Department of Entomology, National Chung-Hsing University, Taichung, Taiwan, ROC.
Pest Manag Sci. 2011 Apr;67(4):466-73. doi: 10.1002/ps.2088. Epub 2011 Jan 11.
The oriental fruit fly, Bactrocera dorsalis (Hendel), is one of the most destructive pests in many Asian countries. An effective strategy to reduce fly density in the field is urgently required. Recently, the doublesex of B. dorsalis (Bddsx(f) ) has been cloned, and RNA interference (RNAi) indicates that it can reduce the offspring in vitro. In this study, a piggyBac-based construct that generates short hairpin RNA (shRNA) against the female-specific region of Bddsx was introduced into the pest to test the RNAi effects on reproductive functions in vivo.
After embryonic injection and backcross, 21 transgenic lines with germline transformation were identified. Genomic DNA analysis showed that the exogenous transgene including short hairpin Bddsx(f) and a DsRed marker had integrated into the genomes of 11 transformed lines. Northern blot analysis indicated the presence of Bddsx(f) short interfering RNA (siRNA) under the control of a U6 promoter in transformed flies. As expected, the specific effects of RNAi led to the delay of egg maturation, and the offspring was significantly reduced. Reverse transcription real-time PCR further demonstrated that in vivo interference not only specifically inhibited the Bddsx(f) transcript but also repressed expression of the downstream yolk protein gene (Bdyp1).
The results clearly indicate that RNAi is heritable through the expression of specific siRNA in early generations of transformed oriental fruit fly. These results can broaden the understanding of sex-related developmental mechanisms in the fly, and also offer a possible molecular approach for pest control in the future.
东方果实蝇（Bactrocera dorsalis （Hendel））是亚洲许多国家破坏性最强的害虫之一。迫切需要采取有效策略降低田间的蝇密度。最近，克隆了双性果蝇的 doublesex （Bddsx(f) ），并通过 RNA 干扰（RNAi）表明它可以减少体外的后代数量。在这项研究中，引入了一种基于 piggyBac 的构建体，该构建体针对 Bddsx 的雌性特异性区域产生短发夹 RNA（shRNA），以测试 RNAi 对体内生殖功能的影响。
经过胚胎注射和回交，鉴定出 21 条具有种系转化的转基因系。基因组 DNA 分析表明，包括短发夹 Bddsx(f) 和 DsRed 标记的外源转基因已整合到 11 条转化系的基因组中。Northern blot 分析表明，在 U6 启动子的控制下，转化果蝇中存在 Bddsx(f) 短干扰 RNA（siRNA）。不出所料，RNAi 的特定作用导致卵成熟延迟，后代明显减少。逆转录实时 PCR 进一步表明，体内干扰不仅特异性抑制 Bddsx(f) 转录本，还抑制下游卵黄蛋白基因（Bdyp1）的表达。
这些结果清楚地表明，通过转化东方果实蝇早期世代中特异性 siRNA 的表达，RNAi 是可遗传的。这些结果可以拓宽对蝇类性别相关发育机制的理解，也为未来的害虫防治提供了一种可能的分子方法。
