太白贝母甾体生物碱生物合成途径的比较转录组学研究

Frequent occurrence of fog and haze has led to an increasing demand of bulbus of Fritillariae cirrhosae, a rare and endangered species recorded in "China Plant Red Data Book" and a herb for moistening Lung. Steroidal alkaloids, the bioactive components, are neither to be largely isolated from Fritillariae cirrhosae due to low level nor chemically synthesized. Their biosynthesis pathway is unclear. F. taipaiensis, one of the major resources of Fritilliariae cirrhosae bulbus, is consistent with F. cirrhosa in diversity and content of steroidal alkaloids. Aiming to identify candiate genes involved in steroidal alkaloid biosynthesis, we analyzed deep sequencing of F. cirrhosa transcriptome and identified candidate genes encoding the corresponding key enzymes, such as cytochrome P450 and so on. Here F. taipaiensis will be detected in variation of expression levels of those enzymes in the different tissues (leaves, stem and bulbs) and at developmental stages by RNA-seq technology, as well as in the relationship between genes expression level of the selected genes and contents of alkaloids by fingerprinting. The genes that co-express the steroidal alkaloids syntheses will be selected by comparing the transcirptome data of F. taipaiensis and F. cirrhosa. They will be cloned and expressed in Saccharomyces cerevisiae, and then the enzymes will be characterized through detecting the enzymatic products. Accuracy of gene expression quantity revealed by RNA-seq will be checked by Real-time PCR method. Finally, the function of the selected genes will be determined. Our project will predict the molecular mechanism of the biosynthetic pathway of steroidal alkaloids in Fritillaria and help to increase the content of steroidal alkaloids by gene engineering.

润肺圣药川贝母资源匮乏，被列入国家三级保护植物名单。近年雾霾天气频发，更加剧了对其资源的需求。活性成分甾体生物碱含量极低，其单体难以通过化学分离或合成方法大量获得。目前关于其生物合成途径尚不明确。太白贝母( F.taipaiensis)是适合低海拔栽种的川贝母基原物种，且甾体生物碱种类含量与之一致。本课题组已完成川贝母转录组测序，获得了大量编码甾体生物碱生物合成的上游基因和下游CYP450基因。本研究利用RNA-seq技术检测不同组织及不同发育时期太白贝母上述候选基因的表达变化，并对太白贝母和川贝母生物碱合成相关基因进行比较，通过对基因表达量与生物碱含量变化进行关联分析，筛选表达模式一致的同源共表达的基因。应用qRT-PCR对基因表达量进行验证。将目的基因在酿酒酵母中表达，通过蛋白异源表达和体外酶促反应完成酶的鉴定。本研究将为甾体生物碱合成途径的阐明和通过生物技术提高生物碱产量奠定基础。

润肺圣药川贝母资源匮乏，被列入国家三级保护植物名单。近年雾霾天气频发，更加剧了对其资源的需求。活性成分甾体生物碱含量极低，其单体难以通过化学分离或合成方法大量获得。本课题组已完成川贝母转录组测序，获得了大量编码甾体生物碱生物合成的上游基因和下游CYP450基因。本研究利用RNA-seq技术检测不同组织及不同发育时期太白贝母上述候选基因的表达变化，项目对测序完成的太白贝母转录组序列进行挖掘，对甾体生物碱合成途径上的7个关键酶——HMGS，HMGR，DXS，DXR，ispH，FPS，CAS以及待选的氧化还原酶包括CYP450超家族酶（CYP90B1、CYP51G1、CYP734A6），DWF5，DWF1，FK等进行功能验证。找到了与川贝母生物碱合成相关的候选基因。通过在大肠杆菌或酿酒酵母中表达这些候选基因，对这些基因的催化活性进行分析确定其功能，为进一步研究甾体生物碱的生物合成奠定了基础。