[关键词]
[摘要]
目的 克隆温郁金Curcuma wenyujin萜类生物合成中的限速酶法呢基焦磷酸合酶(farnesyl pyrophosphate synthase,FPPS)的全长cDNA序列。方法 根据温郁金转录组数据设计特异引物,PCR扩增FPPS全长cDNA序列并对其进行生物信息分析;构建"基因-GFP"融合表达载体,利用农杆菌介导烟草叶片进行亚细胞定位分析;实时荧光定量PCR(qRT-PCR)检测基因在植物组织中的特异性表达。结果 温郁金CwFPPS基因全长1196 bp,包含1个长为1071 bp的开放阅读框,编码356个氨基酸,GenBank登入号为MT489462;CwFPPS编码蛋白属于类异戊二烯生物合成酶超级家族(C1)成员,包含5个保守的功能域,其中2个是富含天冬氨酸(DDXXD)的活性中心;亚细胞定位显示该蛋白位于泡质、细胞膜或细胞核上。qRT-PCR分析表明,CwFPPS基因在根茎中特异表达,相对表达水平是叶片中的13.7倍。结论 克隆获得的温郁金CwFPPS基因全长及其特异表达信息,可为后续进行基因功能鉴定及倍半萜成分的代谢工程研究提供依据。
[Key word]
[Abstract]
Objective To clone the full-length cDNA sequence encoding farnesyl pyrophosphate synthase (FPPS) that is one of the important rate-limiting enzymes involved in terpenoid biosynthesis. Method Based on Curcuma wenyujin transcriptome database, specific primers were designed to amplify the full-length cDNA sequence of FPPS; the bioinformatics of the obtained sequence was further analyzed; GFP-fused constructs were prepared by cloning the full-length coding sequences of CwFPPS and transformed into Agrobacterium tumefaciens GV3101 strain by electroporation systems; the subcellular localization of CwFPPS protein was observed by injecting the mixture of agrobacteria containing the CwFPPS::GFP fusion vector into Nicotiana benthamiana leaves. The real-time quantitative PCR (qRT-PCR) was used to detect the gene expression profiles in different tissues. Results Sanger sequencing showed that the full-length CwFPPS is 1196 bp (GenBank accession number:MT489462), which contains an open reading frame of 1071 bp, encoding 356 amino acids. The CwFPPS encoding protein belongs to the member of the isoprenoid biosynthetic enzyme superfamily (C1) and contains 5 conserved functional domains, 2 of which are rich in aspartic acid (DDXXD) as active zones of FPPS enzyme. Subcellular localization indicated that the protein is located in the cytoplasm, as well as the cell membrane. The CwFPPS was specifically expressed in rhizome and its relative expression level was 13.7-fold compared to that in leaves. Conclusion CwFPPS gene in Curcuma Wenyujin was successfully cloned and its expression pattern in tissues were obtained, which provides a basis for further gene functional identification and metabolic engineering of high-value sesquiterpenoids.
[中图分类号]
R282.12
[基金项目]
温州医科大学人才项目(89220027);浙江省重点实验室项目(2021E10013);大理药业股份有限公司横向课题(KJHX1603);合肥未来药物开发有限公司横向课题(KJHX2008);安徽济人药业横向课题(KJHX2009)