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Nucleotide sequence of Pichia pastoris omega3-fatty acid dehydrogenase and application thereof

A fatty acid dehydrogenase, nucleotide sequence technology, applied in genetic engineering and biological fields, can solve problems such as difficulty in obtaining advanced structures and difficulty

Inactive Publication Date: 2009-05-13
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, due to the difficulty of isolating and identifying membrane-bound proteins [MaKeon, 1981, Methods in Enzymology, 12141-12147; Wang et al., 1988, Plant Physiology and Biochemistry, 26: 777-792], it is difficult to obtain ω 3 -The advanced structure of various membrane-bound protein dehydrogenases including fatty acid dehydrogenase, so for the biological function of fatty acid dehydrogenase, many laboratories have adopted the method of cloning related genes from various organisms and transferring them into Among various receptors, a research method for observing fatty acid changes of receptors in receptors

Method used

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  • Nucleotide sequence of Pichia pastoris omega3-fatty acid dehydrogenase and application thereof
  • Nucleotide sequence of Pichia pastoris omega3-fatty acid dehydrogenase and application thereof
  • Nucleotide sequence of Pichia pastoris omega3-fatty acid dehydrogenase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Isolation of the nucleotide sequence of ω3-fatty acid dehydrogenase from Pichia pastoris

[0048] According to the method provided by A. Adams et al. (A. Adams et al., 2000, Experimental Guidelines for Yeast Genetics Methods, 84-89.) Extract the total DNA from the Pichia pastoris cultured for 36 hours, and take 7 μg as a template for polymerization Enzyme chain reaction. According to the published ω3-fatty acid dehydrogenase homologous sequence histidine conserved region I and III region amino acid sequence design degenerate primers (primer 1 and primer 2), with the above-mentioned extracted DNA as a template in the T-Gradient PCR instrument ( Biometra company) on the PCR amplification, the primers used in the reaction, components and amplification conditions are as follows:

[0049] Primer 1: 5-C(T)TA(TCG)GCA(TCG)CAC(T)GAA(G)TGC(T)GGA(TCG)CA-3

[0050] Primer 2: 5-C(T)TT A(TCG)GG A(G)TC A(TCG)GT A(G)TG C(T)TG A(TCG)A-3

[0051]

[0052]

[0053] Ampl...

Embodiment 2

[0065] Example 2: Homology Search of Isolated Nucleotide Sequences

[0066] The amino acid sequence of the predicted ω3-fatty acid dehydrogenase was searched for homology on Genbank. Most of the obtained homologous sequences were sequences encoding ω3-fatty acid dehydrogenase, and a few of them encoded △ 12 - the sequence of fatty acid dehydrogenase and other dehydrogenases and other enzymes, which has the highest homology with Saccharomyces kluyveri: identity 64%, (attached figure 2 , with figure 2 Shows the amino acid sequence homology comparison diagram of Pichia pastoris ω3-fatty acid dehydrogenase of the present invention and Saccharomyceskluyveri ω3-fatty acid dehydrogenase (AB118663), PPW3: Pichia pastoris ω3-fatty acid dehydrogenase of the present invention Hydrogenase, SKW3: Klebsiella omega 3-fatty acid dehydrogenase. (Shaded portions indicate amino acids that are identical between the two sequences). This shows that the enzyme encoded by the new nucleotide sequen...

Embodiment 3

[0067] Example 3: Construction of Saccharomyces cerevisiae recombinant expression vector

[0068] According to the coding region sequence shown in SEQ ID NO: 1, a pair of gene-specific amplification primers (primer 11 and primer 12) were designed to isolate its potential open reading frame sequence:

[0069] Primer 11: 5-GGCAAGCTTATGTCAAAAGTCACTGTTTCGGG-3' (SEQ ID NO: 11);

[0070] Primer 12: 5'-GCCTCTAGATTAGGTATCC TTAGG-3' (SEQ ID NO: 12);

[0071] The 5' ends of these two primers contain HindIII and XbaI enzyme cutting sites respectively. The amplification conditions and reaction components used are the same as above, and the sequencing result of the amplified product shows that it is consistent with the sequence of 99bp-1346bp shown in SEQ ID NO:1. Then take 50 μl of the PCR product and 1 μl of pYES2.0 for double enzyme digestion, recover the large fragment, and use T4 ligase to ligate overnight in a 4-degree refrigerator. The ligation product was transformed into Escher...

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Abstract

The invention discloses a separating nucleotide sequence or segment, analogue and derivant of coded omega3-fatty acid dehydrogenase with SEQ ID NO:1, which is characterized by the following: the invention also provides connecting sequence of coded delta12-fatty acid dehydrogenase nucleotide sequence and external adjusting sequence, functional expressive carrier and carrier cell organism and succession; the probe corresponding to nucleotide sequence can test relative sequence.

Description

technical field [0001] The invention belongs to the field of biotechnology and genetic engineering, and relates to cloning omega from a kind of yeast—Pichia Pastoris 3 - Fatty acid dehydrogenase gene, specifically Pichia pastoris omega 3 - Nucleotide sequence of fatty acid dehydrogenase and its application. Link the gene directly or with different expression vectors, transfer it into bacteria, yeast, plants or animals, and use it to encode ω 3 - Method and application of fatty acid dehydrogenase to produce unsaturated fatty acid. Background technique [0002] Polyunsaturated fatty acids PUFAs (polyunsaturated fatty acids) are important components of cell membranes and are necessary for the growth of organisms. According to different metabolic pathways and biological activities, PUFAs are mainly divided into two categories: n-6 unsaturated fatty acids and n-3 unsaturated fatty acids. In most fungi and plants, n-6 unsaturated fatty acids are omega 3 - Fatty acid dehydroge...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/04C12N15/63C12N1/19C12P7/64
Inventor 李明春张昕欣魏东盛邢来君周皓
Owner NANKAI UNIV