艾草β-石竹烯合成酶AaCPS基因克隆、亚细胞定位与表达分析

Cloning,Subcellular Localization and Expression Analysis of AaCPS from Artemisia argyi

目的对艾草Artemisia argyiβ-石竹烯合成酶基因(β-caryophyllene synthase,AaCPS)进行全长克隆、亚细胞定位与表达模式分析,为艾草倍半萜生物合成途径中的基因功能解析奠定基础。方法基于艾草转录组数据筛选注释为CPS的基因,采用PCR方法克隆获得目的基因c DNA的全长,对其编码区进行生物信息分析;构建原核表达载体,转化至大肠杆菌中进行诱导表达;构建绿色荧光蛋白(GFP)融合表达载体,利用农杆菌侵染烟草叶片的瞬时表达法进行亚细胞定位分析;运用实时荧光定量PCR(Real-time PCR)技术分析不同采收期(4月、5月、6月、7月)AaCPS基因表达模式,HS-SPME-GC-MS测定β-石竹烯含量并进行相关性分析。结果从艾草中克隆得到的AaCPS基因开放阅读框(Open reading frame,ORF)为1647 bp,编码548个氨基酸,相对分子质量为63667 Da,等电点为5.40,含Terpene_synth和Terpene_synth_C保守结构域。系统进化分析显示AaCPS蛋白与同科植物黄花蒿CPS蛋白的亲缘关系最近。SDS-PAGE结果证实在75000-120000 Da出现目的蛋白条带(包含28132 Da的标签蛋白),说明所获基因能够成功表达出蛋白;亚细胞定位显示其编码蛋白位于细胞质中;实时荧光定量PCR结果表明AaCPS基因表达量呈上升趋势,在7月达到最高。HS-SPME-GC-MS结果显示β-石竹烯含量从4月逐渐升高,在6月达到最高,与4-6月AaCPS基因表达量趋势总体一致。结论通过对AaCPS基因的克隆与分析,为进一步研究该基因在艾草倍半萜物质生物合成途径的功能鉴定奠定基础。

Objective To clone the full-length sequence,subcellular localization,and analyze the expression patterns of theβ-caryophyllene synthase gene(AaCPS)of Artemisia argyi,in order to lay a foundation for the gene function analysis of sesquiterpene biosynthesis pathway in A.argyi.Methods The genes annotated as CPS were selected from the transcriptome data of A.argyi.The full-length cDNA sequence of the target gene was obtained by PCR,and the coding region was analyzed using bioinformatics.A prokaryotic expression vector was constructed and transformed into Escherichia coli competent cells to express recombinant protein.A green fluorescent protein(GFP)fused expression vector was constructed,and the subcellular localization of AaCPS was observed using Agrobacterium tumefaciens transient expression method in tobacco.Real-time quantitative PCR(qRT-PCR)was used to analyze the expression patterns of the AaCPS at different harvest times(April,May,June,July).Determination ofβ-caryophyllene by HSSPME-GC-MS and correlation analysis.Results The AaCPS gene was cloned from A.argyi.The full-length open reading frame(ORF)was 1647 bp,encoding 548 amino acids.The molecular weight was 63667 Da with a theoretical pI of 5.94.AaCPS contained conserved Terpen_synth and Terpen_synth_C domains.Phylogenetic analysis indicated that AaCPS was closely related to the CPS protein of Artemisia annua.SDS-PAGE showed the presence of target protein bands between 75000-120000 Da(containing 28132 Da of the labeled protein),indicating successful expression of the AaCPS protein.The protein was found to be localized in the cytoplasm.As shown by qRT-PCR,the expression of AaCPS gene showed an upward trend,reaching the highest in July.The results of HS-SPME-GC-MS showed that the content ofβ-caryophyllene increased gradually from April to the highest in June.It is consistent with the trend of AaCPS gene expression from April to June.Conclusion Through the cloning and analysis of AaCPS gene,a foundation has been laid for further study of the functional identification of the AaCPS gene in the sesquiterpenes biosynthesis pathway in A.argyi.