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Gene producing glycerin candida NAD+ depending- glycerin3- glycerophosphate dehydrogenase and clone thereof

A phosphate dehydrogenase, Candida technology, applied in genetic engineering, plant genetic improvement, fermentation, etc., can solve problems such as lack of molecular knowledge and genetic background

Inactive Publication Date: 2008-04-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, this yeast has been used in the industrial production of glycerol fermentation, but compared with Saccharomyces cerevisiae, its related molecular knowledge and genetic background are very lacking

Method used

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  • Gene producing glycerin candida NAD+ depending- glycerin3- glycerophosphate dehydrogenase and clone thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: PCR amplification of the glycerol 3-phosphate dehydrogenase gene fragment of Candida glycerinogenes WL2002-5-59 with degenerate primers

[0049] 1. Design of degenerate primers

[0050] Search the sequences of the glycerol 3-phosphate dehydrogenase genes of related yeast and other eukaryotes published in GeneBank, and through the comparison and analysis of amino acid sequences, find the amino acid sequences corresponding to the two conserved regions VVGSGNWGT and GALKNVVA, according to these two conserved regions Design a pair of degenerate primers for the region

[0051] CD-U: 5'-TBRTBGGITCHGGYAAITGGG-3',

[0052] CD-R: 5'-RGCVAIVAYRTTYTTYARIGC-3'.

[0053] Wherein I refers to inosine base; R refers to A or G; Y refers to C or T; H refers to A, C or T; V refers to A, C or G; B refers to C, G or T.

[0054] 2. Extraction of Genomic DNA from Candida glycerinogenes WL2002-5-59

[0055] Chromosomal DNA preparation of Candida glycerologenus WL2002-5-59 was ca...

Embodiment 2

[0059] Embodiment 2: Reverse primer PCR amplifies the full-length sequence of glycerol 3-phosphate dehydrogenase gene

[0060] 1. Design of reverse PCR primers

[0061] Design a pair of reverse PCR primers according to the sequence results obtained by PCR amplification with degenerate primers

[0062] CgI-F: 5′-CCTTATTTCCGTGTTCGTGTTATTAGTG-3′

[0063] CgI-R: 5'-CGCCTTCAATCAATTCTTCATAGAC-3' was used for PCR amplification.

[0064] 2. Preparation of PCR template

[0065] Genomic DNA of C. glycerinogenes WL2002-5-59 was extracted according to the above method, and 5 μg of genomic DNA was digested with restriction enzymes BamH I, Pst I and Sal I respectively, and the digestion effect was checked by agarose electrophoresis. The digested product was extracted with equal volumes of 1:1 phenol and chloroform, then precipitated with 2.5 times the volume of absolute ethanol, and finally dissolved in 50 μL of ultrapure water. In 200 μL ligation system, add 0.1 μg digested DNA, 20 μL ...

Embodiment 3

[0068] Example 3: C. glycerinogenes glycerol 3-phosphate dehydrogenase gene sequence information and homology analysis

[0069] The nucleotide sequence of the Candida glycerologenicum glycerol 3-phosphate dehydrogenase gene of the present invention is 4900 bp in length, and the detailed sequence is shown in SEQ ID NO.1, wherein the open reading frame is at 2082-3246 nucleotides, encoding 388 amino acids , and the internal coding frame does not contain introns.

[0070] The gene sequence of Candida glycerogenes glycerol 3-phosphate dehydrogenase and its encoded protein were analyzed by BSLAT program in Non-redundant GeneBank+EMBL+DDBJ+PDB and Non-redundant GeneBank cdstranslations+PDB+SwissProt+PIR database And protein homology search, it was found that at the amino acid level, it has 70% homology with the glycerol 3-phosphate dehydrogenase gene of Pichia angus, and with the glycerol 3-phosphate dehydrogenase gene of Saccharomyces cerevisiae The genes share 60% homology. It c...

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Abstract

Glycerol fermentation by candida glycerinogenes NAD+ depends on glycerine-3-phosphate dehydrogenase gene and clone thereof and pertains to the microorganism molecular biology field. The invention relates to a new gene order SEQID NO.1. a degenerate primer PCR method and a reverse PCR method are adopted to clone rate-limiting enzyme NAD+ depending glycerine-3-phosphate dehydrogenase gene and lateral regulation sequence thereof from genome DNA of glycerine-producing candida mycoderma WL2002-5-59 which is produced by glycerine. The total length of the gene is 4900bp and a start codon ATG is at 2082bp while a stop codon TAA is at 3246bp. The sequence has no intron but has an 1167bp complete open reading frame which encodes 388 amino acids. The gene has homology as high as 60 percent with saccharomyces cerevisia GPD1 gene and 70 percent with Angus pichia yeast GPD gene. The clone of the gene expands gene resource of microorganism anti-osmotic pressure stress research and lays a foundation for research on molecular mechanism of glycerine-producing candida mycoderma producing glycerine with high yield.

Description

technical field [0001] Glycerologenic Candida NAD + Dependent-glycerol 3-phosphate dehydrogenase gene and its cloning, involving the fields of microbiology, genetic engineering and molecular biology, especially a key enzyme encoding gene NAD in the glycerol synthesis pathway in hyperosmotic glycerologenic Candida tolerant + Dependence-glycerol 3-phosphate dehydrogenase gene and its cloning. Background technique [0002] When the cell is in a high osmotic pressure environment, in order to keep the water and small molecular substances in the cell from leaking out to the environment, it is necessary to increase the intracellular osmotic pressure by synthesizing some biocompatible substances by itself to balance the intracellular and extracellular The pressure difference of the cell can protect and defend the cells. For yeast cells, the biocompatible substances produced under osmotic stress are mainly glycerol, with a small amount of other polyols. NAD + Dependent 3-phosphat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/04
Inventor 诸葛健陈献忠方慧英王正祥饶志明沈微
Owner JIANGNAN UNIV
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