中国农业科学2017，Vol.50Issue(13)：2498-2507,10.DOI:10.3864/j.issn.0578-1752.2017.13.009
飞蝗几丁质脱乙酰基酶基因的分子特性和生物学功能
Molecular Characterization and Biological Function of Chitin Deacetylase Genes in Locusta migratoria
摘要
Abstract
[Objective] Chitin deacetylase (CDA) is a key enzyme involved in chitin metabolism.The objective of this paper is to study the molecular characteristics and biological function of chitin deacetylase genes in Locusta migratoria,and to provide a theoretical basis for screening novel targets for pest control.[Method] Based on the transcriptome of L.migratoria,three cDNA sequences putatively encoded LmCDAs were obtained,and the gene structure was analyzed by comparing with the locust genome database.Alignment was performed with the CDAs from Tribolium castaneum and the functional domains were predicted by BlastP and SMART software.The homologous sequences from T.castaneum,Drosophila melanogaster,Anopheles gambiae,Bombyx mori,Oxya chinensis and Choristoneura fumiferana were selected to perform clustering analysis with LmCDA4,LmCDA5a and LmCDA5b,and phylogenetic tree was constructed by using MEGA 5.02 software with the neighbor-joining (NJ) method.The RT-qPCR was applied to detect the relative expression of LmCDA4 and LmCDA5 in different tissues and developmental stages of the 5th instar nymphs.The RNA interference (RNAi) was performed to study the biological function of LmCDA4 and LmCDA5 during the molting process of L.migratoria.The chitin content was detected by chemical method.The transmission electron microscope (TEM) was applied to study whether the LmCDA4 and LmCDA5 have effects on the cuticle structure.[[Result] The three full length cDNA sequences putatively encoded chitin deacetylase were identified from locust transcriptome database.The bioinformatics analysis showed that they possess the signal peptide,the open reading frame contains two functional domains:chitin binding domain (ChBD) and chitin deacetylase catalytic domain (CDA).Alignment with the CDAs from T.castaneum showed that partial sequences of two CDAs from L.migratoria were different,and the splicing sites are similar to two alternatively spliced variants of TcCDA5 from T.castaneum,which indicated two CDAs were alternatively spliced variants.The phylogenetic analysis showed that three LmCDAs were closely grouped with the CDA4 and CDA5 from other six insect species with high bootstrap value,and named as LmCDA4,LmCDA5a and LmCDA5b,respectively.Tissues expression pattern showed that both LmCDA4 and LmCDA5 were mainly expressed in the integument,foregut and hindgut.Besides,LmCDA5 also had a high expression in the fat body.Expression of CDAs in integuments of different days of 5th instar nymph showed that both of LmCDA4 and LmCDA5 were highly expressed in the 1st and 2rnd days of 5th instar nymph,then decreased gradually.After injection of dsLmCDA4 and dsLmCDA5,the expression of target gene was significantly reduced at 24 h compared with dsGFP injected controls,and the silence efficiency of gene was 98.1％ and 95.6％,respectively.Phenotypic analysis showed no visible phenotypes were observed and both treatment and control insects could molt to adult successfully.Chitin content detection and TEM observation suggested that silencing the expression of both LmCDA4 and LmCDA5 did not affect the chitin content and chitin organization.[[Conclusion] LmCDA4 and LmCDA5 are highly expressed in the integument,foregut and hindgut.Biologic functional analysis suggested both of them are not essential in the molting process of L.migratoria,silencing of their expression showed no effect on locust development and cuticle structure.关键词
飞蝗/几丁质脱乙酰基酶/组织部位/日龄/表型/几丁质含量和排列Key words
Locusta migratoria/chitin deacetylase/tissues/days/phenotype/chitin content and organization引用本文复制引用
于荣荣,丁国伟,刘卫敏,张敏,赵小明,韩鹏飞,马恩波,张建珍..飞蝗几丁质脱乙酰基酶基因的分子特性和生物学功能[J].中国农业科学,2017,50(13):2498-2507,10.基金项目
国家自然科学基金(31672364)、山西省基础研究计划(2015011070)、山西省回国留学人员科研资助项目(2015-007)、山西省高等学校科技创新项目(2017104)、山西省研究生优秀创新项目(02180114092041) （31672364）
