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生物蜡酯普遍存在于自然界生物中,蜡酯对生物生命活动具有储能、保护等诸多功能,在生物生命活动中扮演的角色极为重要[1-3]。昆虫和植物体表的蜡酯层能对抗有害细菌及真菌入侵,同时还能减少体表水分蒸发、疏水御潮、防止紫外辐射、反射阳光辐射、躲避天敌袭击[4-7]。有些昆虫则特化了泌蜡的性状,比如介壳虫,这一类昆虫体表覆盖大量蜡泌物形成的介壳[8],由于蜡酯的保护,常规方法很难达到防治效果,喷洒、涂抹等机械方法和化学治理方法也较为困难不容易操作,且污染环境、耗资大、大量杀伤天敌,介壳虫已成为农林果树危害严重的害虫。如果对蜡酯进行破坏,则易达到介壳虫防治的效果。
不同物种的蜡酯合酶(wax synthase, WS)研究显示,在生物蜡酯合成过程中WS具有关键作用[9-12]。笔者以我国历史悠久的特产资源昆虫—白蜡虫(一种介壳虫)为研究对象,鉴定出白蜡虫WS,实验表明WS在白蜡虫泌蜡中发挥极其重要的作用[13]。近年来,昆虫中对未知基因功能及作用机理研究主要是利用dsRNA诱导的RNAi实现[14],由此本研究欲探索较低成本获取dsRNA,以期建立一种通过原核菌体表达获取大量白蜡虫ws基因dsRNA的方法,从而为介壳虫防治提供一定参考。
用于昆虫实验研究的dsRNA合成方法,主要是试剂盒体外转录以及原核菌体表达获得,其中,dsRNA合成试剂盒价格较高,合成dsRNA的量较少。因此,本研究将选取前期筛选的白蜡虫ws基因片段,采用原核菌体表达合成dsRNA的方法,利用原核表达系统制备dsRNA,以期较低成本获取大量dsRNA。
白蜡虫ws基因RNAi载体构建及原核表达dsRNA
Construction of RNA Interference Vector of Ericerus pela Chavannes) ws Gene and Preparation of dsRNA by Prokaryotic Expression
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摘要:
目的 构建白蜡虫(Ericerus pela)蜡酯合酶(wax synthase,WS)基因干扰载体并建立其体外dsRNA(double-stranded RNA,dsRNA)原核表达体系,低成本大量制备白蜡虫ws基因的dsRNA。 方法 克隆白蜡虫蜡酯合酶基因ws片段,连入L4440载体,将重组质粒转入大肠杆菌HT115感受态细胞,经IPTG诱导获得与目的片段相对应的dsRNA。 结果 白蜡虫ws基因RNA干扰(RNA interference,RNAi)载体成功构建,重组质粒转入HT115感受态细胞经IPTG诱导后菌体成功表达dsRNA,dsRNA的平均获得量1 705 ng·mL-1。 结论 该研究通过原核表达白蜡虫ws基因的dsRNA,为后续利用RNAi实验研究白蜡虫ws基因功能及作用机理奠定基础。 Abstract:Objective This study aims at construct the interference vector of Ericerus pela wax synthase gene and prokaryotic expression system in vitro, and prepare a large number of double-stranded RNA (dsRNA) of E. pela ws gene at low cost. Method The cloned E. pela ws gene fragment was inserted into L4440 vector to construct E. pela ws-L4440 gene interference vector. The recombinant plasmid was transformed into HT115 competent cell, then induced by IPTG to get the dsRNA corresponding to target fragment. Result The interference vector of Ericerus pela ws gene was successfully constructed in vitro, and the dsRNA can also be expressed by HT115 competent cell with transformed recombinant plasmid induced by IPTG. The average production of dsRNA was 1 705 ng·mL-1. Conclusion The expression of the dsRNA of E. pela ws gene by prokaryotic expression system may lay foundation for using RNAi technology to study the function and mechanism of E. pela ws gene. -
Key words:
- Ericerus pela
- / WS
- / L4440
- / prokaryotic expression
- / dsRNA
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