中国农业科学Issue(7)：1321-1329,9.
中华稻蝗几丁质脱乙酰基酶2基因的分子特性和生物学功能
Molecular Characterization and Functional Analysis of Chitin Deacetylase 2 Gene in Oxya chinensis
摘要
Abstract
Objective] In order to provide a theoretical basis for selecting novel target for pest control, molecular characteristics and biological function of chitin deacetylase 2 gene (OcCDA2) from Oxya chinensis were studied.[Method]The cDNA sequences of OcCDA2 were searched from transcriptome of O. chinensis by bioinformatics method, and the conserved domain was anaylzed, the homologous sequences from Tribolium castaneum, Drosophila melanogaster; Anopheles gambiae, Bombyx mori and Choristoneura fumiferana were selected to construct phylogenetic tree. The real-time PCR was applied to detect the relative expression of OcCDA2 in different tissues and developmental stages of the 5th instar nymphs. The RNA interference (RNAi) was performed to study the biological function of the OcCDA2 during molting and development of O. chinensis. [Result]The full length cDNA sequences of two chitin deacetylase genes were identified. The amino acid analysis showed that they possessed the signal peptide, the open reading frame contained three conserved domains: chitin binding domain (ChBD), low-density lipoprotein receptor chass A domain (LDLa) and chitin deacetylase catalytic domain (CDA). The phylogenetic analysis showed that two variants of OcCDA2 clustered with the CDA2s of five insect species, and the two variants differed only in one exon consisting of about 120 nucleotides, the alternatively spliced regions gathered with CDA2a or CDA2b, respectively. Based on sequence alignment and phylogenetic analysis, two variants of OcCDA2 were named as OcCDA2a and OcCDA2b. Tissue-specific expression analysis indicated that both OcCDA2a and OcCDA2b were mainly expressed in the integument, foregut and hindgut. Developmental expression patterns showed that the relative expressions of OcCDA2a and OcCDA2b were higher in the 1st day of 5th instar nymphs, then decreased gradually, and began to ascend before molting. RNAi results showed that the expression of target genes was significantly reduced in 24 h after dsOcCDA2a or dsOcCDA2b was injected into 1st day of 5th instar nymphs, the silence efficiency of genes was 96.72% and 80.43%, respectively. Compared with dsGFP injected controls, the majority of insects injected with dsOcCDA2 and dsOcCDA2a were unable to shed the old cuticle and led to body warping, finally to death. A minority of nymphs died before molting without obvious phenotypes. A few nymphs could develop to the adults, however, the legs appeared strengthless and the movement became slowly. The mortality rates were 87.5% and 94.4%, respectively. While insects injected with dsOcCDA2b alone could successfully molt to adults, and no phenotype was observed.[Conclusion]The OcCDA2 has two alternatively spliced variants, namely OcCDA2a and OcCDA2b. OcCDA2a plays a key role in the development of O. chinensis. The silence of OcCDA2a leads to the ecdysis failure and death of O. chinensis. However, OcCDA2b is not essential to molting process of O. chinensis.关键词
中华稻蝗/几丁质脱乙酰基酶/mRNA表达/RNA干扰/表型Key words
Oxya chinensis/chitin deacetylase/mRNA expression/RNA interference/phenotype引用本文复制引用
于荣荣,丁国伟,郭亚平,马恩波,张建珍..中华稻蝗几丁质脱乙酰基酶2基因的分子特性和生物学功能[J].中国农业科学,2014,(7):1321-1329,9.基金项目
国家自然科学基金项目（31272380，31071980）、教育部博士点专项基金（博导类）（20121401110008）、教育部留学回国人员科研启动基金（20111139）、山西省科学技术发展计划项目 （）
