Rice chlorine ion passage gene OsCLC, coding protein and clone method thereof

A technology of chloride ion channel and cloning method, which is applied in the field of rice chloride ion channel gene OsCLC, its encoded protein and its cloning, and can solve the problem of limited number of plant chloride ion channel gene reports

Inactive Publication Date: 2007-10-17
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, the number of reports on plant chloride channel genes is very limited

Method used

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  • Rice chlorine ion passage gene OsCLC, coding protein and clone method thereof
  • Rice chlorine ion passage gene OsCLC, coding protein and clone method thereof
  • Rice chlorine ion passage gene OsCLC, coding protein and clone method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Cloning and analysis of the full-length coding region of OsCLC

[0026] (1) Preparation of differentiated callus

[0027] Peel off the chaff of mature rice seeds, rinse with 70% ethanol for 1 min, soak in 0.1% mercury liter for 20 min; rinse with sterile water for 4-5 times, inoculate on MS+2, 4-D 2 mg / L induction medium, and place in the dark Culture the induced callus at 26°C; after two weeks, put the callus on the differentiation medium MS+6-BA2mg / L+KT2mg / L+NAA0.2mg / L, collect the differentiated callus, and Stored at -80°C after nitrogen snap freezing.

[0028] (2) Extraction of total RNA

[0029] The total RNA of rice differentiated callus was extracted by the Trizol method, and the total RNA was used as a template, and Oligo(dT) was used 18 As a primer, reverse transcription to synthesize the first strand of cDNA, Oligo(dT) 18 They are:

[0030] The 5' end primer is 5'-CGATGGCGCCAAGGGACCAGTCAT-3',

[0031] The 3' end primer is 5'-TACTAGCGAGCGTCAGAAGACGAG-3', ...

Embodiment 2

[0035] Real-time fluorescent quantitative PCR detection of salt-induced OsCLC gene expression

[0036](1) Sample preparation

[0037] A pair of specific primers were designed according to the sequence of the rice OsActin gene (Genbank: X15865): 5'-end primer (5'-TTG TGA GGG ACA TGA AGG AGA A-3'); 3'-end primer (5'-TAT GAA GGA AGG CTGGAA GAG G-3′) fragment length is 193bp, which is used as an internal reference for real-time fluorescence quantitative PCR. Design a pair of specific primers for the target gene OsCLC, 5'-end primer: 5'-ACA TCG TAT TTC CTG GGC ATT T-3'; 3'-end primer: 5'-CAG TCA TTC TCA TTG ATC CTC C-3' , the fragment length is 179bp. Using the first strand of cDNA synthesized in Example 1 as a template, the PCR reaction program was 94°C, 5min, and the following 30 cycles were performed: 94°C, 20s, 57°C, 20s, 72°C, 20s; finally, 72°C extension for 5min. Then, the two products were ligated into the pMD19-T vector and transformed into DH5α, and positive clones wer...

Embodiment 3

[0049] Mutant functional complementation experiment

[0050] (1) Construction of recombinant plasmids

[0051] Using the first strand of cDNA synthesized in Example 1 as a template, design specific primers: 5' end primer containing Sac I site (5'-AGC TGA GCT CGA TGG CGC CAA GGG ACC A-3'); containing Xbal 3' end primer of I (5'-TAG CTC TAG AGA AGA CGA GGT TGG TGA A-3'). The resulting product was digested with Sac I and Xbal I and inserted into the shuttle vector pYES2, and the recombinant expression plasmid pGAL1::OsCLC (Apr + ), the positive clone obtained by Apr screening, the plasmid was identified as positive by Sac I and Xbal I digestion, and then the lithium acetate method was used [12] Transformed into the Saccharomyces cerevisiae mutant strain YJR040W (gef1::kam), in CM omitted component medium (Ura - ), pGAL1::OsCLC (Ura + ) and pYES2 empty vector transformant yeast colonies, extract yeast DNA as a template, and use the OsCLC gene-specific primers designed in Secti...

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PUM

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Abstract

The present invention relates to rice chlorine ion passage gene OsCLC, its coded protein and clone method. The gene has sequence indicated by SEQ NO 3. The gene of 2429bp is cloned from differentiated rice callus. The opening reading frame of cloned gene encodes protein of 808 amino acid (molecular weight 88KD)which has 11 trans-membrane region indicated by trans-membrane region analysis. Cluster analysis indicates the OsCLC gene is new member of CLC family. The OsCLC gene expression level has positive correlation with salt concentration rise in a certain time range and respond to the salt stress and other environmental stress. The gene can be induced to produce partial complementary GEF1 function in yeast mutant and partially inhibit the sensitivity to NaCl and recover growth function. The gene is sensitive to pH value variety and not sensitive to different cation.

Description

technical field [0001] The invention relates to a rice chloride ion channel gene OsCLC, its encoded protein and its cloning method. Background technique [0002] Rice is the main food crop for more than half of the world's population. The genome sequencing work has been preliminarily completed, and it is a good model for molecular biology research. Salinization is an important factor that limits growth and increases yield in the main cause of crop yield potential not being fully developed—abiotic stress. Death due to carbon starvation However, most of the physiological and molecular biology studies on the mechanism of plant salt tolerance focus on Na + on, involving Cl - studies are few. [0003] Anion channels widely exist in animals, plants and microorganisms, and have important physiological functions, but their research lags behind that of cation channels. As one of the important anion channels, Chloride Channel (CLC) is generally considered to be related to cell tur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/10
Inventor 刘志学康敏华刘特杜喜玲
Owner SHANGHAI UNIV
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