Education Ministry Key Laboratory of Integrated Management of Crop Diseases and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing, China.
Insect Mol Biol. 2020 Oct;29(5):466-476. doi: 10.1111/imb.12656. Epub 2020 Aug 7.
Henosepilachna vigintioctopunctata is a serious pest of Solanaceae and Cucurbitaceae in many Asian countries. RNA interference (RNAi) can effectively reduce transcript abundance in this beetle, offering opportunities to explore the biological function of specific genes. The white gene encodes a half-type ATP-binding cassette transporter that plays an essential role in tryptophan, guanine and uric acid transport across membranes. Mutations that disrupt the function of white are known to cause eye pigmentation phenotypes in many insect species. Here, we found evidence for five white gene paralogues present in H. vigintioctopunctata transcriptome datasets sequenced from a range of developmental stages. We individually knocked down each of the five white genes through the injection of corresponding double-stranded RNAs (dsRNAs) to the fourth-instar larvae to determine whether functional divergence has occurred. We found that injecting 1 μg dswhite3 caused compound eye colour of pupae and adults to develop as red/brown and brown, respectively, compared with black eyes in control beetles. Injection of 2 μg dswhite3 increased RNAi efficacy and produced a clearer eye colour phenotype. At both doses, the ocular diaphragm (a ring of black pigment surrounding each eye) did not change in the white3 RNAi hypomorphs. Moreover, our data revealed that injection of dswhite2 at the fourth-instar larval stage impaired the climbing ability of both male and female adults. Our results confirmed, for the first time, functional divergence of duplicated white genes in an insect species.
二十八星瓢虫是亚洲许多国家茄科和葫芦科植物的重要害虫。RNA 干扰（RNAi）可有效降低该甲虫的转录丰度，为探索特定基因的生物学功能提供了机会。White 基因编码一种半型 ATP 结合盒转运蛋白，在色氨酸、鸟嘌呤和尿酸跨膜运输中发挥重要作用。已知破坏 white 基因功能的突变会导致许多昆虫物种的眼色素表型。在这里，我们在 H. vigintioctopunctata 转录组数据集的测序中发现了存在于 5 个 white 基因的旁系同源物，这些数据集来自一系列发育阶段。我们通过向第四龄幼虫注射相应的双链 RNA（dsRNA）单独敲低了这 5 个 white 基因中的每一个，以确定是否发生了功能分化。我们发现，与对照甲虫的黑色眼睛相比，注射 1μg dswhite3 会导致蛹和成虫的复眼颜色发育为红色/棕色和棕色，而不是黑色。注射 2μg dswhite3 提高了 RNAi 效果，并产生了更清晰的眼睛颜色表型。在这两种剂量下，white3 RNAi 低聚物的眼膈（环绕每只眼睛的黑色色素环）没有变化。此外，我们的数据表明，在第四龄幼虫期注射 dswhite2 会损害雄性和雌性成虫的攀爬能力。我们的研究结果首次证实了昆虫物种中重复的 white 基因的功能分化。
