【作者】 张文宜； 杨明； 张世勇； 陈校辉； 乔慧； 蒋速飞； 熊贻伟； 金舒博； 龚永生； 傅洪拓；

【Author】 Zhang Wenyi;Yang Ming;Zhang Shiyong;Chen Xiaohui;Qiao Hui;Jiang Sufei;Xiong Yiwei;Jin Shubo;Gong Yongsheng;Fu Hongtuo;Ministry of Agriculture, Freshwater Fisheries Research Center Chinese Academy of Fishery Sciences;Nanjing Agricultural University;

【机构】 中国水产科学研究院淡水渔业研究中心农业部淡水渔业和种质资源利用重点实验室； 南京农业大学无锡渔业学院；

【摘要】 本研究从日本沼虾精巢均一化cDNA文库中筛选到一条MnCht1C表达序列片段(expressed sequence tags, EST),该序列具有完整的基因编码区,编码区长度为1 473 bp,编码490个氨基酸。MnCht1C编码的蛋白质具有完整的几丁质酶结构,N端的前23个氨基酸为信号肽,第24～406位氨基酸序列为几丁质酶催化结构域(catalytic domain),第407～433位氨基酸序列为Ser/Thr富含区(S/T-rich linker),其作用在于连接催化结构域与几丁质结合结构域。qPCR分析MnCht1C在日本沼虾各组织和各发育阶段表达情况,研究发现其主要在肝胰腺、肠、眼柄、表皮、精巢5种组织中表达;幼体发育时期不表达,而直至变态时期才开始表达。MnCht1C在日本沼虾整个蜕壳周期的表达呈波动性,蜕壳中期表达水平最高,随着蜕壳这一生理过程的完成,其表达水平也相应地下降直至蜕壳间期无表达水平检出,随着新一轮蜕壳的进行,其表达水平又开始逐渐升高。本研究结果为研究几丁质酶基因在日本沼虾和其他甲壳动物蜕壳及生殖发育中的作用提供参考。更多还原

【Abstract】 In this study, one EST sequence coding MnCht1C was cloned and identified from the Macrobrachium nipponense(M. nipponense) testis c DNA library. The MnCht1C EST had a complete open reading frame(ORF) of 1473 bp, encoding 490 amino acids. The encoded protein by MnCht1C demonstrated complete chitinase structure,with a cleavable signal peptide of 23 amino acid residues in the N-terminal. The encoded protein also comprised catalytic domain and S/T rich linker region to connect the catalytic domain with chitin binding domain, which were on the 24 th～406 th and 407 th～433 th of amino acid sequences, respectively. Expression patterns and tissue distribution of M. nipponense MnCht1C were analyzed by qPCR. The results suggested that MnCht1C was expressed in the tissues of hepatopancreas, gut, eyestalk, cuticle and testis. There were no expression of MnCht1C in the larval stage and MnCht1C has been not expressed until metamorphosis. MnCht1C level fluctuated during the molt period, showing the highest level in ecdysis, then it decreased gradually until non-expression in ecdysis. As a new molt cycle started, the expression level of Mn Cht1C was up-regulated slowly. This study might provide important information for further investigations on the functions of chitinase in molting and reproductive development of M. nipponense and other crustaceans.更多还原