卷叶贝母环阿屯醇合成酶基因的克隆及表达分析

Cloning and expression analysis of cycloartenol synthase gene in Fritillaria cirrhosa

目的对卷叶贝母Fritillaria cirrhosa中参与生物碱合成的关键酶环阿屯醇合成酶(cycloartenol synthase,CAS)基因进行克隆,并对其进行生物信息学和表达分析。方法基于转录组测序结果,通过PCR技术克隆卷叶贝母CAS基因(FcCAS)开放阅读框(open reading frame,ORF)序列,并基于在线工具对cDNA序列进行生物信息学分析。通过荧光定量(q RT-PCR)手段检测FcCAS在野生鳞茎与愈伤组织(通过激素组合刺激获得的组织培养物)中的表达情况并测定总生物碱的含量。结果 FcCAS编码区ORF长为2 271 bp,编码756个氨基酸,并与NCBI上公布的天门冬、芭蕉、油棕等植物CAS蛋白的相似性达80%以上;qRT-PCR与总生物碱含量测定实验表明FcCAS的表达水平与总生物碱含量的变化趋势一致,都是愈伤组织高于野生鳞茎。结论 FcCAS在不同组织状态下表达量差异较大,FcCAS是一个有生物学功能的蛋白质,受激素组合诱导表达,为进一步研究CAS对卷叶贝母生物碱含量的影响和表达调控奠定基础。

Objective To obtain the key enzyme gene involved in alkaloids biosynthesis pathway of Fritillaria cirrhosa, cycloartenol synthase（CAS） gene was cloned, and its bioinformatics analysis and gene expression pattern were also performed. Methods The open reading frame（ORF） of F. cirrhosa CAS（FcCAS） was amplified by PCR based on transcriptome sequencing. The bioinformatics analysis of FcCAS cDNA sequences was carried out by some online tools. Meanwhile, using real-time quantitative PCR（qRT-PCR） to analyze the gene expression patterns between wild and regeneration bulbs. Moreover, the content of total alkaloids in wild and regenerated bulbs were also investigated. Results The results showed that CAS had a length of 2 271 bpORF, which encoding 756 amino acids. The phylogenetic tree analysis showed that FcCAS were highly similar to the corresponding proteins in Asparagus officinalis, Musa acuminate, and Elaeis gunineensis from the NCBI website, and the similarities were more than 80%. The results of qRT-PCR and total alkaloids assay showed that the changing trend of the expression level of FcCAS was consistent with that of the content of total alkaloids, and higher alkaloid accumulation was in regeneration bulbs than wild bulbs. Conclusion The expression of FcCAS gene varied widely in different tissues. These findings suggested that FcCAS was a biological functional protein induced by hormone combination, which laid a theoretical foundation for the improvement of the alkaloid content by using the genetic engineering.