2. Method

[0087] Tissue samples of wild-type Nipponbare seedlings and mature plants under various stress conditions and hormone induction were taken, ground with liquid nitrogen, RNA was extracted, and reverse transcription was performed. The operation steps were as described in Example 1.

[0088] Using rice reference gene (ACTIN, Genbank accession number X16280) as a control, the real-time quantitative PCR analysis of OsHSP18 gene expression level was carried out. OsHSP18 gene quantitative PCR primers are: RT18F and RT18R; ACTIN gene quantitative P...

Embodiment 3

[0103] Example 3: Construction of OsHSP18 Gene Overexpression Recombinant Plasmid

[0104] 1. Reagents

[0105] Plasmid extraction kit EasyPure Plasmid MiniPrep Kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; agarose gel recovery kit EasyPure Quick Gel Extraction Kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; restriction enzymes Pst I, Xba I. T4 ligase was purchased from TaKaRa Company.

[0106] Other imported sub-packages and conventional reagents are the same as in Example 1.

[0107] 2. Agrobacterium strains and plant expression vectors

[0108] The Agrobacterium tumefaciens (Agrobacterium tumefaciens) strain EHA105 used for transgenesis was purchased from Clontech Company; the eukaryotic expression vector pHB was constructed by the laboratory of Professor Yang Hongquan of Shanghai Jiaotong University (Mao et al., Proceedings of the National Academy of Sciences of the United States of America , 2005) provided.

[0109] 3. Medium

...

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Abstract

The invention relates to application of a micromolecular heat shock protein gene improving stress resistance of oryza sativa. The gene improving various stress resistances of oryza sativa is Oryza sativa Heat Shock Protein18 (OsHSP18), with the nucleotide sequence being shown in SEQ ID NO:1 in a sequence table. The full-length coding sequence of the gene in SEQ ID NO:1 is inserted into a eukaryotic vector to obtain a eukaryotic recombinant plasmid over-expressed by the gene OsHSP18, and the eukaryotic recombinant plasmid is transformed into oryza sativa to obtain a transgenic plant over-expressed by the OsHSP18. Experiments show that the expression level of the gene OsHSP18 is obviously raised when oryza sativa is subjected to heat, drought, salt and low temperature stress, and the transgenic oryza sativa over-expressed by the OsHSP18 can enhance the resistance of the plant to various adversities (high temperature, drought, salt and cold damages) and does not have obvious impacts on agronomic traits such as plant height, tillering and the like. Thus, the gene can be applied to improvement of stress resistance of crops.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and particularly relates to the application of a gene for enhancing heat, drought, salt and cold stress resistance of rice in improving crop traits. Background technique [0002] Heat shock proteins (heat shock proteins, HSPs) were discovered in 1962 when studying the salivary glands of Drosophila. , 1988), and play an important role in protein folding, intracellular transport, degradation process and signal transduction pathway, so heat shock proteins are highly conserved and widely distributed in various organisms. In eukaryotes, heat shock proteins are divided into five families according to their molecular weight: HSP100, HSP90, HSP70, HSP60 and small HSP (sHSP) (Vierling, Annual Review of Plant Physiology and Molecular Biology, 1991). Small molecule heat shock proteins sHSPs are a group of proteins with a molecular weight of 12-43kD and a conserved ACD domain (α-crystallin domain) a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/00
Inventor 牛向丽石玮刘永胜黄胜雄彭晓莉毛云
Owner HEFEI UNIV OF TECH
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