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Brassicealongata ehrhart fatty acid elongase and coding gene thereof

A technology of fatty acid elongation enzyme and coding gene, which is applied in the fields of molecular biology and genetic engineering, and can solve problems such as the complex content of erucic acid

Inactive Publication Date: 2013-03-20
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The FAE1 (Fatty Acid Elongase 1) gene is a key gene that regulates erucic acid synthesis. Although James et al. first cloned the FAE1 gene from Arabidopsis thaliana, complete FAE1 genes have been isolated from various cruciferous plants. However, at present, the cloning of FAE1 gene is limited to a few plants of Arabidopsis and Brassica, and Brassicaceae is the main germplasm resource of erucic acid distribution. There are many types of plants and complex erucic acid content. Suitable material for cloning

Method used

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  • Brassicealongata ehrhart fatty acid elongase and coding gene thereof
  • Brassicealongata ehrhart fatty acid elongase and coding gene thereof
  • Brassicealongata ehrhart fatty acid elongase and coding gene thereof

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Experimental program
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Embodiment 1

[0016] Embodiment 1, the cloning of gene BeFAE1

[0017] To clone BeFAE1, the specific steps are as follows:

[0018] 1. Acquisition of BeFAE1 gene

[0019] Brachycarpus mustard was cultivated under normal conditions. After the true leaves grew, the leaves were collected, DNA was extracted from the leaves, polymerase chain reaction was carried out, and the BeFAE1 gene was finally obtained.

[0020] 1. Extraction of Genomic DNA

[0021] Take about 0.2 g of fresh young living plant leaves, grind them fully under liquid nitrogen freezing conditions, transfer them to a 1.5 mL centrifuge tube, add 500 μL of CTAB extract, and bathe in water at 65°C for 45 minutes, during which time they are inverted and mixed 3 times; add 500 μL of Chloroform-isoamyl alcohol with a volume ratio of 24:1, mixed evenly, 12000r·min -1 Centrifuge for 15 min; take the supernatant, add 2 times the volume of absolute ethanol, and put it in the refrigerator at -20°C overnight; 4000r min -1 Centrifuge for...

Embodiment 2

[0031] Example 2, Functional Verification of BeFAE1 Gene

[0032] 1. High expression of BeFAE1 gene in yeast

[0033] In order to demonstrate the coding function of the BeFAE1 gene, the present invention clones the BeFAE1 gene between the BamHI and KpnI sites of pYES2 / NT C from Invitrogen, and transforms the yeast strain InvSc1 to obtain high-level expression.

[0034] The present invention utilizes the multiple cloning sites of the pYES2 / NT C high-efficiency expression vector of Invitrogen Company to realize the high-efficiency expression of BeFAE1. The enzyme cleavage site used in this experiment makes the 5' end of the product expressed by the pYES2 / NT C expression vector add 6 consecutive His fusion proteins, and these 6 consecutive His constitute the specific binding site for i-NTA gel. Therefore, the expression product can be purified by affinity chromatography.

[0035] The pMD-BeFAE1 recombinant vector carrying the BeFAE1 gene and the pYES2 / NT C expression vector wer...

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Abstract

The invention provides a fatty acid elongase derived from brassicealongata ehrhart, and a coding gene thereof (BeFAE1). The gene is introduced in to yeast; and the erucic acid content in the recombinant yeast reaches 1.52+ / -0.13%. The protein and the coding gene thereof have important theoretical and practical significance for the research of a genetic control mechanism of plant erucic acids, the increasement of the erucic acid content and the improvement of related traits, can play an important role in the genetic engineering improvement of the plant erucic acid genes, and have wide application prospects.

Description

technical field [0001] The present invention relates to the fields of molecular biology and genetic engineering, and more specifically relates to Brassicaelongata Ehrhart fatty acid elongase (FAE 1) and its coding gene. Background technique [0002] Erucic acid (C22:1) is a very long chain unsaturated fatty acid, mainly found in the seeds of Brassicaceae and Tropaeolaceae. As a metabolite of plants, erucic acid also affects the growth and development of plants to a certain extent, and participates in the synthesis of biofilm cutin or wax in the interaction between plants and the environment. In terms of nutritional value, erucic acid is an anti-nutritional component of edible oil, which is not easy to be digested and absorbed by the human body, and it is easy to cause diseases such as coronary heart disease and fatty liver. However, in industry, erucic acid and its derivatives, such as erucic acid amide, have good plasticity and hydrophobicity, so they have a wide range of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/63C12N5/10C12N1/19C12N15/09A01H5/00
Inventor 孙小芹杭悦宇李莹李密密庞慧郭建林严琴琴
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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