Rice gene OsHsp40 and application thereof in preparation of transgenic plant with abiotic stress resistance

A technology of abiotic stress and transgenic plants, applied in the fields of plant peptides, plant products, genetic engineering, etc., can solve the problem of unclear roles and achieve the effect of improving the ability

Pending Publication Date: 2021-01-22
SHANGHAI AGROBIOLOGICAL GENE CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, from the published articles, most of the researches on the function of this kind of protein are in dicotyledonous plants such as Arabidopsis, Gramineae, especially important food crop rice, the role of DnaJ / Hsp40-like proteins in adversity stress is still unclear , the mechanism of its function in adversity is still rarely reported. It is still of great significance to clone and study this kind of protein in important food crops, and to find more efficient anti-abiotic stress genes to be applied to rice, the most important food crop in my country.

Method used

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  • Rice gene OsHsp40 and application thereof in preparation of transgenic plant with abiotic stress resistance
  • Rice gene OsHsp40 and application thereof in preparation of transgenic plant with abiotic stress resistance
  • Rice gene OsHsp40 and application thereof in preparation of transgenic plant with abiotic stress resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Isolation and cloning of the OsHsp40 gene

[0051] Vigorously growing rice was excavated outdoors, and total RNA was extracted with TRIzol reagent (GIBCO BRL, USA). It was reverse transcribed into cDNA using reverse transcriptase MLV (Tiangen, China). Using primer F (5'-ATGGCGCTGCAGCTTCAGGCC-3') and primer R (5'-GCTGAGCCCAAGTTCC TTCTTCAG-3'), the full-length coding cDNA (810bp) of the gene was amplified. The PCR reaction conditions were: pre-denaturation at 94°C for 3 min; 35 cycles at 94°C for 30 sec, 60°C for 30 sec, and 72°C for 60 sec; extension at 72°C for 5 min. The amplified PCR product was ligated into pGEM-T vector (Promega, USA), positive clones were screened and sequenced to obtain the cDNA sequence (SEQ ID NO.1) of the OsHsp40 gene. The predicted protein sequence of OsHsp40 was compared with homologous genes, and it was found that the protein encoded by this gene is a family protein including the DnaJ domain, and the 75th-132th amino acid is a c...

Embodiment 2

[0052] Embodiment 2: Construction and genetic transformation of OsHsp40 gene overexpression vector and Crispr / Cas9 knockout vector

[0053] 1) Containing the construction of the target gene expression vector:

[0054] According to the full-length sequence of the OsHsp40 gene (SEQ ID NO.1), primers were designed to amplify the complete coding reading frame, and linker primers were added to the upstream primer and downstream primer respectively, so as to construct the expression vector. Using the amplified product obtained in Example 1 as a template, after performing PCR amplification with the high-fidelity amplification enzyme pfu enzyme (Tiangen, China), the cDNA fragment of the OsHsp40 gene added with the linker was recombined with the plant expression vector pUB06 , constituting a plant expression unit expressed under the maize ubiquitin promoter (see figure 2 ), transformed into Agrobacterium EHA105, and finally carried out the rice callus transformation experiment.

[0...

Embodiment 3

[0076] Example 3: Identification and analysis of transgenic plants overexpressing OsHsp40

[0077]1) Material preparation

[0078] Transgenic T1 generation rice seeds germinated and transplanted in liquid medium (tap water prepared with 1 / 5 MS macroelements). After the seedlings grew for 15 days, the leaves were clipped and quickly put into liquid nitrogen for storage for RNA extraction.

[0079] 2) DNA-free total RNA preparation

[0080] According to the instruction manual of the Plant Leaf RNA Small Extraction Kit provided by Shanghai Quanshijin Biotechnology Co., Ltd. Use Beckman Coulter TMDU 640 UV spectrophotometer to measure RNA concentration. To remove residual DNA in RNA, take 5 μg of each total RNA sample, add 1 μL DNAase I (Invitrogen, USA) and 1 μL 10× reaction buffer, make up the volume to 10 μL, react at room temperature for 30 min, and then add 1 μL 2 mmol L- 1EDTA to terminate the reaction, and finally heated at 70°C for 10 min to inactivate DNAase I.

[...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a gene derived from a rice plant and an application of the gene to improvement of abiotic stress resistance of the plant. The invention discloses a rice gene OsHsp40 for preparing a transgenic plant with abiotic stress resistance. The nucleotide sequence of the rice gene OsHsp40 is as shown in SEQ ID NO.1. The invention further discloses a promoter formed by connecting a separated DNA molecule containing polynucleotide for encoding nucleic acid to the plant. The encoded amino acid sequence of the promoter is as shown in SEQ ID NO.2. The invention also provides the application of the rice gene OsHsp40 in preparation of the transgenic plant with abiotic stress resistance. The OsHsp40 gene has an obvious effect in high temperature resistance and drought resistance of the plant, so that the gene can be combined with an overexpression promoter in the plant and then introduced into a proper expression vector and transformed into a plant host. The abiotic stress resistance of the plant is improved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a gene derived from rice plants and its application in improving the abiotic stress resistance of plants. Background technique [0002] my country is a large agricultural country, and high and stable crop yields are of great significance to ensure food security in our country. However, my country is a country with frequent natural disasters and a vast territory. Various natural climate disasters will occur in different regions from south to north every year. If crops encounter severe natural disasters during the growth process, such as drought, high and low temperature, salinity, heavy metal pollution, and stagnant waterlogging, etc., it will often cause large-scale production reduction. In order to achieve long-term food security and sustainable development, an in-depth understanding of plant abiotic stress biology is one of the important goals of agricultural scien...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/113C12N15/82A01H6/46A01H5/00
CPCC07K14/415C12N15/8273C12N15/8271
Inventor 余舜武邹玉巧杨访问张余李天菲吴金红罗利军
Owner SHANGHAI AGROBIOLOGICAL GENE CENT
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