三疣梭子蟹FAMeT基因克隆及其在蜕皮周期中的表达水平

法尼酸甲基转移酶(farnesoic acid O-methyl transferase, FAMeT)是甲基法尼酯(methyl farnesoate, MF)生物合成途径中最后一步的关键酶，MF是一种类似昆虫保幼激素(juvenile hormone, JH)的倍半萜，在甲壳动物的生长发育及繁殖过程中起到了重要作用。本文克隆得到了三疣梭子蟹FAMeT的全长cDNA序列（GenBank登录号：KC192659），它包括一个201bp 的5非编码区、一个318bp的 3非编码区，和一个825bp的开放阅读框（ORF）,编码274个氨基酸；推导的氨基酸序列与已公布的其他甲壳动物FAMeT进行比对，发现一致性达75%-97%，其中与远海梭子蟹FAMeT的一致性最高；而且该氨基酸序列由两个CF（CPAMD8/FAMeT）区域组成，这两个CF区域是FAMeT的标志，在所有甲壳动物的FAMeT里均有发现，因此推导的氨基酸序列是三疣梭子蟹FAMeT基因。我们运用实时荧光定量PCR(qRT-PCR)的方法分析了其在不同组织中、不同蜕皮周期中的表达量变化，发现FAMeT在三疣梭子蟹的各个组织里均有表达且在胸神经节(Taoracic ganglia)里表达最强；在三疣梭子蟹蜕皮过程中，大颚器FAMeT在D1期表达最强，然后逐渐下降至D4最低。该结果表明FAMeT在三疣梭子蟹蜕皮调控中起着重要的作用。

Cloning and expression analysis of farnesoic acid O-methyl transferase(FAMeT) during molting in Portunus trituberculatus

Farnesoic acid O-methyl transferase (FAMeT) is the enzyme that catalyses methylation of farnesoic acid (FA) to produce isoprenoid methyl farnesoate (MF) at the final step of the biosynthetic pathway. As a sesquiterpenoid analogue of the insect juvenile hormone (JH), MF has been suggested to play a vital role in regulating crustacean growth and reproduction. In this study, the full-length FAMeT cDNA (GeneBank accession number: KC192659) of P. trituberculatus is cloned, it contains a 201bp 5-untranslated region(5-UTR), a 318bp 3-untranslated region (3-UTR) and a 825bp opening reading frame, which encodes 274 amino acid residues. Alignment of deduced amino acid sequence with FAMeT amino acid sequences of other crustaceans revealed that it shares the highest identity with P. Pelagicus among the identities ranged from 75% to 97%. The amino acid residues consist of two copies of CF (CPAMD8/FAMeT) domain, which are the hallmark domain of FAMeT and are present in all crustacean FAMeTs. Realtime PCR was used to quantify the relative expression level of FAMeT in different tissues and molting stages in P. trituberculatus. The results showed that FAMeT was expressed in various tissues. The mRNA level of FAMeT was highest in Taoracic ganglia, followed by gill and mandibular organ (MO). During the molting process, the expression of FAMeT in MO increased to the maximum at D1 stage, then gradually decreased to the minimum at D4 stage. The results suggest that FAMeT plays an important role on molting regulation in P. trituberculatus.