Molecular cloning and sequence analysis of HoCL1 carboxylesterase cDNAs from the midgut of Holotrichia oblita

The peritrophic membrane protein polyclonal antiserum from Trichoplusia ni was used to screen cDNA expression library of the migut of Holotrichia oblita, and a cDNA clone encoding carboxylesterase named HoCL1 was obtained.The sequence analysis indicated that the opening reading frame of HoCL1 is 1599 bp in length encoding 532 amino acid residues of the proteinase with predicted molecular weight 59.5 kDa and p I 4.5.HoCL1 has conserved domains of carboxylesterases with a disulfide bond formation site,a serine activity center,only three cysteine residues and catalytic activity centers of Ser207,Asp333 and His422,and with no nitrogen linked glycosylation sites and oxygen glycosylation sites.Based on the amino acid sequence homology analysis and conserved domain analysis,HoCL1 belongs to B esterase and is mostly similar to the carboxylesterase of Tribolium castaneum ,but the similarity is only 35.2%.In the sequence alignments and phylogenetic tree with other insect carboxylesterases,the sequence conservation of COOH-terminal amino acid in HoCL1 is low,but the amino acid sequence near the N terminus is highly conserved.HoCL1 clusters with the carboxylesterases of T.castaneum and Harmonia axyridis .Cloning and identification of HoCL1 laid the foundation for the study of the expression and function in vivo.