中华蜜蜂apisceranaceranamsra基因的克隆及其表达分析-cloning and expression analysis of apitheraranamsra gene from apitheraranantha cerana.docxVIP

中华蜜蜂(Apis cerana cerana) MsrA 基因的克隆及其表达分析 Analysis of the 5′ flanking region of AccMsrA revealed a group of putative cis-acting elements that are related to the regulation of development and responses to external stimuli. Quantitative real-time PCR (qRT-PCR) detected high levels of AccMsrA mRNA during pupation (fifth-day larvae) and in the head tissue of adults. Immunohistochemical localization showed that AccMsrA was primarily concentrated in the abdomen and intestinal tract wall in fifth-instar larva. Furthermore, the expression of AccMsrA was up-regulated by multiple oxidative stresses, including ultraviolet (UV)-light (30 mj/cm2), heat (43℃) and H2O2 (2 mM). These results indicate that AccMsrA might fulfill an important role in the regulation of insect development and in response to various environmental stresses. The complete ORF sequences of AccMsrA were cloned into the pET30a (+) expression vector. The recombinant expression vectors were transformed into E. coli strain BL21 (DE3) and then induced. The 29 KDa recombinant proteins were separated and purified. Antisera were produced by injecting purified recombinant AccMsrA protein into white mice. The Immunohistochemical localization results revealed that AccMsrA was widely distributed, and high activity was detected in the abdomen and the intestinal tract wall, in fifth-day larvae. This result implies that AccMsrA might be involved in the the growth of segmental ventral organs in pupae. Keywords: Apis cerana cerana? methionine sulfoxide reductase A? Molecular cloning? Expression analysis 4 符 号 说 明 Amp：Ampicillin，氨苄青霉素 Acr：Acrylamide，丙烯酰胺 cDNA：Complementary DNA，互补 DNA Cys：Cys，半胱氨酸 ddH2O：Distilledant deionized water，重蒸水 DEPC：Diethyl pyrocarbonate，焦碳酸二乙酯 dNTP：Deocyribonucleoside triphosphate，脱氧核糖核苷三磷酸 E.coli：Escherichia coli，大肠杆菌 EB：Ethidium bromide，溴化乙锭 EDTA：Ethylene diamine tetraacetic acid，乙二胺四乙酸 iPCR：Inverse Polymerase chain Reaction，反向 PCR IPTG：Isopropyl-β-D-thiogalactoside，异丙基-β-D-硫代半乳糖苷