Chinese Shrimp-o-- Methyltransferase Cloning, Expression Analysis And Functional Studies

Infectious disease has been the biggest problem of the aquaculture of shrimp and fish. To solve this problem, one of the important pathways is to develop new immunology strategies including searching antimicrobial genes with which to raise the ability of shrimp and fish to resist diseases and substitute or decrease the drug usage within the treatment of these diseases.In this study a catechol-O-methyltransferase (COMT) gene has been cloned from the haemocytes of bacteria-infected Chinese shrimp (Fenneropenaeus chinensis) by suppression subtractive hybridisation (SSH) coupled with the SMART cDNA method. Simultaneously, a CD59 gene has also been cloned from the hepatopancreas of bacteria-infected loach (Misgurnus anguillcaudatus) by homology cloning. Expression and function of the two genes were analyzed. The reseach results are as follows.1. The COMT gene was cloned for the first time from crustaceansCOMT (COMT, E.C.2.1.1.6) is a kind of O-methyltransferase (OMT). Although the chemical mechanisms of methyl transfer reactions are identical, OMTs differ in their selectivity to methyl acceptor molecules. In animals, there are two kinds of OMTs studied thoroughly. One of OMTs is farnesoic acid Omethyltransferase (FAMeT). Studies of crustacean FAMeTs indicate that FAMeT catalyses the methylation of farnesoic acid (FA) to produce isoprenoid methyl farnesoate (MF) at the final step of the MF biosynthetic pathway. FAMeT can regulate crustacean growth, reproduction, ovarian development and molting. The other is COMT that can catalyse the transfer of the methyl group from S-adenosine-methionine to the hydroxyl group of catechol compounds such as catecholamines. Thus, COMT plays an important role in the catabolism of endogenous catecholamines such as dopamine and noradrenaline in the brains of animals. It can also inactivate other catechol-type compounds including many catechol-containing xenobiotics and drugs. There is evidence suggesting that COMT may play a role in human emotion and disease.Since human COMT was reported in 1958, COMTs have been found in invertebrates and vertebrates. But crustaceans COMT has no any research report.The cDNA of F. chinensis COMT gene (GenBank no. DQ091255) was cloned by SSH and SMART cDNA cloning methods in this study. Its full-length is 917 bp, which contains a single open reading frame (ORF) of 666 bp encoding a putative COMT protein of 221 amino acids. The predicted protein has a calculated molecular weight of 24572.06 Da and a...