Key Laboratory of Plant Protection Resources and Pest Management, National Ministry of Education, Northwest A&F University, Yangling, Shaanxi, China.
Department of Entomology, The University of Arizona, Tucson, AZ, USA.
Insect Mol Biol. 2016 Feb;25(1):58-71. doi: 10.1111/imb.12198. Epub 2015 Nov 14.
Cantharidin (C(10)H(12)O(4)) is a monoterpene defensive toxin in insects involved in chemical defence as well as in courtship and mating behaviours. It is relatively well known in the medical literature because of its high anticancer activity and as an effective therapy for molluscum contagiosum. However, little is known about its biosynthesis pathway in vivo, and no enzyme involved in cantharidin biosynthesis has been identified. The purpose of this study was to identify the crucial enzyme that is involved in the biosynthesis of cantharidin. Using the homology cloning method, a 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) gene, the rate-limiting enzyme in the mevalonate pathway, was cloned from the blister beetle Epicauta chinensis. Quantitative reverse transcription PCR and gas chromatography methods revealed that the HMGR transcripts had a positive correlation with cantharidin production in the beetles (R = 0.891). RNA interference (RNAi) knockdown of HMGR mRNA expression was achieved by microinjection of a specific double-stranded RNA with more than 90% RNAi efficiency, and an apparent decrease of cantharidin production was observed. Furthermore, the HMGR mRNA was greatly upregulated by exogenous juvenile hormone III (JH III), and cantharidin production was also raised in males; however, when injecting the JH III with RNAi of HMGR mRNA at the same time, cantharidin production did not rise. These results demonstrate that HMGR is an essential enzyme in cantharidin biosynthesis in the blister beetle E. chinensis, which further verifies previous research results demonstrating that cantharidin is synthesized de novo by the mevalonate pathway in blister beetles.
斑蝥素（C(10)H(12)O(4)）是昆虫体内参与化学防御以及求偶和交配行为的单萜防御毒素。由于其高抗癌活性和作为传染性软疣的有效疗法，它在医学文献中相对较为知名。然而，体内斑蝥素生物合成途径知之甚少，也没有鉴定出参与斑蝥素生物合成的酶。本研究旨在鉴定参与斑蝥素生物合成的关键酶。通过同源克隆方法，从斑蝥 Epicauta chinensis 中克隆了甲羟戊酸途径的限速酶 3-羟基-3-甲基戊二酰辅酶 A 还原酶（HMGR）基因。定量反转录 PCR 和气相色谱法显示，HMGR 转录物与甲虫中的斑蝥素产生呈正相关（R=0.891）。通过用具有超过 90%RNAi 效率的特异性双链 RNA 显微注射实现了 HMGR mRNA 表达的 RNAi 敲低，并且观察到斑蝥素产生明显减少。此外，HMGR mRNA 在外源保幼激素 III（JH III）的作用下大大上调，并且雄性中的斑蝥素产量也升高；然而，当同时用 RNAi 的 JH III 注射 HMGR mRNA 时，斑蝥素产量并未上升。这些结果表明，HMGR 是斑蝥 E. chinensis 中斑蝥素生物合成的必需酶，进一步验证了先前的研究结果，表明斑蝥素是通过斑蝥中甲羟戊酸途径从头合成的。
