苹果蠹蛾几丁质脱乙酰基酶2的基因克隆、表达模式和分子特性

Gene Cloning,Expression Pattern and Molecular Characterization of Chitin Deacetylase 2 in Cydia pomonella

【目的】研究苹果蠹蛾(Cydia pomonella)几丁质脱乙酰基酶2(chitin deacetylase 2,CDA2)的分子特性和表达模式,为新型杀虫剂靶标筛选提供理论依据。【方法】基于苹果蠹蛾转录组和基因组数据,通过同源比对获得苹果蠹蛾CDA2的相关信息;新鉴定的CpCDA2a和CpCDA2b与其他昆虫中已鉴定的CDA2氨基酸序列进行多序列比对,并采用MEGA7软件邻接法(neighbor-joining,NJ)构建系统发育树;通过生物信息软件预测CpCDA2a和CpCDA2b两个剪切体的蛋白功能域和三维结构并分析其差异,并通过在线软件对其分子量、等电点和亲/疏水性等蛋白理化性质及糖基化修饰位点等方面进行分析比较;采用荧光定量PCR检测CpCDA2a和CpCDA2b两个剪切体在苹果蠹蛾不同发育时期及其幼虫不同组织的表达情况,以及注射蜕皮激素后其表达量的变化趋势。【结果】获得了苹果蠹蛾CDA2的两个可变剪切体CpCDA2a和CpCDA2b的全长CDS序列及其内含子和外显子位置信息;分析发现两个剪切体的蛋白序列均具有信号肽、几丁质结合域(ChBD)、低密度脂蛋白受体域(LDLa)和几丁质脱乙酰基催化域(CDA)等功能域。多序列比对和聚类分析结果表明,不同昆虫间CDA2差异剪切类型非常类似,相同目昆虫的序列以较高的置信度聚为一支。三维结构模拟与比较发现两个剪切体的ChBD功能域在结构上存在较大差异;理化性质分析显示两个剪切体的亲/疏水性和糖基化位点也存在差异。不同发育时期表达量分析表明,CpCDA2a主要在幼虫期高表达,CpCDA2b主要在蛹的前期和中期高表达,而且在幼虫蜕皮前和蜕皮后CpCDA2a和CpCDA2b表达量均会显著上调;不同组织的表达量分析表明,CpCDA2a和CpCDA2b均在表皮中表达量最高,而CpCDA2b在头部的表达量也较高。对注射蜕皮激素后幼虫中CpCDA2a和CpCDA2b表达量分析显示,与对照相比在注射蜕皮激素后CpCDA2a的表达量在24 h和48 h均有所上调,但差异不显著,而CpCDA2b在24 h和48 h均显著上调。【结论】苹果蠹蛾CDA2的转录本存在两个可变剪切体CpCDA2a和CpCDA2b,根据对其分子特性、发育阶段和组织表达谱、以及注射蜕皮激素后表达动态等方面的综合分析,推测两者均参与苹果蠹蛾蜕皮和新表皮形成过程,但由于可变剪切的序列差异导致的蛋白结构和理化性质上的差异造成了两者在功能上的分化。

【Objective】In order to provide a scientific basis for the selection of novel target for pest control,molecular characterization and expression patterns of chitin deacetylase 2 genes(CpCDA2)of the codling moth(Cydia pomonella)were studied.【Method】The cDNA sequences of chitin deacetylase 2 genes were identified from the transcriptome data of C.pomonella by bioinformatic methods,and the conserved domain was analyzed,the homologous sequences from other insects were selected to construct phylogenetic tree by using neighbor-joining(NJ)method of MEGA7 software.The protein properties including molecular weight(Mw),theoretical isoelectric point(pI),glycosylation site and hydrophilic/hydrophobic properties were analyzed by online software.RT-qPCR was performed to detect the relative expression levels of CpCDA2a and CpCDA2b in different tissues and developmental stages.To explore the functions of these genes in the process of larval molting,the ecdysone(20E)was injected to the 5th instar larvae,and the expression dynamics of them were detected by RT-qPCR.【Result】The full length cDNA sequences of CpCDA2a and CpCDA2b,alternative splicing variants of C.pomonella CDA2,were identified.The amino acid analysis showed that both variants of CpCDA2 possess the signal peptide,and the open reading frames contain three conserved domains,including chitin binding domain(ChBD),low-density lipoprotein receptor class A domain(LDLa)and chitin deacetylase catalytic domain(CDA).The multi-sequence alignment analysis revealed that the CpCDA2a and CpCDA2b are similar to other insect,and the phylogenetic tree showed that the sequences of insect in the same order are clustered into a clade with high confidence.Three-dimensional structure simulation and comparison revealed that the ChBD functional domains of the two splicing variants are different in structure.Physical and chemical properties analysis showed that there are also differences in hydrophilic/hydrophobic properties and glycosylation site of the two splicing variants.Developmental expression patterns showed that the CpCDA2a expression was high in larval stage,CpCDA2b in early and middle stages of pupae,and the expression of CpCDA2a and CpCDA2b was significantly up-regulated before and after larval molting.Tissue-specific expression analysis indicated that the expression of CpCDA2a and CpCDA2b was the highest in integument,and the expression of CpCDA2b in head was also higher.Expression dynamics of the larvae after injection of ecdysone(20E)indicated that both CpCDA2a and CpCDA2b were up-regulated,but with very different levels.【Conclusion】The CpCDA2 has two alternatively spliced variants,namely CpCDA2a and CpCDA2b,which plays a key role in the development of C.pomonella.While,according to the comprehensive analysis of molecular characterization,developmental stages and tissue expression profiles,and expression trends after ecdysone injection,it is deduced that the differences in protein structure and physical and chemical properties of CpCDA2a and CpCDA2b caused by alternative splicing,resulted in the differentiation of function between them.