Glycerol-3- phosphoric desaturase gene relating with glycerol synthesis and uses thereof

A phosphate dehydrogenase and glycerol technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve problems such as the decline in economic benefits of glycerol, and achieve the effect of good application prospects

Inactive Publication Date: 2008-12-10
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Economic Benefits of Glycerol (Glycerol) Pr

Method used

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  • Glycerol-3- phosphoric desaturase gene relating with glycerol synthesis and uses thereof
  • Glycerol-3- phosphoric desaturase gene relating with glycerol synthesis and uses thereof
  • Glycerol-3- phosphoric desaturase gene relating with glycerol synthesis and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Cloning and analysis of the full-length coding regions of DvGPDH1 and DvGPDH2

[0030] Extract total RNA from Salina salina, reverse transcribe the first strand of cDNA, and use the first strand of cDNA as a template to clone into DvGPDH1 by PCR using primers GPDH15'p and GPDH13'p, and clone into DvGPDH2 through primers GPDH25'p and GPDH23'p . In order to further prove the function and application (embodiment three) of these two genes in yeast, by primer GPDH15'p and GPDH25'p, add cloning enzyme point EcoRI and eukaryotic translation conservation sequence Kozak at gene 5' end; Through primer GPDH13 'p and GPDH23'p add clonase site Xho I at the 3' end of the gene.

[0031] The primer sequences are as follows:

[0032] (1) GPDH15'p (introducing EcoR I restriction site and Kozak sequence):

[0033] 5'-AGAATTC AGCATGG TCCTAGGGTCATCACC-3' is the EcoR I recognition site in bold, and the Kozak sequence is underlined;

[0034] (2) GPDH13'p (introduction of Xho ...

Embodiment 2

[0041] Example 2 Protein structure and homology analysis of DvGPDH1 and DvGPDH2

[0042]Using the NCBI (http: / / www.ncbi.nlm.nih.gov / structure / cdd / cdd.shtml) conserved domain analysis system to analyze the proteins encoded by DvGPDH1 and DvGPDH2, the results show that the proteins encoded by the two genes have Unique SERB-GPDH dual domain ( figure 1 ). DvGPDH1 and DvGPDH2 have a GPDH domain of about 400 amino acids at the C-terminus, and compared with the homologous GPDH protein structure in animals and plants, they also have an additional SERB domain of 300 amino acids in length at the N-terminus.

[0043] Homologous analysis of DvGPH1 and DvGPDH2 was carried out by using Vector NTI9.1.0 program. Analysis results show that they have high homology with homologous genes in algae, for example, the homology with Chlamydomonas CrGPDH1 is 45.7% and 45.3%, respectively. The homology with animals or higher plants is relatively low, for example, the homology of yeast ScGpd1p is 19.8...

Embodiment 3

[0045] Example 3 Functional Analysis of DvGPDH1 and DvGPDH2 in Yeast Mutants

[0046] (1) Construction of yeast expression vector

[0047] DvGPDH1 and DvGPDH2 cloned in Example 1 were respectively constructed into yeast expression vector pAJ401 (constitutive strong expression vector) or pYES2 (galactose-inducible expression vector) using the introduced cloning enzyme sites EcoR I and XhoI. In addition, we also constructed vector clones containing only the GPDH domain segment (named DvGPDH1ΔN and DvGPDH2ΔN respectively), and analyzed the function of the gene after the SERB segment was deleted. DvGPDH1ΔN was amplified by primers GPDH1-GPD5’p and GPDH13’p; DvGPDH2ΔN was amplified by primers GPDH2-GPD5’p and GPDH23’p. Use primers GPDH1-GPD5'p and GPDH2-GPD5'p to add cloning enzyme site EcoR I and eukaryotic translation conservation sequence Kozak at the 5' end of the target gene; primers GPDH13'p and GPDH23'p add cloning enzyme at the 3' end of the target gene Point Xho I. The ...

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Abstract

The invention relate to a cDNA sequence of glycerol-3-phosphate dehydrogenase genes DvGPH1 and DvGPDH2 associated with glycerol synthesis, and an amino acid sequence of an encoding protein. The expression vector of the DvGPH1 or DvGPDH2 gene is translated into a yeast gpd1delta cell. The yeast cell shows a salt resistant phenotype on a culture medium containing salts, thereby verifying that the two genes can strengthen glycerol synthesis in the yeast cell and improve the salt resistance of the yeast cell, and simultaneously verifying that the two genes have good application value on improving the glycerol synthesis amount in organisms such as the yeast through genetic engineering means and strengthening the salt resistance of the organisms. As the invention further improves the glycerol-3-phosphate dehydrogenase activity and the capability of catalyzing and synthesizing the glycerol of the two gene products and the salt resistance of a corresponding yeast transformant through the deletion of part of sequences of the DvGPH1 or DvGPDH2 genes, the two deletion type gene derivatives have better prospect in the application fields. In addition, the invention verifies that the expression of the DvGPH1 and DvGPDH2 genes in a dunaliella is subjected to the induction of salt stress, thereby showing that the two genes are the key enzymes for dunaliella synthetic glycerol and further providing the theoretical base for the application of the two genes to the fields.

Description

technical field [0001] The invention relates to a glycerol-3-phosphate dehydrogenase gene related to glycerol synthesis and its application. Background technique [0002] The world population continues to grow and is expected to reach 6 billion by the end of 2050. On the other hand, 20% of the world's arable land is severely salinized, seriously affecting the production of food. The supply of food crops is being threatened by land salinization. It is very important to understand the mechanism of biological salt resistance to improve crop salt tolerance by means of breeding and genetic engineering. [0003] Glycerin is an important light chemical raw material, and the glycerin market is in short supply. With the continuous reduction of petroleum energy and arable land, the production of glycerin by chemical methods has encountered bottlenecks, and the output has also continued to decrease. The economic benefit of producing glycerol (glycerol) by chemical synthesis is cons...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/04C12N15/63C12N1/19
Inventor 宋仁涛许政暟何云霞孟祥宗张男
Owner SHANGHAI UNIV
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