【作者】 王颖；

【导师】 康现江； 赵晓瑜；

【作者基本信息】 河北大学 ， 细胞生物学， 2009， 硕士

【摘要】 中华绒螯蟹(Eriocheir sinensis)是我国名优淡水养殖业的主要品种,也是我国一种重要的经济蟹类,广泛存在于我国南北各地淡水水域中。而普遍发生的1龄蟹性早熟现象严重影响了其产量,给生产带来巨大损失。大多数学者认为中华绒螯蟹性早熟是外部环境和中华绒螯蟹自身生殖内分泌调控共同作用的结果,尤其是性腺抑制激素对性腺成熟起着重要的调节作用。目前,对甲壳动物的性早熟研究主要有两种方法：一种是对胚胎发育、幼体形态及相关组织学和细胞学的研究；另一种是用分子生物学手段在基因水平研究性腺抑制激素。已经测序的性腺抑制激素基因序列均为虾类,尚未见蟹类的性腺抑制激素基因序列的报道。本实验用基因工程的方法来研究中华绒螯蟹的性腺抑制激素。根据已知物种的性腺抑制激素基因序列,选取保守区设计16对引物,依据密码子偏好性及同源树亲缘关系修正引物。利用TRIzol一步法成功地提取了中华绒螯蟹视上神经节总RNA。经过PCR扩增,最终有一条特异引物能够扩增出DNA片段。克隆后测序,得到三条片段的测序结果。分析结果显示,这三条序列互不相同,且均与五种已知性腺抑制激素基因序列较相似,相似性在38%-43%,接近已知性腺抑制激素基因序列间的相似性(39.50%)。而且通过在Swiss-Prot数据库中的比对发现,这三条片段都具有GIH基因结构上的共同特征：类表皮生长因子区域标记(EGF-like domain signature)、2Fe-2S型铁型铁硫结合区域标记(2Fe-2S ferredoxin-type iron-sulfur binding region signature)和C末端胱氨酸结区域标记(C-terminal cysteine knot domain signature)。初步推测,这三条片段均为类性腺抑制激素基因序列的不同位点的片段。更多还原

【Abstract】 Chinese mitten crab (Eriocheir sinensis) is an important economic crab, as one of the famous freshwater species for aquaculture. It exists throughout South and North of freshwater in China. But sexual premature in larvae occurred widespread and influenced the yield badly. The majority of scholars believed that the external environment and theirselves’s regulation of reproductive endocrine plaied a common role on the sexual premature of the crab, in which gonad-inhibiting hormone was important on the regulation of the gonadal maturation.At present, There were two main methods on the sexual premature of crustacean research: One is on embryonic development, larval morphology and histology and cytology of the study; The other is to use molecular biology methods to research gonad-inhibiting hormone in genetic levels. Now, there were five specieses of shrimp gonad-inhibiting hormone were sequenced, and no crab sequence reported.In this paper, genetic engineering technique was used for studying on gonad-inhibiting hormone(GIH), which has an important effect on chinese mitten crab gonad growth. The 16 pairs primers designed according as known conservative and codon usage of gene sequences gonad-inhibiting hormone, and homologous tree of chinese mitten crab.During the experiment, optic ganglion of chinese mitten crab was dissected fristly. Then total RNA was extracted by using the TRIzol reagent. Three DNA fragments were obtained after PCR, cloned and sequenced. The results showed these DNA fragments were similar to the known GIH in 38%-43%. By blasting in Swiss-Prot database there were EGF-like domain signature,2Fe-2S ferredoxin-type iron-sulfur binding region signature and C-terminal cysteine knot domain signature in the DNA fragments as all GIH. So the DNA fragments were presumed to GIH-like fragments of chinese mitten crab.更多还原