摘要
采用RACE-PCR技术结合SMART cDNA合成技术,从银鲫中克隆到Ran的全长cDNA并对其编码区全长进行了原核表达、相应抗体制备及其时空表达特征分析。RT-PCR结果表明,Ran基因除在脑组织的转录水平较低外,其它组织中的转录水平几乎相同;Ran基因在不同发育阶段的胚胎中都有mRNA转录,但其mRNA的量在原肠期以后呈下降趋势。Western blot结果表明,Ran在卵巢和精巢中均高水平表达,在心、脑、肝、脾、肾中有较低水平表达,在肌肉中则不表达。同时检测到Ran在不同胚胎发育阶段均有较强表达。这表明,Ran基因在银鲫中可能起着多种作用,尤其在生殖细胞的发生中具有重要作用。最后,采用免疫耗竭对该基因编码的蛋白在精核解凝中的作用进行了初步研究,结果表明,Ran蛋白可能与精核解凝相关。
Gibel Carp （Carassius auratus gibelio） is a unique species that is able to reproduce by gynogenesis and gonochoristic reproduction. It has two different response mechanisms to homologous or heterologous sperms during fertilization： the heterologoous sperm nucleus keeps in condensation and fails to form male pronucleus, while some of the homologous sperm nuclei can decondense and transform into male pronucleus. The sperm nucleus decondensation after fertilization is a critical early step in fertilization and early embryonic developmental process and a key for elucidating molecular mechanisms underlying gynogenesis of gibel carp. Ran is an abundant GTPase of the Ras supefamily that is highly conserved in eukaryotes. It plays important roles in directing nucleocytoplasmic transport and cell cycle control. In order to study the possible roles of Ran in fish sperm deccndensation and embryonic early development, we cloned a full length cDNA of Ran from gibel carp by using SMART cDNA synthesis and RACE-PCR methods. Moreover, the full length sequence of its coding region was expressed in E. coli （DE3）, and the mulficlonal antibodies against Ran protein expressed in E. coli or that from eggs of gibel carp was prepared. In addition, analyses on expression characterization of Ran in different tissues and at different developmental stages in gibel carp were also performed. RT-PCR results showed that Ran was expressed in all the detected tissues. Although the mRNA level detected in brain was lower than that in other detected tissues, the mRNA of all of the other detected tissues was almost at the same level. The mRNA of Ran was detected in embryos at different developmental stages, whereas the mRNA level was decreased after embryos of gastrula. Western blot results further revealed that Ran protein was strongly expressed in all examined tissues except muscle, with especially robust expression in ovary, testis of gibel carp. At meantime, high level expression of Ran in gibel carp at different stages of embryonic development was detected by Western blot. The widespread and relatively strong expression of Ran in ovary and testis suggests that it has a fundamental role in cells of many tissues, and in many physiological processes, particularly in processes of oogenesis or spermiogenesis. We further investigated the role of Ran in sperm nuclei decondensation in vitro by applying immunodepletion in fish eggs cell-free system. This result shows that sperm nuclei decondensation processes in cell-free system are inhibited obviously after immunodepletion. The apparently ubiquitous expression and abundant RNA levels of Ran gene combined with analyses of immunodepletion suggest that Ran is involved in sperm nuclei decondeusation in cell-free system.
出处
《水产学报》
CAS
CSCD
北大核心
2006年第3期297-304,共8页
Journal of Fisheries of China
基金
国家自然科学基金项目(30130240
30070379)
中国科学院知识创新工程重要方向项目(KSCX2-SW-303)
中国科学院水生生物研究所知识创新工程领域前沿项目(220309)
关键词
银鲫
RAN
时空表达
精核解凝
gibel carp（ Carassius auratus gibelio ）
Ran
temporal and spatial expression
sperm nuclei decondeusation
