卷叶贝母法尼基焦磷酸合酶基因的克隆及表达分析

Cloning and expression analysis of farnesyl diphosphate synthase gene in Fritillaria cirrhosa

为了探究卷叶贝母(Fritillaria cirrhosa)法尼基焦磷酸合酶基因(FcFPPS)是否参与甾类生物碱合成、萜类合成等代谢过程,该研究基于转录组测序结果,通过PCR技术克隆卷叶贝母FPPS基因(FcFPPS)开放阅读框(Open Reading Frame,ORF)序列,运用生物信息学方法对该基因进行分析,预测其编码蛋白的结构与功能,并通过qRT-PCR检测FcFPPS基因在野生鳞茎和再生鳞茎(通过激素组合刺激获得的组织培养物)中的表达情况,以及利用煎煮法测定野生鳞茎和再生鳞茎的总生物碱含量。结果表明:获得了1 059bp的FcFPPS ORF片段,编码352个氨基酸,并与NCBI上公布的麝香百合、虎眼万年青、春兰等植物FPPS蛋白的相似性在85%以上;对FcFPPS蛋白的二级、三级结构预测发现FcFPPS蛋白主要由α螺旋构成;qRT-PCR与总生物碱含量测定结果显示FcFPPS基因的表达水平与总生物碱含量的变化趋势一致,都是再生鳞茎高于野生鳞茎。FcFPPS蛋白质特征区及同源性等生物信息学分析结合qRT-PCR的测定结果证明FcFPPS可能是一个有生物学功能的蛋白质,这为后续利用基因工程手段提高卷叶贝母中生物碱含量奠定了理论基础。

In order to explore whether Fritillaria cirrhosa farnesyl diphosphate synthase gene(FcFPPS)plays some role in the metablic processes for synthesis of sterioid alkaloids and synthesis of terpenoids,in this study,Open Reading Frame(ORF)of FcFPPS was cloned via PCR method based on RNA-Seq.Some bioinformatic methods and softwares were used for the gene structure and function analysis.Quantitative real-time PCR(qRT-PCR)was used for the analysis of gene expression between wild and regeneration bulbs.The total alkaloid content of wild and regenerated bulbs were measured by decoction method.The result showed that the FPPS ORF was 1 059 bp,encoding 352 amino acids.NCBI Blast x search results showed that FcFPPS had the higher amino acids similarity to the corresponding proteins from Ilium longiflorum,Ornithogalum longebracteatum and Cymbidium goeringii.The identities of FcFPPS were more than 85%.The prediction results of FcFPPS protein secondary and tertiary structure showed that the FcFPPS protein was mainly composed ofαhelix.The FcFPPS qRT-PCR results were in parallel with the physiological data of accumulation of alkaloid,both showed higher alkaloid accumulation in regeneration bulbs than wild bulbs.These findings suggest FcFPPS may be a biological functional protein induced by qRT-PCR combination,which establishes a theoretical foundation for the improvement of the alkaloid content by the genetic engineering.