苹果属垂丝海棠MhPPOX1基因克隆及抗缺铁性功能鉴定

Molecular Cloning of MhPPOX1 Gene from Malus halliana and Anti-iron Deficiency Function Identification

原卟啉原氧化酶(Protoporphyrinogen oxidase,PPOX1)是叶绿素生物合成途径中的关键酶,为深入探究苹果PPOX1基因的功能,该研究以苹果砧木垂丝海棠(Malus halliana)为试材,采用PCR方法,克隆MhPPOX1基因,并进行生物信息学分析及功能鉴定;采用农杆菌介导法转化烟草和拟南芥,进一步分析MhPPOX1在缺铁胁迫中的功能,并对转基因烟草与拟南芥进行抗性分析。结果表明:(1)成功克隆获得垂丝海棠MhPPOX1基因片段,经序列比对鉴定为苹果的MhPPOX1基因(序列号:LOC103444480)。MhPPOX1基因的开放阅读框为1644 bp,编码547个氨基酸,等电点为8.98;系统进化树分析表明,苹果属垂丝海棠MhPPOX1与白梨该家族蛋白的亲缘关系最近。(2)成功克隆获得垂丝海棠MhPPOX1启动子序列片段(2016 bp),对该启动子顺式作用元件预测结果显示,MhPPOX1启动子序列中存在干旱、低温、光、生长素以及与叶绿素相关等响应元件。(3)成功构建过表达载体MhPPOX1-pRI101,并成功获得转MhPPOX1基因烟草和拟南芥。(4)qRT-PCR分析表明,垂丝海棠幼苗在缺铁(-Fe)胁迫下植株叶片黄化枯死,且MhPPOX1基因表达量较对照显著升高;转MhPPOX1基因烟草和拟南芥在缺铁胁迫中与野生型相比均生长良好,不易黄化,且缺铁条件下转基因拟南芥和烟草的叶绿素a、叶绿素b总量以及总铁含量明显高于野生型植株,表明MhPPOX1基因过量表达提高了拟南芥和烟草对缺铁胁迫的抗性。研究认为,MhPPOX1基因在植物抵抗缺铁胁迫中可能发挥重要作用。

Protoporphyrinogen oxidase(PPOX1)is a key enzyme in the pathway of chlorophyll biosynthesis,and it is used to explore the function of PPOX1 gene in apple.In this study,Malus halliana,as a apple rootstock was used as the test material.The MhPPOX1 gene fragment was cloned by PCR and bioinformatics analysis and functional identification were conducted.Using the method of agrobacterium mediated genetic transformation of Nicotiana tabacum L.and Arabidopsis thaliana,the function of MhPPOX1 under iron deficiency stress was further analyzed,and resistance analysis of transgenic N.tabacum L.and A.thaliana were conducted.The results show that:(1)the MhPPOX1 gene fragment of M.halliana was successfully cloned and identified as apple's MhPPOX1(serial number:loc103444480).The open reading frame of MhPPOX1 gene is 1644 bp,encoding 547 amino acids,and the isoelectric point is 8.98.Phylogenetic tree analysis showed that the amino acid sequence encoded by MhPPOX1 gene was closely related to the amino acid in Pyrus bretschneideri.(2)Successfully cloning of the MhPPOX1 promoter fragment of M.halliana(2016 bp).The New PLACE database was used to predict and analyze the cis acting elements of the promoter.There were response elements such as drought,low temperature,light,auxin and chlorophyll in MhPPOX1 promoter sequence.(3)The overexpression vector MhPPOX1-pRI101 was successfully constructed,and transgenic N.tabacum L.and A.thaliana were successfully obtained.(4)qRT-PCR analysis showed that under iron deficiency stress(-Fe)the leaves of M.halliana seedlings turned yellow and died,and the expression level of MhPPOX1 gene was significantly higher than that of the control.Compared with wild type,under iron deficiency stress transgenic N.tabacum L.and A.thaliana grew well and not easy yellowing.The total chlorophyll a,chlorophyll b and total iron contents of transgenic N.tabacum L.and A.thaliana were significantly higher than those of wild type plants,which indicated that overexpression of MhPPOX1 gene improved the resistance of N.tabacum L.and A.thaliana to iron deficiency stress.The study suggested that MhPPOX1 gene plays an important role in plant resistance to iron deficiency stress.