家蚕生殖细胞相关基因nanos的克隆表达及在胚胎发育中的表达模式

Cloning and expression of nanos, a gene related to germ stem cell and its expression pattern in embryo development in silkworm

【目的】探讨nanos基因在家蚕Bombyxmori胚胎发育中的表达模式,为进一步研究该基因在家蚕胚胎发育中的功能奠定基础。【方法】根据本实验室提交到GenBank中家蚕nanos的cDNA序列(登录号EF647589)设计引物,扩增出了一条长684bp的编码片段,对该片段进行了克隆和表达,亲和纯化表达的蛋白并免疫新西兰大白兔制备抗体。Western blot检测家蚕早期胚胎nanos的表达情况,荧光定量PCR检测nanos在整个家蚕胚胎发育中的表达情况。【结果】克隆并表达了一条长684bp的编码片段,得到了分子量约33kD的融合蛋白。用制备的抗血清对家蚕早期胚胎蛋白的Western blot检测表明,nanos在此阶段基本是恒定表达。荧光定量PCR结果显示刚产的卵中nanos的表达量最大,第2天开始急剧下降,此后到第10天表达量几乎没有变化。【结论】本实验克隆的nanos是家蚕中的一个同源物,该基因在家蚕胚胎发育中的表达模式与蜜蜂等有很大的不同,反映了昆虫生殖细胞形成机制的多样性。

[ Objective ] To explore the expression pattern of nanos in Bombyx mori embryo and make preparations for the further study on this gene＇s function in silkworm embryo development. [ Methods ] The primers were designed according to the cDNA sequence （GenBank accession no： EF647589 ） of B. mori nartos gene submitted to GenBank database by our laboratory, and used to amplify a fragment of 684 bp. Subsequently, the fragment was cloned and expressed in E. coil （BL21）. The fusion protein was purified by using the affinity chromatography and the purified protein was then used to immunize the New Zealand white rabbit to get the antiserum. The Western blot method was used to explore the expression pattern of nartos in early embryo of B. mori. The expression pattern of nartos through the whole developmental stage of B. mori embryo was studied by real-time PCR. [ Results ] One fragment encoding nartos with 684 bp in length was cloned and expressed, and the fusion protein with the molecular weight about 33 kD was obtained. The Western blot using the antiserum prepared was performed on the proteins from B. mori early embryo, and showed that nanos protein was expressed almost constantly. The real-time PCR analysis on B. mori embryos at different developmental stages showed that the expression level of nartos peaked in newly laid eggs and started to drop dramatically from the second day, since which almost no change was detected till the tenth day after eggs were laid. [ Conclusions ] The gene cloned in this study was a nartos homolog in B. mori. The fact that its expression pattern in embryo development differs greatly from that of its counterpart in honeybee reflects the diversity of mechanisms for the formation of germ cells in insects.