Genes having delta 6 fatty acid dehydrogenase function and use thereof

A gene and nucleotide technology, applied to the gene with the function of △6 fatty acid dehydrogenase and its application field, can solve the problems of lack of fishery resources, increase the production cost of PUFAs, limit the utilization of PUFAs, etc.

Inactive Publication Date: 2009-01-28
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

[0008] (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;
[0009] (3) Due to the need to refine low-quality oils, the pr

Method used

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  • Genes having delta 6 fatty acid dehydrogenase function and use thereof
  • Genes having delta 6 fatty acid dehydrogenase function and use thereof
  • Genes having delta 6 fatty acid dehydrogenase function and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1. Obtaining of RnD6C and RnD6D genes

[0044] 1. Preparation of black currant genomic DNA

[0045] 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

[0063] Embodiment two, the construction of RnD6C, RnD6D gene yeast vector

[0064] From the pGEM-T vector containing the genes RnD6C and RnD6D, use the KpnI and Sac I restriction 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 the yeast expression plasmid pYRnD6C and pYRnD6D were transformed into Escherichia coli DH-5α (preserved by our laboratory) and preserved. Its vector diagram see Figure 5 .

Embodiment 3

[0065] Example 3, Expression of RnD6C and RnD6D genes in yeast

[0066] 1. Transformation of Yeast

[0067] Referring to the method described in Invitrogen's pYES2Kit (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. Using the empty pYES2 plasmid as a control, yeast cells containing each expression plasmid were obtained.

[0068] 2. Induced expression in transformed yeast cells

[0069] Get the yeast single colony transformed with the yeast expression plasmid containing the target gene, inoculate it in 50ml SC-U culture medium (refer to the formula described by Invitrogen company pYES2Kit) in the SC-U culture medium containing 2% raffinose, 250rpm, 28 ℃, culture overnight; add NP-40 (purchased from BBI) (final concentration 1%), exogenous linoleic acid and α-linolenic acid substrates (purchased from...

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Abstract

The invention uses two genes of RnD6C and RnD6D which have complete reading frames and are from ribes nigrum L. DNA to express the RnD6C and RnD6D in a yeast expression system; the GC-MS analysis shows that protein which is coded by the two genes in the yeast can respectively catalyze the external substrates of linoleic acid (LA) and Alpha-linolenic acid (ALA) to respectively generate Gamma-linolenic acid (GLA) and parinaric acid (SDA), and has the function of Delta<6> fatty acid dehydrogenase. The invention can be applied to that the gene engineering technology is adopted and the expression system of lower eukaryotic cells and the plant expression system are used for producing GLA and SDA.

Description

technical field [0001] 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 [0002] Polyunsaturated fatty acids (PUFAs) refer to straight-chain fatty acids containing two or more double bonds and a carbon chain length of 18-22 carbon atoms, mainly including linoleic acid (LA, 18 :2Δ 9，12 ), γ-linolenic acid (γ-linolenic acid, GLA, 18:3, Δ 6，9，12 ), ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/04C12N15/82C12P7/40C12P7/44
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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