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Marine microbial cold shock protein gene csp, its encoded protein and its application

A technology of marine microorganisms and cold shock proteins, applied in the field of genetic engineering

Active Publication Date: 2020-09-29
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In recent years, the research on CSP gene to improve plant stress resistance has become popular, but most of the gene sources of research focus on known microorganisms such as Escherichia coli and Bacillus subtilis, while 99% of marine environment microorganisms are not cultivable. There is no report on the isolation of CSP genes from microorganisms in the marine environment

Method used

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  • Marine microbial cold shock protein gene csp, its encoded protein and its application
  • Marine microbial cold shock protein gene csp, its encoded protein and its application
  • Marine microbial cold shock protein gene csp, its encoded protein and its application

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Experimental program
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Effect test

Embodiment 1

[0027] Cloning and analysis of embodiment 1CSP gene

[0028] (1) Collection of marine microorganisms: seawater is taken from the sea area of ​​Heishijiao, Dalian, China, and clean seawater that can be directly used for aquaculture is obtained through a sand filter device. The clean seawater is suction filtered, and the microorganisms are left on the filter membrane, and then gently scraped with a clean blade and rinsed with seawater for centrifugation to obtain a mixed sample of marine microorganisms. The mixed sample of marine microorganisms was quickly stored at 4°C and used for subsequent DNA extraction experiments as soon as possible. Combining physical and chemical cell lysis and various enzyme digestion methods, marine microbial metagenomic DNA with good quality and high purity is extracted and purified from marine microbial samples.

[0029] (2) Merge primer design: compare the homology of the CSP family gene sequences of microorganisms such as Escherichia coli, Bacill...

Embodiment 2

[0054] The acquisition of embodiment 2 transgenic Arabidopsis thaliana

[0055] (1) CSP gene plant expression vector construction

[0056] ①Plasmid mini-extraction kit from Sangon Bioengineering Co., Ltd. was used to extract the pMD18-CSP plasmid and the empty plasmid of the plant binary expression vector pTF 101, respectively. See the instructions for specific methods.

[0057] ②Using SmaI and SacI two restriction endonucleases to perform double enzyme digestion reaction on pMD18-CSP, pTF101-35s and pTF101-ubi plasmids to linearize them, and recover the CSP gene fragment and the large fragment of pTF101 vector.

[0058] ③ Ligate the recovered and purified plant expression vectors pTF101-35S and pTF101-ubi with the coding region of the CSP gene by using T4 ligase. Recombinant vectors pTF101-35S-CSP and pTF101-ubi-CSP were obtained respectively (the structural maps are respectively Figure 4 A and Figure 4 B).

[0059] ④The connection solution was transformed into Escheric...

Embodiment 3

[0069] Physiological detection of transgenic Arabidopsis under the drought stress condition of embodiment 3

[0070] Transgenic Arabidopsis thaliana after screening were transplanted into square pots, and two Arabidopsis overexpressing CSP were planted in each square pot, and were used for the next physiological test after 5 weeks of growth. For control wild-type Arabidopsis thaliana, the same culture method as above was adopted, without Basta solution being sprayed, and physiological tests were carried out at the same time as the experimental group after 5 weeks of growth.

[0071]Select transgenic and wild-type Arabidopsis experiments with consistent growth conditions. In this experiment, the harsh watering method was used to simulate the drought under natural conditions, that is, all the plants were watered with sufficient water before the drought treatment, and no watering was performed after that. On the 0th day, the 3rd day, the 6th day, the 9th day and the 12th day of ...

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Abstract

The invention discloses a cryoprotein gene CSP, an encoded protein and the application thereof. The cryoprotein gene CSP disclosed by the invention is derived from marine microbial metagenome and hasa base sequence shown as SEQ ID NO:1. The encoded protein CSP is a type of highly conserved nuclear acid binding proteins, can serve as an RNA molecular chaperone, and participates in cellular physiological activities such as transcription, translation, growth and development, abiotic response stress and the like. According to the characteristic of improving drought endurance and cold resistance of transgenic plants, the cryoprotein gene CSP can be applied to molecular breeding by virtue of a transgenic technology, so that the yield loss of crops during abiotic stress is reduced.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a cold shock protein gene CSP derived from a marine microbial metagenome, its encoded protein and the application of the gene in improving plant stress resistance. Background technique [0002] Abiotic stresses such as drought and low temperature seriously affect the growth of crops and bring immeasurable losses to my country's grain output. Improving the tolerance of crops to abiotic stress through traditional breeding methods is slow and takes a long time, but the above-mentioned problems are solved by genetic engineering, which greatly speeds up the breeding process and effectively makes up for the shortcomings of traditional breeding methods. [0003] Cold shock proteins (CSPs) are a class of highly conserved nucleic acid-binding proteins that widely exist in microorganisms, animals, and plants. The cold shock protein CspA was first discovered in Esche...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/63C12N1/21C07K14/245A01H5/00A01H6/20A01H6/46C12R1/01
CPCC07K14/245C12N15/8273
Inventor 苏乔丁风鹅曹钰雪徐永盛
Owner DALIAN UNIV OF TECH
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