摘要
目的:对牛蒡中的醇氧化酶基因AlAOX1进行分析,为后续研究醇氧化酶基因的作用提供理论基础。方法:基于课题组前期转录组数据筛选出的AlAOX1基因,对AlAOX1进行基因克隆、生物信息学分析、系统发育分析与亚细胞定位分析,明确其基因特性和理化性质,通过实时荧光定量聚合酶链式反应(Real-time PCR)分析牛蒡子萌发过程和不同组织中的基因表达情况,并对不同非生物胁迫条件下的基因表达情况进行分析。结果:成功克隆出AlAOX1基因,并上传至国家生物技术信息中心(NCBI)数据库。AlAOX1基因的序列长度为2214 bp,编码737个氨基酸。AlAOX1蛋白具有亲水性,属于稳定蛋白、无跨膜结构与信号肽,包含75个磷酸位点。系统进化树分析表明该蛋白与菊科植物聚为一支,其中与刺苞菜蓟中的同源蛋白相似性最高。亚细胞定位分析表明AlAOX1定位于叶绿体。Real-time PCR分析表明,AlAOX1基因在种子萌发过程中与不同组织中的表达量存在差异,在第9天的幼苗和根中的表达量最高,分别为(25.62±3.24)(P<0.01)和(1.70±0.11)(P<0.01),显著高于空白组;非生物胁迫实验结果表明,牛蒡根在高盐胁迫(200 mmol·L^(-1))、干旱胁迫(25%PEG)和低温胁迫(4℃)处理下,分别在24、24、12 h表达量达到最高,分别为(2.63±0.93)(P<0.01),(1.58±0.21)(P<0.05)和(2.51±0.44)(P<0.01),均显著高于对照组,具有抗非生物胁迫的作用。结论:该研究成功克隆出AlAOX1基因,确定了AlAOX1基因的分子特征与表达特性,推测其在牛蒡的生长发育及非生物胁迫中发挥调控作用,为进一步研究醇氧化酶基因在牛蒡中的具体作用奠定理论基础。
Objective To analyze the alcohol oxidase gene AlAOX1 in Arctium lappa and lay a theoretical foundation for the subsequent study of the role of the alcohol oxidase gene.Methods The AlAOX1 gene was screened based on the pre-transcriptomic data of our group.In this paper,gene cloning,bioinformatics analysis,phylogenetic analysis,and subcellular localization analysis were performed to clarify the gene characteristics and physicochemical properties of AlAOX1,and the gene expression of A.lappa in the germination process and different tissues was analyzed by Real-time polymerase chain reaction(Real-time PCR),and the gene expression of A.lappa was analyzed under different abiotic stresses.Results The AlAOX1 gene was successfully cloned and uploaded to the National Center of Biotechnology Information(NCBI)database.The sequence length of the AlAOX1 gene was 2214 bp,encoding 737 amino acids.The AlAOX1 protein is hydrophilic and belongs to the category of stabilized proteins,non-transmembrane structure,and signal peptide,and it contains 75 phosphorylation sites.Phylogenetic tree analysis showed that this protein clustered into a clade with Asteraceae,with the highest similarity to homologous proteins in Cynara cardunculus.Subcellular localization analysis showed that AlAOX1 was localized in chloroplasts.The Real-time PCR analysis showed that the expression of the AlAOX1 gene varied with different tissues during seed germination,and the highest expression was found in seedlings and roots at day 9,reaching(25.62±3.24)(P<0.01)and(1.70±0.11)(P<0.01),respectively,which were significantly higher than the control group.The results of abiotic stress experiments showed that A.lappa roots were treated with high salt stress(200 mmol·L^(-1)),drought stress(25%PEG),and low-temperature stress(4℃)at 24,24,12 h,respectively,at(2.63±0.93)(P<0.01),(1.58±0.21)(P<0.05),and(2.51±0.44)(P<0.01),all significantly higher than the control group,which possessed the effect of resistance to abiotic stresses.Conclusion In this study,the AlAOX1 gene was successfully cloned,and the molecular characterization and expression properties of the AlAOX1 gene were determined.It was hypothesized that it plays a regulatory role in the growth and development of A.lappa,as well as in abiotic stress,which lays a theoretical foundation for further research on the specific roles of alcohol oxidase genes in A.lappa.
作者
薛嘉宁
赵容
于莹
高铭泽
韩依霖
XUE Jianing;ZHAO Rong;YU Ying;GAO Mingze;HAN Yilin(School of Pharmacy,Liaoning University of Traditional Chinese Medicine,Dalian 116600,China)
出处
《中国实验方剂学杂志》
北大核心
2025年第23期173-180,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
辽宁省教育厅高校基本科研项目(LJ212410162046)
辽宁省科技厅项目(2023-MSLH-185)。
关键词
牛蒡
醇氧化酶
基因克隆
表达分析
亚细胞定位
Arctium lappa
alcohol oxidase
gene cloning
expression analysis
subcellular localization
