Liao Y F, Wang Y, Huang Y, Zha S F, Liu J J, Wang Z K, Yin Y P, Liao Y F, Wang Y
School of Life Science, Chongqing University, Chongqing Engineering Research Center for Fungal Insecticides, Chongqing, China.
Clinical Medicine College of Tianjin Medical University, Tianjin, China.
Arch Insect Biochem Physiol. 2015 Jul;89(3):127-37. doi: 10.1002/arch.21229. Epub 2015 Mar 14.
Cantharidin is a biomolecule with a role in host defense that can also be used as an anticancer drug. The in vivo biosynthetic pathway for cantharidin has been the subject of debate for several decades and the mechanism is not yet completely understood. To study the biosynthetic pathway of cantharidin in blister beetles, Mylabris cichori, a full-length MenA (McMenA) cDNA was cloned based on the partial sequence of the MenA gene from a suppression subtractive hybridization (SSH) library of male and female adult M. cichorii. The cDNA was 1264 base pairs (bp) with an open reading frame of 1026 bp nucleotides encoding a 341 amino acid protein. Analysis of the McMenA amino acid sequence showed that the aspartate rich motif N/DDxxD represented binding sites for prenyl diphosphate via a Mg(2+) ion. Phylogenetic analysis showed that McMenA was most closely related to MenA of Tribolium castaneum, and the amino acid sequence similarity was 86%. The expression pattern of McMenA in adults was analyzed using RT-qPCR, and we found that the highest expression of McMenA occurred during 22-25 days in the sex-separate breeding males, while the lowest expression occurred in females at the same time. Injection with a specific double-strand RNA (dsRNA) of McMenA led to a significant reduction of McMenA mRNA levels after 24 h. Cantharidin and ATP concentrations dropped around the same time. Together, our data showed that the McMenA gene might be involved in cantharidin biosynthesis.
斑蝥素是一种在宿主防御中发挥作用的生物分子，也可用作抗癌药物。几十年来，斑蝥素的体内生物合成途径一直是争论的焦点，其机制尚未完全明了。为了研究芫菁科昆虫大斑芫菁中斑蝥素的生物合成途径，基于从大斑芫菁雌雄成虫抑制性消减杂交（SSH）文库中获得的MenA基因部分序列，克隆了全长的MenA（McMenA）cDNA。该cDNA为1264个碱基对（bp），开放阅读框为1026个bp核苷酸，编码一个341个氨基酸的蛋白质。对McMenA氨基酸序列的分析表明，富含天冬氨酸的基序N/DDxxD代表通过Mg(2+)离子与异戊二烯二磷酸的结合位点。系统发育分析表明，McMenA与赤拟谷盗的MenA关系最为密切，氨基酸序列相似度为86%。利用RT-qPCR分析了McMenA在成虫中的表达模式，我们发现McMenA在雌雄分开饲养的雄性成虫22-25天时表达最高，而在同一时期雌性成虫中表达最低。注射McMenA的特异性双链RNA（dsRNA）导致24小时后McMenA mRNA水平显著降低。斑蝥素和ATP浓度在同一时间下降。总之，我们的数据表明McMenA基因可能参与斑蝥素的生物合成。
