家蚕Bmmγ基因的克隆，表达与功能的初步研究.docVIP

  家蚕 Bmmγ基因的克隆，表达与功能的初 步研究# 刘敏，聂作明，吕正兵，陈健，盛清，张耀洲** 5 10 15 20 25 30 35 （浙江理工大学生命科学学院，浙江杭州 310018） 摘要：E(spl)mγ 基因是果蝇中 Notch 信号通路的下游调控靶基因，与果蝇的神经发育调节 相关，在家蚕和人类中都发现了 Notch 途径的保守靶基因序列。家蚕 Bmmγ (Bombyx mori E(spl)mγ )是果蝇 E(spl)mγ 的同源基因，GenBank 登录号为 AB474582，ORF 框为 747bp， 编码 249 个氨基酸残基，预测分子量为 27.91kDa。经生物信息学分析发现该蛋白与大红斑 蝶 E(spl)mγ 蛋白的同源性最高。克隆表达并纯化该蛋白，免疫新西兰大白兔获得多克隆抗 体，用该抗体检测了 Bmmγ 蛋白在家蚕各时期和组织中的分布及进行亚细胞定位。分别采 用不同时间和梯度浓度的 Notch 信号途径阻断剂 DAPT 处理家蚕 BmN 细胞，检测 Bmmγ 基因的转录水平变化。结果显示，随着阻断剂浓度的增加和处理时间的延长，Bmmγ 基因 的转录水平呈现下降的趋势，因此推测家蚕 Bmmγ 基因是家蚕 Notch 信号途径的靶基因， 有类似在果蝇中的功能，与调控神经细胞的发育相关。 关键词：生物化学与分子生物学；家蚕；Bmmγ 基因；Notch 信号途径；表达谱；免疫组化 中图分类号：Q71 Cloning, expression profiling and preliminary functional analysis of Bmmγ gene from silkworm, Bombyx mori Liu Min, Nie Zuoming, Lv Zhengbing, Chen Jian, Sheng Qing, Zhang Yaozhou (College of Life Sciences, Zhejiang sci-tech University) Abstract: E(spl)mγ gene in Drosophila is the target gene of Notch pathway, and involved in Drosophila’s neural development. Conserved target genes of Notch pathway were also found in the silkworm, human, et al. Bmmγ (Bombyx mori E(spl)mγ) is a homolog of Drosophila E(spl)mγ gene, and its GenBank accession number is AB474582. The size of the ORF is 747bp, encoding 249 amino acid residues with the predicted molecular of 27.9kDa. According to bioinformatic analysis, the protein has the best similar homologs in Danaus plexippus. The ORF of Bmmγ was cloned into expression vector and expressed. The recombinant protein was purified and used to immunize New Zealand rabbit for polyclonal antibody preparing. Using the antibody, the expression profiling analysis was preformed at different stages and tissues of silkworm as well as subcellular localization. Notch signaling pathway was blocked by using different times and different concentration of DAPT to treat BmN cells. The results showed that the higher the concentration of the blocking agent and longer processing time, the lower Bmmγ gene transcription levels, showing that sil