Asperugo procumbens delta6-fatty acid desaturase ApD6D gene family as well as recombinant expression vector and application thereof

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

Inactive Publication Date: 2015-03-25
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • 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 important achievements, but it is still troubled by complicated operation, high cost and high price

Method used

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  • Asperugo procumbens delta6-fatty acid desaturase ApD6D gene family as well as recombinant expression vector and application thereof
  • Asperugo procumbens delta6-fatty acid desaturase ApD6D gene family as well as recombinant expression vector and application thereof
  • Asperugo procumbens delta6-fatty acid desaturase ApD6D gene family as well as recombinant expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the cloning of grass grass D6D (ApD6D) gene family

[0037] (1) Extraction of the total DNA and total RNA of the grass grass genome

[0038] The young leaves of the grass plant 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 Grass grass 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 as detected by spectrophotometry, which could be used for PCR amplification.

[0039] 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 grass grass were used as materials , respectively, using a small amount of pla...

Embodiment 2

[0053] Embodiment 2, the bioinformatics analysis of ApD6D 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) The structure and nucleic acid characteristics of the gene family of ApD6D:

[0056] ApD6D1 as figure 2 As shown, the results show that the ApD6D1 gene is 1768bp, and the mRNA is 1768bp (excluding the poly(A) tail, the same below), where A1 is the transcription start site, 5'UTR (untranslated region) is 161bp, and there is C 1768 It is poly(A) tailing site, 3'UTR is 257bp, and ...

Embodiment 3

[0068] Example 3, ApD6D gene family expression organ-specific fluorescent quantitative PCR detection

[0069] With the total RNA of root (Ro), stem (St), leaf (Le), bud (Bu), flower (Fl), early seed (ES), middle seed (MS) and late seed (LS) of rough grass, Then treat 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 ApD6D1 and ApD6D2 are FApD6D1S and RApD6D1S, fApD6D2S and rApD6D2S, respectively. The primer sequences are shown in Table 2.

[0070] Fluorescent quanti...

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Abstract

The invention discloses an asperugo procumbens delta6-fatty acid desaturase ApD6D gene family as well as a recombinant expression vector and application thereof. The ApD6D gene family comprises two members ApD6D1 and ApD6D2, has delta6-fatty acid desaturase activity, and is capable of catalyzing linoleic acid to form gamma-linolenic acid (GLA), and catalyzing alpha-linolenic acid (ALA) to form therapic acid (SDA); after ApD6D1 and ApD6D2 senses are transferred into asperugo procumbens, the GLA and SDA content in transgenic asperugo procumbens seeds is greatly improved; after the ApD6D1 and ApD6D2 are transferred into cabbage type rape in a sense manner, the GLA and the SDA, which are not included in non-transgenic rape seeds are accumulated in the transgenic rape seeds; new resource materials can be created by using the ApD6D gene family; and the asperugo procumbens delta6-fatty acid desaturase ApD6D gene family can be applied to industrial production of the GLA and the SDA, or can be directly applied to production of health edible oil rich in GLA and SDA.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to the ApD6D gene family of grass grass Δ6-fatty acid desaturase, and also relates to the recombinant expression vector and application of the ApD6D 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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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/63C12N9/02C12P7/64
Inventor 柴友荣柴成燕练剑平付春王瑞韩发周雪荣马赑
Owner SOUTHWEST UNIVERSITY
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