白蜡虫蜡酯合酶基因dsRNA的合成及其在Sf9中的表达

发布时间：2019-04-22 20:45

【摘要】：白蜡虫(Ericerus pela Chavannes)二龄雄虫分泌的白蜡,是重要的工业原料,广泛地应用于化工、食品、医药、化妆品等行业。蜡酯合酶(wax synthase,WS)在白蜡生物合成途径中催化长链脂肪醇和脂酰辅酶A生成酯,是蜡酯生物合成的关键酶。本研究根据实验室前期获得的ws全长序列,扩增其cDNA全长,用试剂盒体外转录了ws基因的dsRNA,注射于白蜡虫雌成虫体内,并用荧光定量PCR检测ws基因的干涉效果,为验证ws的生理功能奠定了基础;同时利用Bac-to-Bac表达系统构建了ws基因的pFastBac~(TM)HT B表达载体,转化大肠杆菌DH10Bac~(TM),经抗性和蓝白斑筛选获得重组杆粒rBacmid/EpelWS,转染Sf9(Spodoptera frugiperda)细胞,在Sf9细胞中初步进行表达,免疫荧光观测并用蛋白免疫印迹验证WS的表达。主要研究结果如下:(1)成功用试剂盒体外转录了ws基因的dsRNA,检测结果表明,dsRNA已成功合成。(2)荧光定量PCR结果表明,注射ws基因的dsRNA一周后的试验组白蜡虫与经gfp基因的dsRNA处理的对照组相比,试验组体内ws mRNA转录量明显下调,为后续功能的研究奠定了基础。(3)利用Bac-to-Bac表达系统成功构建了WS表达载体,PCR鉴定及测序结果表明,目的片段已插入载体中。获得了重组杆粒rBacmid/EpelWS,转染Sf9细胞后收获了P3病毒,蛋白免疫印迹表明:被P3病毒感染后的Sf9细胞总蛋白中,存在目的蛋白产生的杂交反应,WS被成功表达。本研究体外转录了白蜡虫ws基因的dsRNA,并在昆虫细胞中成功表达了WS,为白蜡虫ws的生理功能及WS的生化特性的进一步研究奠定了重要基础。
[Abstract]:White wax secreted by the second instar male worm of white wax worm (Ericerus pela Chavannes) is an important industrial raw material. It is widely used in chemical industry, food, medicine, cosmetics and other industries. Wax ester synthase (wax synthase,WS) catalyzes long chain fatty alcohol and fatty acyl coenzyme A to form esters in the biosynthetic pathway of white wax, which is the key enzyme in the biosynthesis of wax esters. In this study, according to the full-length ws sequence obtained earlier in the laboratory, the full-length cDNA was amplified. The dsRNA, of ws gene was transcribed in vitro and injected into the female adult of Wax Alba, and the interference effect of ws gene was detected by fluorescence quantitative PCR (FQ-PCR). It laid a foundation for verifying the physiological function of ws. At the same time, the pFastBac~ (TM) HT B expression vector of ws gene was constructed by Bac-to-Bac expression system and transformed into E. coli DH10Bac~ (TM),. The recombinant plasmid rBacmid/EpelWS, was transfected into Sf9 (Spodoptera frugiperda) cells by resistance and blue-white spot screening. The expression of WS was detected by immunofluorescence and Western blot. The expression of WS was detected by immunoblotting in Sf9 cells. The main results are as follows: (1) the dsRNA, detection results of ws gene were successfully transcribed with a kit in vitro. (2) the results of fluorescence quantitative PCR showed that dsRNA had been successfully synthesized. One week after injection of dsRNA with ws gene, the amount of ws mRNA transcript in the experimental group was significantly down-regulated compared with that in the control group treated with dsRNA of gfp gene. The results of PCR identification and sequencing showed that the target fragment had been inserted into the vector. (3) the WS expression vector was successfully constructed by Bac-to-Bac expression system, and the result of PCR identification and sequencing showed that the target fragment had been inserted into the vector. P3 virus was harvested after transfection of recombinant baculovirus rBacmid/EpelWS, into Sf9 cells. Western blot showed that there was hybridization reaction of target protein in the total protein of Sf9 cells infected with P3 virus and WS was expressed successfully. In this study, we transcribed the dsRNA, of white wax worm ws gene in vitro and successfully expressed WS, in insect cells, which laid an important foundation for the further study of the physiological function of ws and the biochemical characteristics of WS.
【学位授予单位】：中国林业科学研究院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S899.1

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