褐飞虱flightin的原核表达及其在褐飞虱中差异表达检测
Prokaryotic expression of Nilaparvata lugens flightin gene and its differential expression in the insect
张小琴 胡玉琼 张传溪
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作者单位：浙江大学昆虫科学研究所
中文关键词：褐飞虱，flightin，原核表达，多克隆抗体，翅型差异, 发育差异
英文关键词：the brown planthopper, flightin, prokaryotic expression, polyclonal antibody, wing dimorphism，developmental expression
中文摘要：Flightin最早发现于果蝇的间接飞行肌中，并且定位于粗肌丝。这种蛋白对维持肌节的结构和功能起到了重要的作用，但其在具有长短翅型分化的褐飞虱的不同翅型间差异并不清楚。本研究以长翅型雌虫褐飞虱cDNA为模板，通过PCR扩增得到褐飞虱flightin基因ORF全长，将其连接到表达载体pGEX-6P-1 中以与谷胱甘肽S-转移酶（GST）融合表达。将表达载体转入大肠杆菌表达株Rosseta，在不同温度、不同浓度IPTG的条件下诱导表达flightin，得到了最优表达条件，获得了高水平可溶性表达。在用GST抗体进行Western blotting验证GST-flightin融合重组蛋白表达的正确性后，我们通过GST柱纯化了的GST- flightin，进而用纯化后的蛋白免疫新西兰兔制备了高特异性的多克隆抗体。最后，我们用制备的多克隆抗体检测了长、短翅型雌成虫和不同发育阶段的褐飞虱体内flightin的表达差异。结果显示，flightin仅在长翅型成虫中表达，在短翅型雌成虫中未检测到其明显表达，而且flightin只在成虫期表达。本研究为进一步研究褐飞虱的flightin与其它蛋白互作、翅肌发育和翅型分化打下了基础。
英文摘要：Flightin, located in thick filament, was originally found in indirect flight muscle of Drosophila melanogaster. It is important for structural integrity and sarcomere function. The difference of the muscle components between macropterous and brachypterous adults of the brown planthopper (BPH, Nilaparvata lugens) was unknown yet. We amplified flightin gene from cDNA of macropterous female adults and inserted it into the expression vector pGEX-6P-1 for fusion expression with gultathione S transferase (GST).The recombinant vector was then tranformed into Escherichia coli Rosseta. After optimization of the expression conditions of different temperatures and concentrations of IPTG, high level expression of flightin was archieved. The soluble expressed GST-flightin was purified and was further used to immune rabbit for preparing the polyclonal antibody against flightin. Using the prepared antibody, we detected that flightin expression in different developmental stages and in two wing forms. The results showed that flightin was uniquely expressed in adult stage and only expressed in macropterous female adults, no positive bands were detected in eggs, nymphs and brachypterous female adults. This study laid a basis for the study of wing muscle development and wing dimorphism，as well as for research of the interaction of flightin with other proteins in BPH.