昆虫学报 ›› 2023, Vol. 66 ›› Issue (1): 27-36.doi: 10.16380/j.kcxb.2023.01.004

• 研究论文 • 上一篇    下一篇

RNAi介导的HvCDA1基因沉默影响茄二十八星瓢虫存活和发育

王亚洁1, 刘卓琦1, 潘广2, 邱宝利1, 潘慧鹏1,3,*, 杨春晓3,*   

  1. (1. 华南农业大学植物保护学院, 广东省生物农药创制与应用重点实验室, 广州 510642; 2. 广州国家现代农业产业科技创新中心,
    广州 510520; 3. 华南农业大学, 亚热带农业生物资源保护与利用国家重点实验室, 广州 510642)
  • 出版日期:2023-01-20 发布日期:2023-03-04

RNAi-mediated silencing of HvCDA1 affects the survival and development of the 28-spotted ladybeetle, Henosepilachna vigintioctopunctata (Coleoptera: Coccinellidae)#br#

WANG Ya-Jie1, LIU Zhuo-Qi1, PAN Guang2, QIU Bao-Li1, PAN Hui-Peng1,3,*, YANG Chun-Xiao3,*   

  1. (1. Key Laboratory of BioPesticide Innovation and Application of Guangdong Province, College of Plant Protection, South China Agricultural University, Guangzhou 510642, China; 2. National Modern Agricultural Industry Science and Technology Innovation Center of Guangzhou, Guangzhou 510520, China; 3. State Key Laboratory for Conservation and Utilization of Subtropical AgroBioresources, South China Agricultural University, Guangzhou 510642, China)
  • Online:2023-01-20 Published:2023-03-04

摘要: 【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HvCDA1基因的表达量显著的高于其他组织中的。1龄幼虫取食50 ng/μL dsHvCDA1溶液浸泡处理的茄子叶片后,幼虫蜕皮后其枝刺不能直立,角质层不能正常形成,且出现取食障碍直至死亡;饲喂50, 200和400 ng/μL dsHvCDA1溶液浸泡处理的茄子叶片后48 h时,死亡率分别高达84%, 94%和100%。与对照组(饲喂dsGFP溶液浸泡处理的茄子叶片)相比,饲喂1龄幼虫50 ng/μL dsHvCDA1溶液浸泡处理的茄子叶片后48和96 h时,其HvCDA1基因的表达量分别下降了38.63%和79.00%。另外,4龄幼虫单头饲喂50, 200和400 ng dsHvCDA1溶液,可分别导致29%, 40%和66%的幼虫化蛹后呈畸形且不能正常羽化。与对照组(饲喂200 ng/头dsGFP溶液)相比,饲喂4龄幼虫200 ng/头dsHvCDA1溶液后48和96 h时,其HvCDA1基因的表达量分别下降了52.99%和70.09%。【结论】上述结果表明,HvCDA1基因在茄二十八星瓢虫的存活和发育中起重要作用。当该基因表达受阻,会负面影响茄二十八星瓢虫的蜕皮、化蛹以及羽化,最终死亡。本研究结果表明HvCDA1可作为1个潜在的防治二十八星瓢虫的RNAi靶标基因。

关键词:  , 茄二十八星瓢虫,  , HvCDA1, RNAi, 存活, 发育

Abstract: 【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.

Key words: Henosepilachna vigintioctopunctata, HvCDA1, RNAi, survival, development