家蚕动力蛋白轻链8基因克隆及其对重力的响应

Cloning and Response to Gravity of The Silkworm Dynein Light Chain 8 Gene

克隆家蚕动力蛋白轻链8(dynein light chain 8,Dlc8)基因开放阅读框架并对其序列进行分析.探讨Dlc8基因在家蚕胚胎、头、丝腺、中肠、皮肤、血液、脂肪和马氏管等组织中的分布.在细胞水平,应用RT-PCR和实时定量RT-PCR方法分析大梯度强磁场(large gradient high magneticfield,LGHMF)重力模拟环境(0g、1g、2g)对家蚕Dlc8基因表达的影响.在整体水平,应用实时定量RT-PCR方法分析Dlc8基因在家蚕胚胎反转期和整个胚胎期对LGHMF模拟失重环境的响应.克隆的家蚕Dlc8基因开放阅读框架长度为270bp,编码89个氨基酸.家蚕Dlc8基因推导的氨基酸序列与拟南芥(Arabidopsis thaliana)、果蝇(Drosophila melanogaster)、线虫(Caenorhabditis elegans)、热带爪蟾(Xenopus tropicalis)、小鼠(Mus musculus)、人类(Homosapiens)等6个物种Dlc8基因的氨基酸序列同源性分别为67%、96%、91%、95%、92%、92%.信号肽分析结果显示,该蛋白质为非分泌蛋白,不存在糖基磷脂酰肌醇锚定位点.家蚕Dlc8分子质量与等电点分别为10.34ku和6.81.Dlc8基因在家蚕的胚胎、头、丝腺、中肠、皮肤、血液、脂肪、马氏管中稳定表达.在细胞水平,家蚕Dlc8基因表达对重力变化较敏感,对磁场变化不敏感.在整体水平,Dlc8基因在家蚕胚胎发育的不同时期对重力的响应不同.整个胚胎发育期Dlc8基因在模拟失重条件下表达量与对照组接近.家蚕Dlc8基因可以作为重力生物学效应研究的分子靶标.该研究为深入探讨家蚕Dlc8基因重力生物学效应机制奠定了基础.

To study the changes of the silkworm dynein light chain 8 （Dlc8） gene expression pattern in different gravitational environments. The open reading frame sequence of Dlc8 gene was amplified and analyzed. The distribution ofDlc8 gene was investigated in embryo, head, silk gland, midgut, cuticle, blood, fat, tuba rnalpighii of silkworm respectively. The effects of simulated gravitational environments （0 g, 1 g, 2 g） produced by large gradient high magnetic field （LGHMF） on expression of the silkworm Dlc8 gene were tested by RT-PCR and real time RT-PCR in BmN cells. Expression of the silkworm Dlc8 gene in simulated weightless environment was tested by real time RT-PCR in inverse period and in total embryo period of silkworm. The Dlc8 gene with 270 bp coding 323 aa was successfully amplified. The homology rates of amino acid sequences of Dlc8 gene between silkworm and Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans, Xenopus trapicalis, Mus musculus, Homo sapiens were 67%, 96%, 91%, 95%, 92%, 92%, respectively. Signal peptide analysis showed that Dlc8 was not a secretion protein. There was not glycosyl-phosphatidyl inositol anchor site in Dlc8 amino acid sequence. Molecular mass and isoelectric point of Dlc8 were 10.34 ku and 6.81, respectively. Dlc8 gene is conservative in embryo, head, silk gland, midgut, cuticle, blood, fat, tuba malpighii of silkworm. Dlc8 gene is more sensitive to gravity than magnetic field in BmN cells. There are different responses to gravity on Dlc8 gene expression in different period of silkworm embryo. There was no significant change of Dlc8 gene expression between simulated weightless and control groups. The results showed that Dlc8 gene could be the molecular target to study gravity bioeffect. This research may contribute to reveal the mechanism of gravity bioeffect of the silkworm Dlc8 gene.