Department of Plant Pathology, IFAS, Citrus Research and Education Center, University of Florida, Lake Alfred, FL, USA.
Department of Plant Pathology, IFAS, Citrus Research and Education Center, University of Florida, Lake Alfred, FL, USA.
Insect Biochem Mol Biol. 2018 Oct;101:131-143. doi: 10.1016/j.ibmb.2018.09.003. Epub 2018 Sep 8.
Plant piercing sucking insects mainly feed on phloem sap containing a high amount of sucrose. To enhance the absorption of sucrose from the midgut, sucrose hydrolase digests sucrose into glucose and fructose. In this study, a sucrose hydrolase homolog (DcSuh) was identified and targeted in Diaphorina citri, the vector of huanglongbing (HLB), by RNA interference (RNAi). In silico analysis revealed the presence of an Aamy domain in the DcSUH protein, which is characteristic of the glycoside hydrolase family 13 (GH13). Phylogenetic analysis showed DcSuh was closely related to the sucrose hydrolase of other Hemiptera members. The highest gene expression levels of DcSuh was found in the 4th and 5th instar nymphs. dsRNA-mediated RNAi of DcSuh was achieved through topical feeding. Our results showed that application of 0.2 μL of 500 ng μL (100 ng) dsRNA-DcSuh was sufficient to repress the expression of the targeted gene and cause nymph mortality and reduce adult lifespan. The reduction in gene expression, mortality, and lifespan was dose-dependent. In agreement with the gene expression results, treatment with dsRNA-DcSuh significantly reduced sucrose hydrolase activity in treated nymphs and emerged adults from treated nymphs. Interestingly, some emerged adults from treated nymphs showed a swollen abdomen phenotype, indicating that these insects were under osmotic stress. Although the percentage of swollen abdomens was low, their incidence was significantly correlated with the concentration of applied dsRNA-DcSuh. Metabolomic analyses using GC-MS showed an accumulation of sucrose and a reduction in fructose, glucose and trehalose in treated nymphs, confirming the inhibition of sucrose hydrolase activity. Additionally, most of the secondary metabolites were reduced in the treated nymphs, indicating a reduction in the biological activities in D. citri and that they are under stress. Our findings indicate that sucrose hydrolase might be a potential target for effective RNAi control of D. citri.
植物刺吸式昆虫主要以含有高浓度蔗糖的韧皮部汁液为食。为了增强从中肠吸收蔗糖的能力，蔗糖水解酶将蔗糖分解为葡萄糖和果糖。本研究通过 RNA 干扰（RNAi）鉴定并靶向定位了柑橘木虱（Diaphorina citri）中的蔗糖水解酶同源物（DcSuh），该同源物是黄龙病（HLB）的传播媒介。计算机分析表明，DcSUH 蛋白中存在 Aamy 结构域，这是糖苷水解酶家族 13（GH13）的特征。系统发育分析表明，DcSuh 与其他半翅目成员的蔗糖水解酶密切相关。DcSuh 的基因表达水平在 4 龄和 5 龄若虫中最高。通过局部喂食实现了 DcSuh 的 dsRNA 介导的 RNAi。研究结果表明，应用 0.2μL、500ng μL（100ng）dsRNA-DcSuh 即可充分抑制靶基因表达，并导致若虫死亡和减少成虫寿命。基因表达减少、死亡率和寿命与剂量呈正相关。与基因表达结果一致，dsRNA-DcSuh 处理显著降低了处理过的若虫和成虫中的蔗糖水解酶活性。有趣的是，一些处理过的若虫发育成的成虫出现腹部肿胀表型，表明这些昆虫受到渗透胁迫。尽管腹部肿胀的成虫比例较低，但它们的发生率与应用的 dsRNA-DcSuh 浓度显著相关。使用 GC-MS 进行的代谢组学分析表明，处理过的若虫中蔗糖积累，果糖、葡萄糖和海藻糖减少，证实了蔗糖水解酶活性的抑制。此外，处理过的若虫中的大多数次生代谢物减少，表明 D. citri 的生物活性降低，它们处于应激状态。研究结果表明，蔗糖水解酶可能是柑橘木虱有效 RNAi 控制的潜在靶标。
