蛇足石杉中石杉碱甲合成相关基因HsPKS1的克隆及表达分析  

Cloning and expression analysis of HsPKS1 gene related to synthesis of huperzine A in Huperzia serrata

作　　者：沈玉 吴昊 吴德财 姚诗晴 李欣媛 戴亮芳[1] 罗向东[1] SHEN Yu;WU Hao;WU Decai;YAO Shiqing;LI Xinyuan;DAI Liangfang;LUO Xiangdong(College of Life Sciences,Jiangxi Normal University,Nanchang 330022,China)

机构地区：[1]江西师范大学生命科学学院,江西南昌330022

出　　处：《中草药》2024年第18期6324-6334,共11页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(32060074);江西省自然科学基金资助项目(20232ACB2050009)。

摘　　要：目的克隆蛇足石杉Huperzia serrata中石杉碱甲合成相关基因HsPKS1,解析其功能,为揭示石杉碱甲生物合成机制奠定基础。方法基于蛇足石杉转录组及qRT-PCR,筛选并克隆HsPKS1基因,利用生物学信息分析预测其功能;采用同源重组方法构建基因表达载体,遗传转化至烟草中进行亚细胞定位和表达分析,利用广泛靶向代谢组分析HsPKS1基因参与的生物合成通路的作用机制。结果qRT-PCR和HPLC结果表明不同组织间HsPKS1基因相对表达量与其石杉碱甲含量呈正相关。基因克隆、测序及生物信息学分析表明,HsPKS1基因开放阅读框为1200 bp,编码399个氨基酸,编码蛋白具有酰基转移酶活性区域及多个辅酶A的结合位点。系统进化树分析表明,HsPKS1蛋白与25个物种的聚酮合酶均具有较高的同源性,其中与江南卷柏的亲缘关系最近。亚细胞定位结果表明HsPKS1蛋白在细胞核和细胞质均有表达。广泛靶向代谢组分析发现,HsPKS1基因可以显著影响烟草次生代谢产物的积累,并引起合成黄酮类的前体化合物和异喹啉类生物碱化合物的物质变化。结论成功克隆并获得HsPKS1基因序列,分析了基因表达模式、生物学功能及亚细胞定位,揭示了该基因在类黄酮和生物碱合成等方面起重要作用,为研究和揭示HupA合成代谢通路的分子机制提供科学依据。Objective To clone HsPKS1,a gene related to the synthesis of huperzine A(HupA)in Huperzia serrata,and to analyze its function,laying the foundation for revealing the mechanism of HupA biosynthesis.Methods Based on the transcriptome analysis and qRT-PCR verification,the HsPKS1 gene in H.serrata was identified and cloned.The gene expression vectors were constructed using the homologous recombination method,and then transferred it into tobacco(Nicotiana benthamiana L.)for subcellular localization and gene expression analysis.Subsequently,the action mechanism of HsPKS1 gene involved in the related biosynthetic pathway were further analyzed by widely target metabolome analysis.Results The results of qRT-PCR and HPLC showed that the relative expression of HsPKS1 gene was positively correlated with the content of HupA among the different tissues.Gene cloning,sequencing and bioinformatics analyses showed that the open reading frame of HsPKS1 gene was 1200 bp,encoding 399 amino acids.And the encoded protein had an acyltransferase active region and multiple coenzyme A binding sites.Phylogenetic tree analysis showed that the HsPKS1 protein had high homology with the polyketide synthases of 25 species,and it has the closest genetic distance to Moellendorf's Spikemoss Herb.Subcellular localisation experiments showed that HsPKS1 protein was located in the nucleus and cytoplasm.Widely targeted metabolome analyses showed that the HsPKS1 gene could significantly affect the accumulation of tobacco secondary metabolites and induced changes in the synthesis of precursor compounds of flavonoids and isoquinoline alkaloid.Conclusion The HsPKS1 gene sequence was successfully cloned and obtained,and the gene pattern,biological function and subcellular localization were analyzed.These results revealed that the HsPKS1gene play an important role in flavonoids and alkaloids synthesis,which will provide more scientific evidence for revealing the molecular mechanism of HupA biosynthetic pathway.

关 键 词：蛇足石杉 聚酮合酶 基因克隆 烟草瞬时表达 广泛靶向代谢组 

分 类 号：R286.12[医药卫生—中药学]