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Atriplex canescens glycogen synthetase kinase 3 gene clone and its application

A technology encoding genes and amino acids, applied in the field of real-time fluorescent quantitative PCR

Inactive Publication Date: 2015-02-25
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Meanwhile, the expression of SGG / GSK3 gene is induced by NaCl and ABA, but not KCl, so this gene may be involved in responding to abiotic stress (Piao et al., 2001)
However, in the current numerous studies, there is no research report on the physiological functions of the Atriopteris abatrix AcGSK3 gene in stress resistance and other aspects.

Method used

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  • Atriplex canescens glycogen synthetase kinase 3 gene clone and its application
  • Atriplex canescens glycogen synthetase kinase 3 gene clone and its application
  • Atriplex canescens glycogen synthetase kinase 3 gene clone and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Acquisition and sequence analysis of the full-length cDNA of AcGKS3 gene

[0040] 1. Stress treatment of Amarina tetraptera:

[0041] The four-winged Amarina plants grown for three months at -10°C were transplanted into an artificial climate incubator, and after 10 days of normal culture, the plants recovered and survived, and new leaves were issued, growing robustly. Transfer the plants to hydroponic conditions containing nutrients, and after two days of normal growth, use 500mL of NaCl400mM salt solution to hydroponically treat the plants of Amarina tetraptera. After 4 days of stress treatment, pick the leaves, clean the tender stems and young roots Quick-frozen in liquid nitrogen and stored at -80°C for storage.

[0042] 2. Construction of cDNA library:

[0043] Total RNA ( figure 1 , figure 2 ), and then use Invitrogen's 'cap antibody module' to enrich the single-stranded cDNA containing the complete cap structure, and follow the The Full-Length cD...

Embodiment 2

[0059] Example 2: Sequence Analysis of AcGSK3 Gene

[0060] After sequencing analysis, the full-length cDNA sequence of the AcGSK3 gene is 1101bp, including an open reading frame of 339bp, encoding a total of 113 amino acid residues. According to DNAMAN analysis, the isoelectric point is 9.69. Generally, the protein is positively charged, and the protein secondary structure contains 57 coils, 56 helices, no folding, 6 antigenic peptides, protein contains 40 hydrophilic amino acids, 73 hydrophobic amino acids, according to the analysis of Hphob / Kyte&Doolittle of Expasy protscale, the possible surface area of ​​the globulin is the middle and C-terminal. PSORT Prediction predicts the possible localization of proteins in order of cytoplasm and microbodies. This is the main feature of GSK3 family proteins. The analysis showed that the full-length cDNA sequence of AcGSK3 gene was obtained, and the accession number on GenBank was KJ027006.

[0061] Using NCBI (http: / / www.ncbi.nlm....

Embodiment 3

[0062] Example 3: Transformation and detection of recombinant yeast

[0063] Saccharomyces cerevisiae strain INVSc1 is uridine auxotroph (Ura - ) model strain, in the yeast basic medium (SC-Ura) lacking uric acid - ), the yeast strain can barely grow and reproduce. The yeast expression vector (pYES2-DEST52) contains the URA3 gene, and the expression of this gene can make the yeast transformant in SC-Ura - Normal growth on medium. Therefore, SC-Ura - The selection medium allows selection of positive and non-positive yeast transformants. Transform vector plasmids pYES-AcGSK3 and pYES-DEST52 into yeast competent strain INVSc1 by lithium acetate method, and spread the transformed yeast liquid on SC-Ura - On the solid selection medium, use untransformed yeast as a control to culture. After 2 days, except for the empty yeast that does not grow at all, the yeast transformed with the two plasmids can grow and have colonies, indicating that the yeast transformation is successful. ...

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Abstract

The invention relates to an Atriplex canescens glycogen synthetase kinase gene clone and its application, and belongs to the technical field of plant gene engineering. A plant resistance related protein AcGSK3 is a protein composed of an amino acid sequence represented by SEQ ID NO:2 in a sequence table; and the coding sequence of the coding gene AcGSK3 of the plant resistance related protein AcGSK3 is one of a coding sequence with the nucleotide sequence represented by 346-684th deoxynucleotides at 5'-terminal of an amino acid sequence represented by SEQ ID NO:1 in the sequence table, and a nucleotide molecule for coding the protein sequence of the SEQ ID NO:2 in the sequence table. The invention also relates to a recombinant expression vector containing the coding gene AcGSK3 of the plant resistance related protein AcGSK3, and a recombinant bacterium containing the coding gene AcGSK3 of the plant resistance related protein AcGSK3. A result of qRT-PCR detection of the original plant Atriplex canescens shows that the gene improves the expression under NaCl and PEG stress. AcGSK3 is introduced into Saccharomyces cerevisiae to obtain transgenic Saccharomyces cerevisiae, and stress treatment is carried out, so the result shows that the AcGSK3 can improve the drought (sorbitol) and salt (NaCl) resistance of yeast.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to the use of Saccharomyces cerevisiae INVSc1 bacterial strain to express the protein AcGSK3 related to plant stress resistance (especially salt and drought), and simultaneously adopt real-time fluorescent quantitative PCR (real-time PCR, Real-Time-PCR ,) method to study the function of the gene to abiotic stress NaCl and PEG. Background technique [0002] At present, adversity stress is one of the most important factors and challenges affecting grain production in agricultural production. Crops will be affected by various adverse factors throughout the growing season, such as biotic stress such as pests and diseases, and abiotic stress such as salt, alkali, high temperature, low temperature, drought, etc. These stresses limit the growth and development of plants, and eventually It leads to the reduction of crop yield and quality, and directly affects o...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/81C12N1/19C12R1/865
CPCC12N9/12C12Y207/11013
Inventor 潘洪玉李敬涛余刚孙新华刘金亮刘言志张祥辉贾承国
Owner JILIN UNIV
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