关键词: 仁扇舟蛾, 化学感受蛋白, 分子克隆, 序列分析, 时空表达

Abstract: [Objective]In this study, a novel cDNA encoding the chemosensory protein3 (CresCSP3) from Clostera restitura was cloned and its characteristics and its expression in different developmental stages, different age of days and different tissues, in order to identify the role of CSP in insects chemical sensory system.[Method]The full length cDNA sequence of the covering coding region of CresCSP3 was cloned by using transcriptional sequence and RACE technique, and the signal peptide, isoelectric point, molecular mass, and three-dimensional structure of the signal peptide were predicted by using online tools. The homology was compared by using BLAST, the phylogenetic tree was constructed using a neighbor-joining method with MEGA 6.0 software, and the affinity between CresCSP3 and CSPs from other 12 lepidopteran species was analyzed. The expression profiles of CresCSP3 in every instar larval, at different ages after emergence and in different tissues were assayed using real-time quantitative PCR (qRT-PCR).[Result]A new chemosensory protein was cloned from C. restitura named CresCSP3. The full length of CresCSP3 cDNA is 545 bp, containing a 61 bp non-coding district in 5' end, a 100 bp non-coding district in 3' end, and a 384 bp open reading frame encoding a putative 127 amino acids with a molecular mass of 14.34 kD and an electric point of 5.75. The deduced amino acid sequence possesses a putative signal peptide of 18 amino acids. The three-dimensional structure of CresCSP3 is composed of 6 α-helices, forming a hydrophobic cavity, which conform to the typical CSPs structural model. The amino acid sequence of CresCSP3 shows the highest identity (61%) with the CSPs of Plutella xylostella, and the lowest identity with Ectropis oblique hypulina CSP2 with only 27%. Phylogenetic tree analysis showed that CresCSP3 was not clustered into one branch with any other CSPs among the 12 lepidopteran species. The qRT-PCR analysis revealed that the CresCSP3 was expressed in the whole development stages and it was predominantly expressed in 4th instar. For adults, CresCSP3 was distributed in the both male and female antennae, but the expression pattern differed:the CresCSP3 was predominantly expressed in 3-4 days old male adults but it was highly identified in 4-5 days old female adults. It also showed that CresCSP3 was expressed in heads, antennae, feet and wings while highly expressed in antennae both in male and female.[Conclusion]The sequence characteristics of nucleotides and amino acids of CresCSP3 were clarified. CresCSP3 had a distant relationship with CSPs from other lepidopteran insects, which reveals the diversity of chemosensory proteins family. The expression patterns of CresCSP3 in different developmental stages indicated that CresCSP3 might participate in the host localization during larval feeding. The expression patterns of CresCSP3 in the antennae of male and female adults at different ages showed that CSP3 was involved in mating and spouse localization. Besides, it might also be involved in host localization during female laying. The expression of CresCSP3 was the highest in the antennae, which may probably be because antennae are the main structure to feel the external volatile odor molecules. The wings, feet and head were also involved in the perception of the external environment to some extent. CresCSP3 was widely distributed in the insects, indicating that it may play an important role in identifying the complex chemical signals in the environment so that the insect can react accurately.

Key words: Clostera restitura, chemosensory protein (CSP), molecular cloning, sequence analysis, expression profile