HE Shi-bao, YANG Cheng-fei, SHANG Sha, WANG Ling-yan, TANG Wen-chao, ZHU Yong
Objective:The objective is to clone the cDNA sequence of Bmtol from silkworm Bombyx mori, predict the protein structure, explore its expression profiles in different tissues and at different time of JHA treatment of silkworm, and to provide a fundamental evidence for the future study of the physiological function of this gene.Method:The full-length cDNA sequence of Bmtol was cloned from the entire tissues of silkworm by RT-PCR and submitted to the GenBank database. Physiochemical properties and structure characteristics of the deduced amino acid sequence were analyzed by multiple bioinformatics methods. Phylogenetic tree between BmTOL and its homologous JHBP from other insects was constructed using neighbor-joining of MEGA5.0. The expression levels of Bmtol mRNA of different tissues at day-3 in 5th instar were detected by qPCR.Result:The full-length cDNA sequence of Bmtol was obtained (GenBank is KY681053). The open reading frame (ORF) of Bmtol is 759 bp, encoding a protein of 252 aa with an estimated molecular weight of 27.72 kDa and pI of 6.16. The encoding protein has a signal peptide and no transmembrance structure, possesses a JHBP superfamily domain between 25~251 residues, exists hydrophobic regions in N-terminus and contains two conserved cysteins, suggesting that it is the core part of the family proteins to bind ligand. Subcellular localization results showed that BmTOL was located on the secretory pathway about endoplasmic reticulum, golgi and plasmalemma which belongs to the secretory protein. BmTOL possesses three α-helixs. The thirty-fourth bit Cys and the forty-fourth bit Cys form a disulfide bond in the α1 helix and the N-terminus, which forms the core part of the BmTOL protein binding to the ligand. The results of amino acid sequence alignment showed that the amino acid sequence of BmTOL of silkworm was significantly different from TO of other insect. It was homologous to DmTO with 25.10% amino acid sequence identity, and had 19.69% sequence identity with MsTO, and had 25.78% sequence identity with AgTOL-2, and had 23.53% sequence identity with AaTO, and had 28.17% sequence identity with PrTOL, and had 23.05% sequence identity with AmTOL, and had 21.18% sequence identity with EpTO1. Phylogenetic analysis revealed that BmTOL,EpTO1, MsTO, AmTOL, DmTO and PrTOL were clustered into one group, AaTO, AgTOL-2 and BmTOL were clustered into the other group. Besides, the results of qPCR showed that the Bmtol transcripts were higher expressed in the head, epidermis and testis of the day-3 in 5th instar.The Bmtol gene expression was not significantly different in the head at 0 h, 24 h, 48 h, 72 h and 96 h in 5th silkworm by JHBP treatment.Conclusion:BmTOL belongs to the JHBP family with a JHBP domain. The results of expression profiling and JHA treatment suggested that BmTOL belongs to juvenile hormone binding protein (JHBP) and is involved in not only juvenile hormone binding recognition, but also other physiological functions.
