Molecular Characterization and Function of Chitinase 10 Gene (OcCht10) from Oxya chinensis
Objective] The objectives of this study are to obtain cDNA sequence of chitinase 10 gene (OcCht10) from Oxya chinensis, analyze its functional domain and phylogenetic relationship with chitinases from other known insect species, investigate its expression patterns and biological function during molting process, and to provide a new candidate gene for pest control.[Method]cDNA fragments of OcCht10 were searched from O. chinensis’ transcriptome database. After blast analysis, the cDNA sequence of OcCht10 was assembled and translated, the functional domains of OcCht10 were predicted by bioinformatics methods. Phylogenetic analysis was performed with other insect chitinase 10 amino acid sequences. The first-stranded cDNAs were synthesized by using RNA isolated from integument of each day of 5th instar nymphs and various tissues of the 6th day in 5th instar nymphs. Reverse transcription quantitative PCR (qPCR) was carried out to analyze the gene expression patterns. Biological function of OcCht10 was studied by RNA interference method. The dsRNA primers were designed for dsOcCht10 synthesis in vitro. The dsRNAs were injected into the 2nd day of 5th instar nymphs for RNA interference, integument was dissected for silencing efficiency detection at 24 h after injection by using qPCR method. The phenotype was carefully observed and mortality was calculated till control insects molted to adults.[Result]The obtained cDNA (9 318 bp) of OcCht10 contained an open reading frame of 8 613 bp, encoding 2 870 amino acid residues and a non-coding region of 705 bp at 3′end. There were about 500 bp lost in 5′end. The deduced amino acid sequence included five chitinase catalytic domains and six chitin binding domains. Phylogenetic analysis showed that OcCht10 belonged to chitinase group Ⅱ, the genes from this group were crucial for insect molting based on references. Tissue specific expression analysis of OcCht10 showed that it was predominately expressed in the integument, foregut and hindgut, which developed from ectoderm. The results suggested that OcCht10 may be involved in chitin metabolism of insect integument. Developmental expression patterns showed that OcCht10 was highly expressed before and after molting stages, lower in middle stages of 5th instar nymphs, which implied that OcCht10 could digest chitin of integument during molting process. RNA interference results indicated that the corresponding transcript level was silenced by 70% after OcCht10 dsRNA injection. Compared with the dsGFP injected control group, the nymphs injected with OcCht10 dsRNA displayed slow development and failed to detach old cuticle during molting, the mortality reached 100%.[Conclusion] The partial cDNA sequence of OcCht10 was obtained from O. chinensis, the mRNA expression of OcCht10 was higher in the integument before molting; OcCht10 is involved in O. chinensis molting process, and dsOcCht10 injection can effectively silence mRNA expression of this gene and result in the block of ecdysis and even death of O. chinensis.
国家科技期刊平台
NSTL
万方数据
维普
