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Cloning and Function Identification of the Promoter of Δ6-Fatty Acid Desaturase Gene from Mucor sp.

Author: MaoRuoYu
Tutor: HuangJianZhong
School: Fujian Normal University
Course: Microbiology
Keywords: gamma - linolenic acid △ 6 fatty acid desaturase Promoter Enhancer Reporter gene
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 24
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Abstract


Gamma-linolenic acid (GLA) is one polyunsaturated fatty acid is widely distributed in nature and the human body has an important physiological role. Δ6-fatty acid desaturase organisms synthesize GLA key rate-limiting enzyme for further study of the molecular mechanisms of its transcriptional regulation by the convergence of the first PCR (LA-PCR) amplified from the Mucor sp.EIM-10 in 1291 bp Δ6-fatty acid desaturase gene 5 'flanking region of a single product, the sequence of multiple transcription factors, promoter database analysis and application of a variety of computer software and more promoter-line forecast website analysis, we found that the eukaryotic promoter sequence having the basic structural characteristics, such as having a CAAT box, TATA box, GC box, and many other components. In this experiment, constructed shuttle vector PYGFP, PYESMCD6 build the promoter function test vector PYMCD6. The cloned Δ6-fatty acid desaturase gene promoter insert PYMCD6, recombinant expression plasmid PYMD6PMCD6. Further fermentation experiments showed that after adding the substrate linoleic acid (LA), comprising the Δ6-fatty acid desaturase promoter region of the recombinant yeast fatty acid methyl ester fractions the GLA Sinfeng, Δ6-fatty acid desaturase proved gene 5 'flanking region can start Δ6-fatty acid desaturase gene expression. Eukaryotic promoter region has a variety of regulatory elements, and the type of genes having the type of upstream promoter element, its position is not the same, which makes the type of control mechanism for the type of gene expression, respectively. The promoter function tests carrier PYMCD6 of different lengths obtained by subcloning technique the Mucor sp.EIM-10Δ6-of fatty acid desaturase gene promoter fragment and connected to the Qian Wenyi built to transform S. cerevisiae uracil-deficient strains. Positive clones were screened after the fermentation test and analyze different length transcription promoter fragment according to the rate of substrate conversion function. The results showed that contain different length promoter deletion fragments (in addition to -121 bp), the recombinant expression vector recombinant yeast fatty acid methyl ester fractions are able to appear GLA new peak, the substrate conversion rate gradually decreases with the reduction in the length of the promoter. However, if the lack of -919 bp ~-784bp fragment of the promoter substrate conversion rate dropped significantly and is very unstable. This shows that the -919 bp ~-784bp region is very likely to have the enhancer effect. In order to further determine the promoter region -919 bp ~-784bp enhancer effect by overlap extension PCR the Mucor sp.EIM-10Δ6-Fatty Acid desaturase gene promoter -919 ~ -784 bp fragment and only has a very The low conversion rate of -434 to -1 bp fragment fusion and fusion fragment into the Qian Wenyi the promoter function tests carrier PYMCD6 of build recombinant plasmid PYOL94MCD6, transforming Saccharomyces cerevisiae uracil deficient strain INVScl is fermentation experiments. The results showed that, after adding the substrate linoleic acid (LA), comprising start promoter region -919 ~ -784 bp and -434 ~ -1 bp fusion fragment of the recombinant yeast fatty acid methyl ester fractions the GLA Sinfeng, transforming rate of more than 60%, significantly higher than the enhancer function of the promoter region -919 ~ -784 bp fragment containing the promoter region -434 to -1 bp conversion rate to make the authentication.

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