摘要
几丁质脱乙酰基酶(CDAs)是调控昆虫生长发育过程中的蜕皮生理、组织生长和抗病免疫等的重要酶类。利用cDNA末端快速扩增(RACE)法克隆了家蚕CDAs基因的cDNA全长序列,鉴定出BmCDA基因mRNA存在2种剪接体,命名为BmCDA1和BmCDA2,外显子遗漏是这2种剪接体mRNA的主要剪接模式。生物信息学分析表明,BmCDA基因由6个外显子和5个内含子组成;推测BmCDA1和BmCDA2编码氨基酸序列都包含几丁质脱乙酰基结构域、几丁质结合(ChBD)结构域和低密度脂蛋白受体A类(LDLa)结构域等3个功能域,但二者的ChBD结构域有部分氨基酸残基不同。基于功能域基序和系统进化分析,昆虫CDA蛋白可分成5大类,BmCDA1和BmCDA2属于第1大类,是具有全部3个功能域的典型昆虫CDA蛋白。荧光定量PCR分析表明BmCDA表达不仅具有发育时期特异性,而且具有组织特异性:在幼虫和蛹蜕皮前后的表达量最高,蜕皮后第2天表达量最低;在幼虫蜕皮时发生激剧变化的表皮、气管和中肠等组织中的表达量显著高于变化较小的中部丝腺和脂肪体等组织;BmCDA1主要在表达量高的发育时期和组织中表达,BmCDA2主要在表达量低的组织中表达。研究结果有助于进一步解析昆虫CDAs基因的功能和调控机制。
Chitin deacetylases （CDAs） are important enzymes that regulate ecdysis physiology,organ formation and anti-pathogen immunity during insect development.The full length cDNA of CDAs in Bombyx mori was cloned using rapid amplification of cDNA ends （RACE） method and its mRNA was identified to have two splicing variants,designated as BmCDA1 and BmCDA2,both of which were formed predominantly by exon skipping.Bioinformatic analysis to gene structure of BmCDAs revealed that BmCDAs are composed of six exons and five introns.The deduced amino acid sequence of both BmCDA1 and BmCDA2 contain chitin deacetylase domain （CDA）,chitin binding domain （ChBD） and low-density lipoprotein receptor class A （LDLa） domain.However,partial divergence of the amino acid residues of BmCDA1 and BmCDA2 was discovered in the chitin binding domain.Insect CDA-like proteins are classified into five orthologous groups based on phylogenetic analysis and the presence of functional domains.BmCDA1 and BmCDA2 belonged to Group Ⅰ,which are typical insect CDA proteins comp-rising of all three functional domains.Real time PCR analysis revealed that expression of BmCDAs varied not only at different developmental stages but also in different tissues.Its expression level was the highest before and after molting/ecdysis of the larvae and pupae,and was the lowest on the second day after molting of the larvae.During ecdysis,the expression level of BmCDA in tissues like epidermis,trachea,and midgut etc which have dramatic morphological changes was obviously higher than that in tissues like middle silk gland and fat body which have little morphological change.Besides,BmCDA1 was predominantly expressed at the developmental stages or in the tissues which had high expression levels of BmCDAs,while BmCDA2 was predominantly expressed in tissues which had low expression levels of BmCDAs.These results would facilitate further investigations on functions and regulatory mechanism of CDA genes.
出处
《蚕业科学》
CAS
CSCD
北大核心
2010年第6期921-929,共9页
ACTA SERICOLOGICA SINICA
基金
国家重点基础研究发展计划"973"项目(No.2005CB121-003)
浙江省自然科学基金项目(No.Y3080289)
浙江省科技计划项目(No.2009C32069)
