RNAi介导的HvCDA1基因沉默影响茄二十八星瓢虫存活和发育

【目的】茄二十八星瓢虫Henosepilachna vigintioctopunctata是茄科(Solanaceae)植物上的重要害虫。昆虫体内几丁质脱乙酰酶1(chitin deacetylase 1, CDA1)催化N-乙酰氨基-D-葡萄糖胺脱去乙酰基，促使几丁质转化为壳聚糖，控制昆虫体内几丁质纤维有序堆积，并维持角质层结构的完整性。抑制虫体中CDA1基因的表达会抑制壳聚糖的合成，影响昆虫表皮结构的形成，使昆虫不能正常发育而亡。【方法】利用RT-qPCR方法测定HvCDA1基因在茄二十八星瓢虫不同发育阶段(卵、1-4龄幼虫和预蛹)和4龄幼虫不同组织(表皮、脂肪体、中肠和马氏管)中的表达模式。通过饲喂茄二十八星瓢虫1龄幼虫不同浓度dsHvCDA1溶液浸泡处理1 min的茄子叶片后及直接饲喂4龄幼虫不同浓度dsHvCDA1溶液，探究沉默茄二十八星瓢虫HvCDA1基因对其幼虫存活和发育以及HvCDA1基因表达量的影响。【结果】发育表达谱结果表明，HvCDA1在茄二十八星瓢虫的各发育阶段均有表达，但在1龄末和2龄末幼虫中的表达量最高。组织表达谱结果显示，在茄二十八星瓢虫4龄幼虫的表皮中H...

RNAi-mediated silencing ofHvCDA1 affects the survival and development of the 28-spotted ladybeetle,Henosepilachna vigintioctopunctata(Coleoptera: Coccinellidae)#br#

【Aim】The 28-spotted ladybird, Henosepilachna vigintioctopunctata is an important pest of the Solanaceae plants. Chitin deacetylase 1 (CDA1) in insects catalyzes the deacetylation of N-acetylamino-D-glucosamine, facilitating the conversion of chitin to chitosan. Chitosan controls the orderly stacking of chitin fibers in insects and maintains the structural integrity of the cuticle. The silencing of CDA1 gene in insects inhibits the synthesis of chitosan which impairs the formation of cutiular structure and development, and eventually leads to death. 【Methods】The expression patterns of HvCDA1 in different developmental stages (egg, 1st－4th instar larvae and prepupa) and different tissues (cuticle, fat body, midgut and Malpighian tubules) of the 4th instar larvae of H. vigintioctopunctata were analyzed by RT-qPCR. The silencing of HvCDA1 was achieved by the oral feedings of eggplant leaves soaked in different concentrations of dsHvCDA1 solution for 1 min in the 1st instar larvae and direct oral feeding of different concentrations of dsHvCDA1 solution in the 4th instar larvae of H. vigintioctopunctata, and the RNAi-mediated silencing effects on the larval survival and development, and expression level of HvCDA1 were investigated.【Results】 Developmental expression profiling revealed that HvCDA1 was expressed across various developmental stages of H. vigintioctopunctata, with the highest expression level in the late 1st and late 2nd instar larvae. Tissue-specific expression profiling exhibited that the expression level of HvCDA1 in the cuticle of the 4th instar larvae was significantly higher than those in the other tissues. After feeding of eggplant leaves soaked in 50 ng/μL of dsHvCDA1 solution, the 1st instar larvae spines could not stand uprightly after molting, the cuticle could not form normally, and showed hindered feeding until death. The 1st instar larvae at 48 after being fed with the eggplant leaves soaked in dsHvCDA1 solution at the concentrations of 50, 200 and 400 ng/μL had the mortality rates of 84%, 94% and 100%, respectively. The oral feeding of the eggplant leaves soaked in 50 ng/μL of dsHvCDA1 solution reduced the expression levels of HvCDA1 by 38.63% and 79.00% in the 1st instar larvae at 48 and 96 h, respectively, as compared to the control (oral feeding of the eggplant leaves soaked in 50 ng/μL of dsGFP solution). On the other hand, ingestion of 50, 200 and 400 ng/individual of dsHvCDA1 solution by the 4th instar larvae resulted in 29%, 40% and 66% of larvae being deformed and unable to emerge normally after pupation, respectively. Similarly, the expression levels of HvCDA1 in the 4th instar larvae at 48 and 96 h after being fed with 200 ng/individual of dsHvCDA1 solution were significantly reduced by 52.99% and 70.09%, respectively, as compared to those in the control (fed with 200 ng/individual of dsGFP solution). 【Conclusion】 These results indicate that HvCDA1 plays an important role in the survival and development of H. vigintioctopunctata. The silencing of HvCDA1 negatively affects the ecdysis, pupation and emergence of H. vigintioctopunctata, and eventually leads to death. Collectively, the results from this study suggest that HvCDA1 can be a potential target gene for the RNAi-mediated control of H. vigintioctopunctata.