家蚕BmYki-1基因鉴定与表达特征

Identification and Expression Analysis of BmYki-1 in the Silkworm(Bombyx mori)

【目的】Hippo信号通路中的核心激酶通过磷酸化作用于转录共激活因子Yki来调控下游基因的表达,在控制器官大小、细胞增殖与凋亡等方面起关键作用。家蚕(Bombyx mori)作为鳞翅目模式昆虫,对其Hippo通路研究较少。论文旨在通过鉴定家蚕BmYki-1并对其序列信息和表达特征进行分析,为进一步研究中肠中Hippo信号通路提供基础,更好地了解Hippo信号通路在肠道稳态维持及组织损伤修复方面的调控机制。【方法】利用NCBI数据库及家蚕基因组数据库等相关信息,以哺乳动物和果蝇的Yki蛋白序列为依据,对家蚕Yki基因的同源序列进行鉴定;以家蚕大造品系为研究材料,利用PCR和c DNA末端快速克隆技术克隆获得家蚕BmYki两种可变剪切体的全长序列BmYki-1和BmYki-2,并基于生物信息学方法对两种剪切体的基因序列特征及蛋白结构特征进行分析;重点对BmYki-1在家蚕中肠组织中的功能进行探索;利用半定量PCR检测家蚕L5D3不同组织及中肠各个龄期BmYki-1表达情况;构建BmYki-1过表达载体并转染家蚕胚胎细胞系对其进行亚细胞定位分析;实时荧光定量PCR(q RT-PCR)检测用博来霉素损伤的家蚕中肠中BmYki-1的表达情况。【结果】家蚕中存在两种可变剪切形式的BmYki,分别为BmYki-1和BmYki-2,在家蚕基因组数据库中对应的注释号分别为BGIBMGA0082-TA和BGIBMGA0081-TA,其开放阅读框(ORF)分别为1 329和1 227 bp,分别编码442和408个氨基酸,其蛋白质分子量分别为48和44.1 k D,等电点分别为5.18和5.50,在139—171、220—252氨基酸处均各有1个WW结构域。多物种进化树表明,家蚕BmYki-1与同为鳞翅目的昆虫脐橙螟的亲缘关系最近,与鞘翅目、膜翅目昆虫较近,与双翅目昆虫较远,而与脊椎动物的亲缘关系最远。结果显示BmYki-1在家蚕中肠和丝腺中特异表达,中肠不同龄期表达谱分析结构显示表达量从2眠开始上调,在5龄的前中期表达水平较高。亚细胞定位试验表明BmYki-1蛋白在家蚕胚胎细胞系中主要存在于细胞核中,但在细胞质中也有分布。与对照相比,用博来霉素损伤的家蚕中肠中BmYki-1的表达量显著上调。【结论】获得了BmYki-1全长序列及时空表达特征,亚细胞定位BmYki-1蛋白主要在细胞核中,推测其在家蚕中肠细胞生长发育及损伤修复中起重要作用。

【Objective】Yki, a transcriptional coactivator, is phosphorylated and activated by upstream kinases cascade of the Hippo signaling pathway, and then regulates the downstream genes expression to control organ size, proliferation and apoptosis of cell. As a model of Lepidopteran insect, little is known about the Hippo signaling pathway involved in the silkworm（Bombyx mori）. The objective of this study is to identify B. mori BmYki-1 and analyze its sequence information and express characteristics, laying the foundation for further study of Hippo signaling pathways in midgut, which will help us better understand Hippo signaling pathways in intestinal regulatory mechanism of steady maintaining and tissue damage repairing.【Method】Based on mammals and drosophila Yki protein sequence, B. mori homologous Yki gene sequences was identified using NCBI databases and silkworm genome database. B. mori Dazao strain being research material, full-length sequence of two Yki spliced variants were cloned by PCR and RACE. According to bioinformatics methods, gene sequences and protein structure characteristics of the two spliced variants were analyzed. Here, function of BmYki-1 in B. mori intestinal tissue was focused. The spatial-temporal expression profile of BmYki-1 was investigated by semi-quantitative RT-PCR. BmYki-1 was constructed into a transit expression vector, and transfected into B. mori embryonic cell line to analyze the subcellular location of BmYki-1. q RT-PCR was utilized to detect the expression of BmYki-1 in B. mori intestinal after treated with bleomycin.【Result】There are two alternatively spliced forms of BmYki, namely BmYki-1 and BmYki-2, whose Gene ID are BGIBMGA0082-TA and BGIBMGA0081-TA in silkworm genome database. The ORF of the two spliceosomes consist of 1 329 and 1 227 bp, encoding 442 and 408 amino acids, with 48 and 44.1 k D protein molecular weight and 5.18 and 5.50 isoelectric point, respectively. In addition, both spliceosomes have two WW domains from 139 th to 171 th and 220 th to 252 th amino acids. A multi species phylogenetic tree demonstrated that BmYki-1 was most closely related to Amyelois transitella, which is Lepidoptera insect like B. mori, and was comparatively closely related to Coleoptera and Hymenoptera insects. However, BmYki-1 had a distant relationship with that of Diptera insect, and furthest with vertebrates. Expression profile showed that BmYki-1 was specifically expressed in the midgut and silk gland, and the expression was significantly up-regulated from 2nd molting stage in the midgut and reached the top at early-to-mid stage of 5th instar. Overexpressed BmYki-1 in Bm E cells was mainly distributed in the nucleus. BmYki-1 was significantly up-regulated in the midgut treated with bleomycin compared to the control.【Conclusion】The BmYki-1 c DNA was cloned and its expression patterns in B. mori were analyzed. The subcellular location of BmYki-1 was investigated and mainly located in nucleus. BmYki-1 may play a critical role in the development and repair of damaged midgut tissue of B. mori.