The Full-length CDNA Cloning And Expression Analysis Of ELOVLm And ELOVL6 Of Eriocheir Sinensis

Elongation of long chain fatty acids(ELOVL)which participating in the extension of carbon chain fatty acid, is the key enzyme in the process of fatty acid synthesis, so this paper is in order to explore the ability of fatty acid synthesis of Eriocheir sinensis and its synthesis process and so on, preliminarily exploring the ELOVL extended process of fatty acid for Eriocheir sinensis.This paper used the reverse transcription PCR(RT-PCR) technology and the rapid amplification cDNA end(RACE) technology, cloning ELOVLm cDNA(GenBank accession number: KR005628) and ELOVL6 cDNA(GenBank accession number: KT779219) ofEriocheir sinensis.Among them, ELOVLm sequence is 2089 bp,whose open reading frame(ORF) is 1065 bp, encodind 354 amino acids and ELOVL6 sequence is 2247 bp,whose open reading frame is 1139 bp, encoding 290 amino acids.Both sequences have characteristics of ELOVL family: several transmembrane areas, high conservative histidine HXXHH clusters and a few of conservative districts, at the same time ELOVL6 has a typical endoplasmic reticulum retention of signal KXKXX.Through real-time fluorescent quantitative PCR(qPCR) technology to study the two genes’ expression of different organizations and when feeding different fat sources in Eriocheir sinensis. Results showed that both genes were expressed in tissues such as hepatopancreas, intestine, muscle, stomach, heart and gill,meanwhile it was expressed highest in hepatopancreas which was significantly higher than other tissues(P<0.05).The fact is that ELOVL gene expressed secondly in intestine, which lower than hepatopancreas, at the same time ELOVL6 expressed little or trace in other organizations. Results in different feeding group showed that it was detected in four edible parts including hepatopancreas, muscle, ovary and testis. The expression level in hepatopancreas, ovarian of ELOVLm and in hepatopancreas of ELOVL6 appeared significantly increased(P<0.05) when the ratio of fish oil(rich in n-3 PUFA)decreased, soybean oil(lack of n-3 PUFA) increased in feed, but the performance is not obvious in testis and muscles(P>0.05).All the above results showed that theexpression of ELOVL was influenced by feed fat levels in Eriocheir sinensis.It was through constructing eukaryotic expression vector and inducing expression of ELOVL gene in Saccharomyces cerevisiae to explore funcation of ELOVL.Results showed that it was successfully constructed of recombinant expression vector pYES-ELOVL6. Then detected by GC–MS after induction training,it contained only four endogenous fatty acids— C16:0, C16:1-7, C18:0 and C18:1 n–9in Saccharomyces cerevisiae. Its biochemical function characterized by heterologous expression showing that it caused reduction of C16:0 and C16:1n-7 and increasion of C18:0 and C18:1n-9, while produced new product C18:1n-7. The evidences above showed that E.sinensis ELOVL6 could catalyze the production of C16 long-chain fatty acids including both saturated and monounsaturated fatty acids.Studies have shown that ELOVL of E. sinensis itself have a certain activity to extend the enzyme, at the same time it was affected by the feed fat levels,which laid a foundation for PUFA synthesis pathways, regulatory mechanism of fatty acid, HUFA biosynthesis and regulating mechanism research.