桑树1-氨基环丙烷-1-羧酸氧化酶基因(MnACO)启动子功能分析

Functional Analysis of 1-Aminocyclopropane-1-carboxylate Oxidase Gene's Promoter in Mulberry

1-氨基环丙烷-1-羧酸氧化酶(ACO)作为关键酶,能够催化1-氨基环丙烷-1-羧酸(ACC)形成乙烯。为探究桑树MnACO基因在桑树生长发育和抵御外界胁迫中的功能,本研究构建了p MnACO::GUS的植物表达载体并转化拟南芥。采用GUS组织染色法鉴定转基因拟南芥不同生长阶段及胁迫处理后的GUS活性。通过PCR克隆得到MnACO1和MnACO2启动子片段,它们分别为1518 bp和1429 bp,启动子区域有大量的TATA-box、CAAT-box和其他响应外界刺激的顺式作用元件。GUS活性分析显示MnACO启动子能驱动GUS在拟南芥中表达;MnACO1启动子能驱动GUS在拟南芥的根、叶片、花瓣、花药、花丝、柱头以及果荚中表达,且活性较MnACO2强;MnACO2启动子不能驱动GUS在果荚中无表达。转MnACO1和MnACO2植株经不同逆境处理后GUS表达活性不同,转MnACO1植株的GUS活性随处理延长时间而减弱,转MnACO2植株GUS活性随处理时间延长而增强。q RT-PCR检测2周苗龄的桑幼苗在经过胁迫处理后Ma ACO1和Ma ACO2的基因表达量,发现Ma ACO基因的表达模式与MnACO启动子GUS活性变化趋势一致。本研究结果表明,MnACO为诱导型启动子,MnACO1兼具组成型启动子特性,MnACO2兼具组织特异型启动子特性。MnACO1在转基因植株中对胁迫响应能力更强,预示着可将其用来调控改良桑树品种抗逆性靶基因;Ma ACO2可能与果实成熟有关,可将其启动子作为果实特异性启动子对桑椹品质进行合理改良。

1-Aminocyclopropane-1-carboxylate oxidase(ACO)as a key enzyme catalyzes the reaction from1-Aminocyclopropane-1-carboxylate(ACC)to ethylene.In order to explore the function of MnACO in mulberry growth and developmentand resisting external stresses,we constructed pMnACO::GUS fusion and transformed it into Arabidopsis thaliana,and used GUShistochemical staining to identify GUS activities of transgenic Arabidopsis thaliana treated by different stresses in differentgrowth stages.MnACO1and MnACO2promoter fragments obtained by PCR were1518bp and1429bp,respectively.There werelots of cis-acting elements such as TATA-box,CAAT-box and others responded to external stimuli.MnACO promoter could driveGUS to express in Arabidopsis thaliana;MnACO1promoter could induce GUS expressed in the root,leaf,flower petal,anther,filament,stigma and silique of Arabidopsis thaliana and their activities were higher than those of MnACO2;while,MnACO2promoter could not drive GUS expression in silique.The pMnACO1::GUS and pMnACO2::GUS transgenetic plants had differentactivities in different treatments.The GUS activities of the pMnACO1::GUS transgenetic plants were weakened,while those ofthe pMnACO2::GUS transgenetic plants were strengthened with the elongation of stress treatment time.Detection of MaACOexpression in stress treatment of2-week-old seedlings by qRT-PCR,indicated that the trends of expression pattern of MaACO and its GUS activity changes were almost the same.This research indicated that MnACO is inducible promoter,MnACO1has thecharacteristic of constitutive promoter,and MnACO2has the characteristic of tissue-specific promoter.MnACO1had more abilityto respond to stresses in the transgenic plant,indicating it can be used to regulate the target gene of improving mulberry stressresistance.MaACO2might be relevant to fruit maturation,thus its promoter could be used as fruit-specific promoter to improvethe quality of mulberry fruit.