Huang Chun-Yen, Huang Chia Chia, Dai Shu-Mei, Chang Cheng
Biotechnology Centre, National Chung-Hsing University, Taichung, Taiwan.
Department of Entomology, National Chung-Hsing University, Taichung, Taiwan.
Pest Manag Sci. 2016 Feb;72(2):280-8. doi: 10.1002/ps.3990. Epub 2015 Mar 3.
The oriental fruit fly, Bactrocera dorsalis (Hendel), a very destructive insect pest in many areas of Asia, including Taiwan, can cause significant damage by ovipositing in and larval feeding on many kinds of fruit. A female lethal system, combining the splicing property of doublesex (dsx) with the toxicity of ricin A chain (RTA), has been developed. In this system, a modified RTA is separated by Bddsx intron 3; the expressed RNA can only be spliced in females, with toxic effects, whereas the immature RTA in males is harmless.
Two RTA-Bddsx constructs, clone BE 24-7 and clone CF 26-21, containing Bddsx intron 3 and its flanking exonic sequences, with four nucleotides at the 5'-end and five nucleotides at the 3'-end, correctly spliced in a sex-specific manner. Wild-type and modified RTAs expressed in an Escherichia coli system retained their ability to suppress protein synthesis: 90.4% for Ricin-WT, 71.3% for Ricin-LERQ and 58.0% for Ricin-FEGQ. Embryonic injection of Acp-CF26-21, the RTA-Bddsx gene driven by the actin 5C promoter, resulted in a significant increase in male percentage in the eclosed adults.
Our results indicate that the RTA-Bddsx hybrid system offers a novel and promising approach for oriental fruit fly control.
东方果实蝇，即橘小实蝇（Bactrocera dorsalis (Hendel)），是亚洲许多地区（包括台湾）极具破坏性的害虫，它通过在多种果实中产卵及幼虫取食造成严重损害。一种雌性致死系统已被开发出来，该系统将双性基因（doublesex，dsx）的剪接特性与蓖麻毒素A链（Ricin A chain，RTA）的毒性相结合。在这个系统中，一个经过修饰的RTA被Bddsx内含子3隔开；所表达的RNA仅在雌性中进行剪接并产生毒性作用，而雄性中未成熟的RTA则无害。
两个RTA - Bddsx构建体，克隆BE 24 - 7和克隆CF 26 - 21，包含Bddsx内含子3及其侧翼外显子序列，在5'端有四个核苷酸，3'端有五个核苷酸，以性别特异性方式正确剪接。在大肠杆菌系统中表达的野生型和修饰型RTA保留了抑制蛋白质合成的能力：蓖麻毒素野生型（Ricin - WT）为90.4%，蓖麻毒素LERQ型（Ricin - LERQ）为71.3%，蓖麻毒素FEGQ型（Ricin - FEGQ）为58.0%。用肌动蛋白5C启动子驱动的RTA - Bddsx基因Acp - CF26 - 21进行胚胎注射，导致羽化成虫中雄性比例显著增加。
我们的结果表明，RTA - Bddsx杂交系统为控制东方果实蝇提供了一种新颖且有前景的方法。
