中文摘要：

为探究几丁质酶基因在三疣梭子蟹蜕皮过程中的生理作用,本研究通过转录组测序和RACE技术克隆了三疣梭子蟹几丁质酶基因（Pt Chi）c DNA全长（登录号：KF914663）,并通过荧光定量PCR（qRT-PCR）技术研究了该基因在三疣梭子蟹不同组织及不同蜕皮阶段的表达情况。结果表明：（1）Pt Chi基因c DNA全长2 200 bp,包括5＇非编码区（5＇-UTR）16 bp、3＇非编码区（3＇-UTR）714 bp和开放阅读框1 470 bp,编码489个氨基酸,预测蛋白质分子量和等电点为53.97 ku和4.76。（2）Blast P结果显示,Pt Chi推导氨基酸序列与已知甲壳动物Chi-3的一致性为61%～96%,系统进化树分析表明Pt Chi与其他甲壳动物Chi-3聚为一支。（3）qRTPCR结果显示Pt Chi基因在C期三疣梭子蟹肝胰腺中表达水平最高,胃、大颚器、心脏和眼柄中Pt Chi-mRNA表达水平依次降低,在其他组织中Pt Chi-mRNA表达量最低,且表达水平无显著差异。（4）不同蜕皮阶段,Pt Chi在肝胰腺、肠、胃和大颚器4种组织中的表达变化模式有所不同,肝胰腺中Pt Chi-mRNA表达水平在AB期最高,C期最低,暗示Pt Chi可能参与三疣梭子蟹蜕皮后期对病原体的免疫防御;肠中的Pt Chi-mRNA表达水平在E期最高,C期最低,推测Pt Chi参与蜕皮过程中肠道围食膜的分解和免疫功能;胃中Pt Chi表达水平在C期最高,暗示其参与了食物消化。以上结果表明,本研究克隆的Pt Chi可能为甲壳动物Chi-3型,其准确生理学功能及其在蜕皮过程中的调控机制有待进一步深入研究。

英文摘要：

The swimming crab,Portunus trituberculatus is a commercially important mariculture species distributed in east coast regions of China,Japan and Korea. Its high nutritional value and increasing market demands have promoted the quick development of pond-culture in east coastal waters of China since the 1990 s. However,the molting death syndrome（ M DS） and other disease problems are two limiting factors for the sustainable development of this crab aquaculture. Chitinase has been reported to participate in the molting regulation and immune defence for crustacean species,and the further study of crustacean chitinase will not only enhance our understanding for the mechanism of molting activities,but also provide valuable information for the control of M DS in the aquaculture of P. trituberculatus. Unfortunately,to date,no available information could be found on the chitinase of P. trituberculatus. The present study was therefore conducted to clone full length c DNA of a chitinase（ Genbank accession number： KF914663） for P.trituberculatus by transcriptome sequencing and RACE（ rapid amplification of c DNA ends）. Relative gene expression levels of Pt Chi（ P. trituberculatus Chi） gene in various tissues were then detected by qRT-PCR（ quantitative real-time PCR） during the molting cycle. The results showed that：（ 1） The full length of Pt Chi c DNA was 2 200 bp,including a 1 470 bp ORF（ open reading frame） which encoded 489 amino acid residues,a 16 bp 5＇-UTR and a 714 bp 3＇-UTR,while its calculated molecular weight and isoelectric point were 53. 97 ku and 4. 76,respectively.（ 2） Homologous analysis using Blastp showed that the predicted amino acid sequence of Pt Chi shared 61%- 96% identity with the type of Chi-3 of other crustaceans,and Pt Chi was clustered with crustaceans Chi-3s in phylogenetic tree.（ 3） Pt Chi had the highest gene expression levels in hepatopancreas,then decreased in the order of stomach mandibular organ heart eyestalk,while the lowest expression levels were fo