Microula sikkimensis delta 6-fatty acid desaturase MsD6D gene family as well as recombinant expression vector and application of MsD6D gene family

A gene family and expression vector technology, applied in the field of genetic engineering, can solve the problems of high price, high cost and complicated operation

Inactive Publication Date: 2015-02-18
SOUTHWEST UNIVERSITY
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  • Description
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  • Application Information

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Problems solved by technology

Cultivating algae and fungi to produce PUFA is an important research hotspot, and has made impo

Method used

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  • Microula sikkimensis delta 6-fatty acid desaturase MsD6D gene family as well as recombinant expression vector and application of MsD6D gene family
  • Microula sikkimensis delta 6-fatty acid desaturase MsD6D gene family as well as recombinant expression vector and application of MsD6D gene family
  • Microula sikkimensis delta 6-fatty acid desaturase MsD6D gene family as well as recombinant expression vector and application of MsD6D gene family

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Experimental program
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Embodiment 1

[0035] Embodiment 1, the cloning of microspore grass D6D (MsD6D) gene family

[0036] (1) Extraction of total DNA and total RNA of Microporus genome

[0037] The young leaves of Microporus plants were taken, and the total genomic DNA was extracted by the cetyltrimethylammonium bromide (CTAB) method, and the quality and concentration of the nucleic acid samples were evaluated by 1.0% agarose gel electrophoresis and spectrophotometry. The results showed that the total DNA of the extracted Microspore genome had good integrity, the average molecular weight was slightly larger than the 23kb band of λ-HindIII DNA Marker, the RNA was completely digested, and the purity was high by spectrophotometry, which could be used for PCR amplification.

[0038] At the same time, the roots (Ro), stems (St), leaves (Le), buds (Bu), flowers (Fl), early seeds (ES), middle seeds (MS) and late seeds (LS) of Microporus For materials, total RNA was extracted with a small amount of plant tissue RNA ext...

Embodiment 2

[0053] Embodiment 2, the bioinformatics analysis of MsD6D gene family

[0054] Perform gene and protein annotation, sequence alignment, open reading frame (ORF) search and translation, parameter analysis on Vector NTI Advance 9.0, and perform BLAST and protein on http: / / www.ncbi.nlm.nih.gov / CDD search, protein structure analysis at http: / / bip.weizmann.ac.il / and bioinformatics sites with links such as www.expasy.org at http: / / prodes.toulouse.inra.fr / multalin / multalin.html and http: / / www.ebi.ac.uk / clustalw / for multiple alignment and phylogenetic analysis of gene and protein sequences.

[0055] (1) Structure and nucleic acid characteristics of the gene family of MsD6D

[0056] MsD6D1 as figure 2 As shown in middle A, the result shows that the MsD6D1 gene is 1818bp, and the longest mRNA is 1818bp (excluding the poly(A) tail, the same below), where A1 and A5 are the transcription start sites, and the longest 5'UTR (untranslated region) 132bp, G 1668 、C 1694 、C 1753 、C 1...

Embodiment 3

[0068] Example 3, MsD6D gene family expression organ-specific fluorescent quantitative PCR detection

[0069] The total RNA of roots (Ro), stems (St), leaves (Le), buds (Bu), flowers (Fl), early seeds (ES), middle seeds (MS) and late seeds (LS) of Microporus , and then treated with RNase-free DNase I according to its instructions to eliminate DNA contamination. Take 1 μg of total RNA from each organ, and use PrimeScript in a 20 μl system TM RT reagent Kit with gDNA Eraser for reverse transcription to obtain the first strand of total cDNA for gene expression detection. Fluorescent quantitative PCR detection was carried out on the CFX96 quantitative PCR instrument, and the kit was Premix Ex Taq TM II (Tli RNaseH Plus) ROX plus uses the primer combination F25S and R25S for internal standard amplification. The primer combinations for MsD6D1 and MsD6D2 are FMsD6D1S and RMsD6D1S, FMsD6D2S and RMsD6D2S, respectively. The primer sequences are shown in Table 2.

[0070] Fluoresc...

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Abstract

The invention discloses a microula sikkimensis delta 6-fatty acid desaturase MsD6D gene family as well as a recombinant expression vector and the application of the MsD6D gene family. The MsD6D gene family comprises two members MsD6D1 and MsD6D2, has the delta 6-fatty acid desaturase activity, and is capable of catalyzing linoleic acid to form gamma-linolenic acid (GLA) and also catalyzing alpha-linolenic acid (ALA) to form stearidonic acid (SDA); after the sense transformation of the MsD6D1 and the MsD6D2 into the microula sikkimensis, the content of GLA and the content of SDA in the GMO (Genetically Modified Organism) seeds are greatly increased; after the transformation of the MsD6D1 and the MsD6D2 into cabbage type rape, GLA and SDA that the non-GMO rape seeds do not have are accumulated in the GMO rape seeds; as a result, new resource materials can be created by use of the MsD6D gene family, and the new resource materials can be used for industrial extraction of GLA and SDA or directly used for producing health type edible oil rich in GLA and SDA.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to the MsD6D gene family of microporus Δ6-fatty acid desaturase, and also relates to the recombinant expression vector and application of the MsD6D gene family. Background technique [0002] Polyunsaturated fatty acids (Polyunsaturated Fatty Acids, PUFA) are unsaturated fatty acids containing more than two double bonds between carbon atoms, and its metabolic pathway is based on linoleic acid (Linoleic acid, LA; C18:3Δ 9,12 ,n-6) as the initial substrate, under the catalysis of a series of fatty acid desaturase (fatty acid dehydrogenase) and fatty acid elongase, successively generate γ-linolenic acid (γ-linolenic acid, GLA; C18:3Δ 6,9,12 ,n-6), α-linolenic acid (ALA; C18:3Δ 9,12,15 , n-3), stearidonic acid (stearidonic acid, SDA, octadecatetraenoic acid, OTA, C18:4Δ 6,9,12,15 ,n-3), double homogamma-linolenic acid (DHLG), arachidonic acid (ARA), eicosapentaenoic acid (...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/82A01H5/00
Inventor 柴友荣柴成燕付春韩发王瑞练剑平马赑周雪荣
Owner SOUTHWEST UNIVERSITY
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