【作者】 夏来新；

【导师】 程汉华； 周荣家；

【作者基本信息】 武汉大学 ， 细胞生物学， 2004， 硕士

【摘要】 很多机制参与了脊椎动物的性别发育这个复杂的过程。然而,参与性别决定和分化的基因目前很多都未知。黄鳝是具有天然的性反转的淡水鱼类,在它的一生中会经历先雌后雄的性别发育过程,其分子机理目前仍不清楚。我们从它的性腺中,克隆到人破骨细胞刺激因子Ostf(osteoclast-stimulating factor)的同源基因。聚类分析表明该基因和其它物种Ostf基因有很强的同源性。它编码一个含214个氨基酸的多肽,其中含有SH3 domain(c-Src homology 3 domain),ankyrin repeats和N端的脯氨酸富集区,这提示它可能参与了细胞内的信号传导。随后我们研究了它的表达特征,尽管该基因是广谱表达的,但是它在黄鳝性腺中的表达却只局限于生殖上皮。黄鳝的生殖上皮中含有双向分化潜能的生殖细胞,暗示着该基因可能参与了黄鳝的性反转。 β-actin是actin家族的一员,β-actin在维持细胞结构,细胞内运动,细胞分裂等细胞生理活动方面发挥着重要的作用。我们克隆了黄鳝的β-actin基因。它的cDNA全长1860bp,编码375个氨基酸,在脊椎动物中不同物种的β-actin基因之间的序列同源性超过了98%。基于目前已知的全部鱼类的β-actin cds和3’UTR,我们分别构建了进化树。结果它们都一致支持将鱼类的β-actin基因划分为四类。黄鳝的β-actin基因属于其中的第二类。同时RT-PCR表明我们克隆的黄鳝的β-actin基因在睾丸、卵巢、心、肝、脾、脑等组织中广谱表达。 BAC(细菌人工染色体)文库在分析复杂的基因组结构、功能方面有着非常重要的作用。黄鳝基因组很小,只有600Mb,我们设法构建它的基因组BAC文库。基因组DNA用BamHI部分消化,然后通过脉冲场电泳两次选择回收,电洗脱,最后克隆到pBeloBAC11的衍生型载体plndigoBAC-5中。初步结果,得到了3000个克隆,平均插入片段110kb,空斑率12%。尽管最终高覆盖率的文库没有最终完成,但是这为以后构建基因组覆盖的BAC文库提供了相应的技术基础。更多还原

【Abstract】 Sexual development in vertebrates is a complex process. Several mechanisms were used in determination of the development of the male or female of vertebrates. The genes for determination of sequential hermaphrodite sex are unknown. We identified a homologue of human osteoclast-stimulating factor (OSF) from the rice field eel, a teleost with characteristic of naturally sex transformation from female, via intersex to male during its life. The rice field eel OSF-like cDNA encoded a peptide of 214 amino acids that contains a c-Src homology 3 domain, proline-rich region and ankyrin repeats, suggesting that it was potentially involved in cell signaling. The gene was clustered into the OSF gene group of all the other vertebrates. Although expressed in the three kinds of gonads and also in other tissues, in gonads of all the three sexes, OSF-like expression was restricted to the gonadal germinal epithelium, from where bipotential gonia (oogonia or spermatogonia) will differentiate, suggesting that the OSF-like may be potentially involved in sexual differentiation, besides other roles as a regulator in development.β-actin is a member of the actin family of genes, which play important roles in maintaining cytoskeletal structure, cell motility, cell division, intracellular movements and contractile processes. We report here the identification of a β-actin cDNA of the rice field eel, a teleost fish with a characteristic of natural sex reversal. The cDNA sequence of this gene was 1860 bp in length, which codes a 375 amino acid protein. Amino acid identities of the 6-actin between the rice field eel and other vertebrates including human, chicken, and other fishes, were more than 98%. Phylogenetic trees including all β-actin cds or 3’ UTR of teleost fishes were constructed respectively, which indicate that teleost β-actin can be classified into four types, and the β-actin cloned from the rice field eel here is type II. RT-PCR showed expression of the rice field eel β-actin in testis, ovotestis, ovary, heart, liver, spleen and brain, suggesting ubiquitous expression pattern.BAC (bacterial artificial chromosome) library is a crucial tool for modern structural, functional, and comparative genomics analysis of large and complex-genome organisms. Here we try to construct a BAC library of rice field eel genome, which is only 600 Mb. This genome DNA was partially digested withenzyme BamH I, subjected to twice size selections, electroeluted and cloned into the pIndigoBAC-5 which is derived from pBeloBACll. Finally, we obtained about 3000 clones with an average insert size 110kb and 12% empty clones, which provides technique base for further construction of the BAC library of the rice field eel.更多还原