紫苏磷脂酸磷酸水解酶基因(PfPAH1)的鉴定及功能分析  

Identification and Functional Analysis of Phosphatidic Acid Phosphohydrolase Gene(PfPAH1)from Perilla frutescens

作　　者：董书言 邢志 尹苗 王尧 周雅莉 黄旭升 王超[1] 王计平[1] 李润植[1] DONG Shuyan;XING Zhi;YIN Miao;WANG Yao;ZHOU Yali;HUANG Xusheng;WANG Chao;WANG Jiping;LI Runzhi(Institute of Molecular Agriculture and Bioenergy,Shanxi Agricultural University,Taigu Shanxi 030801,China)

机构地区：[1]山西农业大学分子农业与生物能源研究所,山西太谷030801

出　　处：《西北农业学报》2024年第4期695-706,共12页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家自然科学基金(31201266);山西省基础研究计划(自由探索类)(20210302123418)。

摘　　要：本文基于紫苏基因组数据筛选获得两条PfPAH1基因序列,分别命名为PfPAH1-1和PfPAH1-2。应用生物信息学工具分析PfPAH1s蛋白理化性质、功能结构域及系统进化。定量PCR(qRT-PCR)检测PfPAH1s在紫苏根、茎、叶、花及不同发育时期种子的表达谱。进一步克隆到高表达的PfPAH1-1基因的编码序列并进行烟草瞬时转化以鉴定其功能。结果表明,PfPAH1-1和PfPAH1-2基因的开放阅读框(ORF)长度均为2715 bp,编码904个氨基酸残基,含有Lipin_N、Lipin_mid和属于HAD_like超家族的LNS2三个保守结构域。系统进化分析显示紫苏PfPAH1-1蛋白与唇形科植物一串红的SsPAH1亲缘关系最近,其次是葡萄和芥菜。PfPAH1-2蛋白则与唇形科的芝麻SiPAH1亲缘关系最近,其次为油橄榄,紫苏和芝麻均为油料作物,推测PfPAH1-2和SiPAH1可能具有相似功能。qRT-PCR分析表明PfPAH1-1和PfPAH1-2基因均在开花后40 d种子中表达量最高,预示着其可能参与油脂合成积累。烟草瞬时转化揭示过表达PfPAH1-1基因烟草叶片总脂肪酸及中性脂含量均高于野生型烟草,然而磷脂含量降低。This enzymatic process plays an important role in plant lipid metabolism.In this study,two PfPAH1 genes(PfPAH1-1 and PfPAH1-2)were retrieved from the perilla genome database.Bioinformatics tools were used to analyze the physicochemical properties,functional domains and phylogenetic tree of PfPAH1 proteins.Quantative PCR(qRT-PCR)was employed to examine the expression profiles of the PfPAH1 genes in perilla roots,stems,leaves,flowers and seeds at different development stages.The coding sequence of the highly-expressed PfPAH1-1 gene was cloned and and further used for transient transformation in tobacco to characterize PfPAH1-1 function.The results showed that the open reading frame(ORF)of PfPAH1-1 and PfPAH1-2 gene sequences were 2715 bp in length,encoding 904 amino acid residues.The PfPAH1 protein contained three conservative domains(Lipin_N,Lipin_mid and LNS2)the typical characteristics of the HAD_like superfamily.Phylogenetic tree analysis indicated that PfPAH1-1 protein exhibited the closest genetic relationship with SsPAH1 from Salvia splendens of Labiatae,followed by PAH1s from Vitis vinifera and Capsella rubella,Similarly,PfPAH1-2 protein demonstrated the closest genetic relationship with SiPAH1 from Sesamum indicum of Labiatae,followed by PAH1s from Olea europaea and Capsicum annuum.This suggested that PfPAH1-2 and SiPAH1 genes,which have similar functions,are present in oil crops from the same family.qRT-PCR analysis indicated that the highest expression levels of PfPAH1-1 and PfPAH1-2 genes were detected in the developing seeds at 40 days after flowering,suggesting their crucial role in lipid biosynthesis.The evaluation of transiently transformed tobacco plants showed higher contents of total fatty acids and neutral lipids in the PfPAH1-1 transgenic leaves compared to wild tobacco leaves,while the content of phospholipids was lower in the transgenics.

关 键 词：紫苏 磷脂酸磷酸水解酶 基因克隆 表达特性 烟草瞬时表达 

分 类 号：S565.9[农业科学—作物学]