Crambe filiformis fatty acid elongase and coding gene thereof

A fatty acid elongase, encoding gene technology, applied in the fields of molecular biology and genetic engineering, can solve the problem of complex erucic acid content

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, the cloning of the FAE1 gene is currently limited to a few plants of the Arabidopsis thaliana and Brassica, and the Brassicaceae is the main germplasm resource for the distribution of erucic acid. There are many types of plants and complex erucic acid content. FAE1 Suitable materials for gene cloning

Method used

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  • Crambe filiformis fatty acid elongase and coding gene thereof
  • Crambe filiformis fatty acid elongase and coding gene thereof
  • Crambe filiformis fatty acid elongase and coding gene thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1, the cloning of gene CfFAE1

[0017] Clone CfFAE1, the specific steps are as follows:

[0018] 1. Acquisition of CfFAE1 gene

[0019] C. filiformis 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 finally the CfFAE1 gene was obtained.

[0020] 1. Extraction of C. filiformis 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...

Embodiment 2

[0031] Example 2, Functional Verification of CfFAE1 Gene

[0032] 1. High expression of CfFAE1 gene in yeast

[0033] In order to demonstrate the coding function of the CfFAE1 gene, the present invention clones the CfFAE1 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 CfFAE1. 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-CfFAE1 recombinant vector carrying the CfFAE1 gene and the pYES2 / NT C expression vector were...

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Abstract

The invention provides a fatty acid elongase derived from Crambe filiformis and a coding gene thereof CfFAE1. When the gene is introduced into yeast, the recombinant yeast erucic acid content is up to 0.76+ / -0.14%. The protein and the coding gene thereof provided by the invention are of great theoretical and practical importance in the research on a plant erucic acid gene regulation mechanism, the increase of plant erucic acid content and the improvement of correlated characters, and can play important roles in the gene engineering improvement of the plant erucic acid gene, thereby having wide application prospects.

Description

technical field [0001] The present invention relates to the fields of molecular biology and genetic engineering, more specifically, relates to Crambe filiformis Jacq. 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 is likely to cause diseases such as coronary heart disease and fatty liver. However, in industry, erucic acid and its derivatives, erucamide, etc. have Good plasticity and hydrophobicity, so it has a wide range of uses, and is an impo...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N15/63C12N5/10C12N1/19C12N15/09A01H5/00
Inventor 李密密杭悦宇孙小芹庞慧郭建林李莹严琴琴
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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