Effects Of Δ6-and Δ9-desaturase During The Fatty Acid Synthesis Process Of The Chinese Mitten Crab(eriocheir Sinensis) And The Preliminary Studies Of Elongation Factor EF-1δ And EF-2

We cloned Δ6-and Δ9-desaturase genes from Eriocheir sinensis (Chinese mittencrab) using reverse transcriptase polymerase chain reaction (RT-PCR) andrapid-amplification of cDNA ends (RACE) for the first time. The full-length cDNAsequence of Δ6-desaturase was2278bp which coded442amino acid residues andΔ9-desaturase was2419bp which coded347amino acid residues. Gene expressionanalysis via real-time quantitative polymerase chain reaction revealed that the fatty acidΔ6-and Δ9-desaturase transcripts are widely distributed in various tissues, with highexpression levels in the hepatopancreas and cranial ganglia. This study focuses on thenutritional regulation of genes involved in the HUFA biosynthetic pathway in Chinesemitten crab. A feeding trial was performed whereby crablets were fed for238d withfour different diets: control diet without oil lipids (added with3%basic lipid of thefundamental diets); fish oil diet (FO; added with3%of the fundamental diets); soybeanoil diet (SO; added with3%of the fundamental diets); and FO/SO diet (1:1; added with3%of the fundamental diets). The hepatopancreas of crabs sampled at168d and238dto determine the effects on fatty acid desaturase mRNA expression. The results showthat the expression of fatty acid Δ6-desaturase is higher in the hepatopancreas of crabsfed with SO diet than those fed with FO diet. Furthermore, gene expression increasedby2.45-fold in the hepatopancreas of crabs fed with SO after238d than those fed after168d but remained steady for those fed with FO after238d. A significantly increasingtrend was also observed in the hepatopancreas of Δ9-desaturase at the megalopa stage tothe maturity stage of the Chinese mitten crab. We determined the effects ofΔ9-desaturase mRNA expression in the hepatopancreas after the crabs were fed with different diets for168days. Results showed that the FO diet elicited a strong increasingeffect on the amount of Δ9-desaturase genes compared with control and SO diets.Elongation factor (EF) is a protein factor which play roles in the peptide chainelongation in the process of protein synthesis, including elongation factor1(EF-1) andelongation factor2(EF-2). Elongation factor1consists of four subunits, β, γ and,and plays a key role in protein translation process. In this study, we cloned EF-1andEF-2genes from Eriocheir sinensis using reverse transcriptase polymerase chainreaction (RT-PCR) and rapid-amplification of cDNA ends (RACE). The full-lengthcDNA sequence of EF-1was933bp which coded263amino acid residues and EF-2was2752bp which coded846amino acid residues. And comparation results showedthat the nucleotide homology of EF-1was70%similar to Xenopus laevis’ and aminoacid homology of EF-1was54%similar to Danaus plexippus’, using BLASTN andBLASTX software. The phylogenetic analysis based on amino acid sequence shows thatEF-1has highest similarity with EF-1of Lepeophtheirus salmonis. Comparationresults which were used by BLASTX software showed that the nucleotide homology ofEF-2was88%similar to Libinia emarginata’s and amino acid homology of EF-2washigher than90%similar to others’ crustaceans. The phylogenetic analysis based onamino acid sequence showed that EF-2had highest similarity with EF-2of L.emarginata and clustered with other crustaceans. The expression of the gene in differenttissues and stage of E. sinensis was analyzed by real-time fluorescent quantitative PCR.The result showed the EF-1mRNA was mainly detected in muscle and small amountin testis, hepatopancreas and trace in heart, ovary, stomach, intestine and gill. EF-1mRNA was detected with high volume in muscles compared to hepatopancreas and gillin different developmental states of the crab, and display significant difference atP<0.05. It also have significant difference at P<0.05between muscles in differentdevelopmental states, and it was detected the highest expression in precocious crab,followed by mature crab and by a lower expression of crablet; And there is nosignificant difference expression between hepatopancreas and gill tissues in differentdevelopmental states. The result showed that EF-2mRNA was mainly detected inmuscle and small amount in testis, hepatopancreas and trace in heart, ovary, stomach,intestine and gill. We examined the expression of the EF-2in hepatopancreas, gills andmuscles in different developmental status of crablet, precocious crab and normal maturecrab; and also examined the expression of EF-2in hepatopancreas and gills in crablet which were exposed to different pH, the results showed that pH stress can induce theexpression of EF-2.