关键词: 家蚕, nanos, 胚胎发育, 原始生殖细胞, 荧光定量PCR, Western blot

Abstract: 【Objective】 To explore the expression pattern of nanos in Bombyx mori embryo and make preparations for the further study on this genes function in silkworm embryo development. 【Methods】The primers were designed according to the cDNA sequence (GenBank accession no: EF647589) of B. mori nanos 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. coli (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 nanos in early embryo of B. mori. The expression pattern of nanos through the whole developmental stage of B. mori embryo was studied by real-time PCR. 【Results】 One fragment encoding nanos 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 nanos 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 nanos 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.

Key words: Bombyx mori, nanos, embryo development, primordial germ cells, fluorescence quantitative PCR, Western blot