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Rocket salad 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: 2013-01-23
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 materials for gene cloning

Method used

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  • Rocket salad fatty acid elongase and coding gene thereof
  • Rocket salad fatty acid elongase and coding gene thereof
  • Rocket salad fatty acid elongase and coding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1, the cloning of gene EvFAE1

[0017] To clone EvFAE1, the specific steps are as follows:

[0018] 1. Acquisition of EvFAE1 gene

[0019] Arugula was cultivated under normal conditions, and after the true leaves grew, the leaves were collected, DNA was extracted from the leaves, polymerase chain reaction was carried out, and the EvFAE1 gene was finally obtained.

[0020] 1. Extraction of arugula 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 EvFAE1 Gene

[0032] 1. High expression of EvFAE1 gene in yeast

[0033] In order to demonstrate the coding function of the EvFAE1 gene, the present invention clones the EvFAE1 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 EvFAE1. 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 of i-NTA gel. Therefore, the expression product can be purified by affinity chromatography.

[0035] The pMD-EvFAE1 recombinant vector carrying the EvFAE1 gene and the pYES2 / NT C expression vector were...

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Abstract

The invention provides a rocket salad fatty acid elongase and a coding gene EvFAE1 thereof. The gene is introduced into yeast, and the content of recombination yeast erucic acid reaches 2.50+ / -0.06%. The protein and coding gene have important theoretical and practical significance for studying a plant erucic acid genetic regulation mechanism, and improving the erucic acid content of plants and correlated characters, plays an important role in the erucic acid genetic engineering improvement and is wide in the application prospect.

Description

technical field [0001] The invention relates to the field of molecular biology and genetic engineering, more specifically, relates to aruga (Eruca vesicaria subsp. sativa (Miller) Thellung) fatty acid elongase (FAE1) 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 hav...

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 郭建林杭悦宇孙小芹庞慧李莹李密密严琴琴
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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