Genes with functions of delta6 fatty acid desaturase and application thereof

A gene and nucleotide sequence technology, which is applied to genes with Δ6 fatty acid dehydrogenase function and its application fields, can solve the problems of limiting the utilization of PUFAs, increasing the production cost of PUFAs, and no reports.

Inactive Publication Date: 2010-12-22
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] (2) The limited natural marine fishery resources are lack of fishery resources due to overfishing, and the inherent fishy smell and oxidative instability of fish oil limit the further utilization of PUFAs;
[0010] (3) Due to the need to refine low-quality oils, the production cost of PUFAs is greatly increased
Furthermore, although borage [5] has Δ 6 Fatty acid dehydrogenase gene has been reported abroad, but the two genes of this application are derived from the Δ 6 Fatty acid dehydrogenase gene, so far not reported at home and abroad

Method used

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  • Genes with functions of delta6 fatty acid desaturase and application thereof
  • Genes with functions of delta6 fatty acid desaturase and application thereof
  • Genes with functions of delta6 fatty acid desaturase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1. Obtaining of RnD6C and RnD6D genes

[0045] 1. Preparation of black currant genomic DNA

[0046] Using the live plant of black tea currant (Ribes nigrum L.) grown in Beijing Botanical Garden as the material, take its young leaves (about 100mg), add steel balls (diameter 5mm) into a 7ml Ep tube, and freeze in liquid nitrogen for 20 -30min, high-speed crushing on the vortex machine, repeat the operation 2-3 times until the material is completely crushed. Add 1-2ml of preheated CTAB extract solution (see "Refined Molecular Biology Experiment Guide" 2001, Science Press, translated by Yan Ziying and Wang Hailin), and mix well. 65°C water bath for 30min. Add an equal volume of chloroform, and gently extract for about 5 minutes. Centrifuge at 12000rpm room temperature for 10min. Take the supernatant, add 1 / 2 volume of isopropanol to mix well, and place at room temperature for 10 min to precipitate DNA. The precipitated DNA was picked out with a Tip, washed t...

Embodiment 2

[0064] Embodiment two, the construction of RnD6C, RnD6D gene yeast vector

[0065] From the pGEM-T vector containing the genes RnD6C and RnD6D, use the Kpn I and Sac I enzyme cutting sites to obtain the RnD6C and RnD6D genes after double cutting, and directionally clone them into the yeast expression vector pYES2 (purchased from Ivitrogen Company) to obtain yeast expression Plasmids pYRnD6C and pYRnD6D, transformed into Escherichia coli DH-5α (preserved by our laboratory) were preserved. Its vector diagram see Figure 5 .

Embodiment 3

[0066] Example 3, Expression of RnD6C and RnD6D genes in yeast

[0067] 1. Transformation of Yeast

[0068] Referring to the method described in Invitrogen's pYES2 Kit (Cat# V285-20), the yeast expression plasmids pYRnD6C and pYRnD6D of the above-mentioned chimeric genes were transformed into Saccharomyces cerevisiae auxotrophic strain INV Sc I (purchased from Invitrogen) using lithium acetate. , with the empty pYES2 plasmid as a control, yeast cells containing each expression plasmid were obtained.

[0069] 2. Induced expression in transformed yeast cells

[0070] Get the yeast single colony transformed with the yeast expression plasmid containing the gene of interest, and inoculate it in 50ml SC-U culture fluid (with reference to the formula described in Invitrogen's pYES2 Kit) containing 2% raffinose SC-U culture fluid, 250rpm, 28°C, Cultivate overnight; add NP-40 (purchased from BBI Company) (final concentration 1%), exogenous linoleic acid and α-linolenic acid substrate...

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Abstract

The invention utilizes two genes RnD6C and RnD6D which root in DNA of Ribes nigrum L. and have complete reading frames and expresses the genes in a yeast expression system. GC-MS analysis shows that the protein coded by the two genes in the yeast can respectively catalyze applied substrates linoleic acid (LA) and alpha-linoleic acid (ALA) to respectively generate gamma-linoleic acid (GLA) and stearidonic acid (SDA). The genes have the functions of delta6 fatty acid desaturase. The genes can be applied to gene engineering technology to produce GLA and SDA by using a lower eukaryotic cell expression system and a plant expression system.

Description

[0001] This application is a divisional application of the patent application whose application number is 2007101194771. technical field [0002] The present invention relates to plant coding gene and its application. Specifically, it involves two genes with complete reading frames, RnD6C and RnD6D, which are respectively derived from two DNA sequences of black currant (Ribes nigrum L.), and two genes with complete reading frames cloned on this basis. gene sequence. The present invention also relates to the gene encoded with Δ 6 Polypeptide with fatty acid dehydrogenase function, lower eukaryotic cell expression vector and plant expression vector containing the DNA sequence, host cell and application of the gene to transform lower eukaryotic organisms and plants to produce GLA and SDA respectively. Background technique [0003] Polyunsaturated fatty acids (PUFAs) refer to straight-chain fatty acids containing two or more double bonds and a carbon chain length of 18-22 carb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/04C12N15/63C12N15/82C12N1/13C12N1/19C12N5/10C12P7/64
Inventor 胡赞民宋丽英张岩胡军尹维波陈宇红
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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