【作者】 王晓菲；

【导师】 柳峰松； 刘保忠； 谢松；

【作者基本信息】 河北大学 ， 水生生物学， 2011， 硕士

【摘要】 贝类养殖是我国海水养殖业中重要的一部分。贝类人工大规模养殖中关键环节是早期幼虫能否正常发育，这对幼虫变态和变态后生长率、存活率有很大影响，直接影响到生产效益，因此了解幼虫发育机制对于生产实践具有重要意义。长牡蛎(Crassostrea gigas)在我国沿海均有分布，以广东、福建较多，为南方沿海主要养殖品种之一，是重要的经济贝类。本文以长牡蛎幼虫为研究对象，分别对长牡蛎幼虫发育过程中神经发育相关的钙结合蛋白(CgCalbin)和初生壳形成相关的酪氨酸酶(CgTyrosinase)两个基因进行了克隆，分析了基因在牡蛎幼虫四个发育时期的时空表达特征，解析了可能的功能。从长牡蛎幼虫cDNA文库中获得CgCalbin序列，进行了序列分析比对并构建了进化树。通过Real time PCR发现该基因mRNA表达量在幼虫的担轮期及成体肝胰脏中最高。整装原位杂交结果显示CgCalbin的mRNA主要存在于担轮期幼虫的神经节部位，推断该基因与长牡蛎幼虫神经发育相关。根据D形幼虫与稚贝的消减文库信息，利用染色体步移法以及3’ RACE法在幼虫中克隆到CgTyrosinase全长cDNA序列。通过Real time PCR发现该基因在稚贝中表达最高，提示该基因可能与变态过程、早期贝壳形成有关。通过对上述两种基因的研究，分别探讨了长牡蛎幼虫神经发育相关、初生壳形成相关的基因及在幼虫中的表达分析，为研究贝类生长发育调控的分子机理提供了新的依据。更多还原

【Abstract】 Bivalve culture is one of most widely aquaculture in China. Early larvae have greatinfluence on larval metamorphosis and growth rate after metamorphosis. If the early larvaehave normal growth, it can bring great production efficiency. Larval development is a keyphase in large scale bivalve culture industry. So investigation of molecular mechanism inlarval development is very important for production practice.As an important economic bivalve, Crassostrea gigas distributes in the coastal areasand it is the main breeding species for the southern coast. In this paper, we studied CgCalbingene which is a EF-hand Calcium-binding protein and CgTyrosinase gene which is relatedto the formation of prodissconchⅠ. We obtained sequences of the two genes, and analyzedtemporal and spatial expression in C. gigas larvae and possible functions of CgCalbin generespectively.CgCalbin sequence was obtained from larvae cDNA library of oyster. Sequence wasthen analyzed and phylogenetic tree was constructed. The real time PCR analysis revealedthat CgCalbin has the highest mRNA expression level in trochophore, and then theD-shaped larvae, the spat, and has the lowest level in veliger. The different tissue analysisshowed that CgCalbin has a very high mRNA expression level in liver, gonad and gill, andhas a very low level in mantle and muscle. The result of whole mount in situ hybridizationshowed that CgCalbin was located at the position of ganglion in trochophore stage,indicating CgCalbin may plays a role in neuromodulation.EST sequence was selected from the subtranctive library of D-shape larvae and the spat.The full-length of CgTyrosinase was cloned by genome walking and RACE method. Thesequence of CgTyrosinase was analysed, and the real time PCR revealed that the mRNAlevel is highest in the spat. This indicates that the gene plays an important role in themetamorphosis and formation of shell in larvae.In conclusion, the two genes (CgCalbin, CgTyrosinase) associated withneuromodulation and prodissconchⅠformation, have been characterized and analyzed respectively. The investigation will be helpful for studying the molecular mechanism ofgrowth and development in bivalve larvae.更多还原