眼斑芫菁在斑蝥素合成期的抑制性消减杂交文库构建及分析

Construction and Analyses of Suppression Subtractive Hybridization(SSH)Library of Mylabris cichorii at the Stage of Cantharidin Synthesis

斑蝥素是芫菁科昆虫合成的一种倍半萜类物质,对于治疗肿瘤和其他疾病有特别的疗效.通过构建抑制性消减杂交文库(SSH),筛选眼斑芫菁在斑蝥素合成期的差异表达基因.用斑点杂交对消减文库中的1 500个克隆进行筛查,130条特异表达序列标签(ESTs)进行分析. 34条(26%)ESTs与NCBI中标注的假设基因有很高的同源性;45条(35%)ESTs与NR数据库中的序列没有同源性,可能代表新的基因;51条(39%)ESTs与已知基因的序列同源性很高,这些基因参与了生物调控、应激反应和解毒、信号转导、代谢与合成.最后通过实时定量逆转录PCR对文库中的9个上调表达基因进行验证.对眼斑芫菁在斑蝥素合成期的表达进行研究,为最终阐明斑蝥素的生物合成提供帮助.

Cantharidin,a kind of terpene anhydride synthesized by blister beetles,has special efficacy to cure several cancers and some other diseases.In this study,Suppression subtractive hybridization(SSH)was used to discriminate the differentially expressed genes at the stage of cantharidin synthesis in Mylabris cichorii.A total of 1500 clones from the subtracted cDNA library have been screened by dot blotting and 130 unique expressed sequence tags(ESTs)have been analyzed.Among these,34 ESTs(26%)have significant similarity to NCBI annotated hypothetical genes,45 ESTs(35%)have low similarity in the NR database or represent novel genes,and 51 ESTs(39%)have significant similarity to known genes involved in biological regulation,stress response and detoxification,signal transduction,and metabolism and biogenesis.It has been confirmed that the up-regulation of 9 selected genes with quantitative real-time reverse transcription PCR analysis.The results of this study provide transcription profiles of M.cichorii at the stage of cantharidin synthesis,which will be very helpful for deciphering biosynthesis pathway of cantharidin.