紫花苜蓿MsLEA4启动子的克隆及表达分析

Cloning and Expression Analysis of MsLEA4 Promoter from Medicago Sativa

为了深入研究紫花苜蓿(Medicago sativa)MsLEA4基因的功能,本试验采用染色体步移技术从紫花苜蓿基因组中扩增出MsLEA4启动子序列,构建GUS植物超表达载体,并根据序列分析结果对紫花苜蓿进行相应逆境胁迫。结果表明:紫花苜蓿MsLEA4启动子含有响应脱落酸(abscisic acid,ABA)调控的顺式作用元件ABRE,响应赤霉素(gibberellin,GA)调控的顺式作用元件P-box,参与胚乳合成的顺式作用元件GCN4和Skn-1,以及涉及光调控和光周期的顺式作用元件;外源ABA、GA、持续光照以及黑暗均能诱导MsLEA4基因的表达;GUS基因只在拟南芥花和荚果中表达。因此,MsLEA4基因能参与紫花苜蓿的逆境调控,与该基因启动子序列中所含的一系列顺式作用元件相关。本研究为进一步探索MsLEA4基因在紫花苜蓿逆境胁迫、光调控以及组织特异性表达中的作用奠定了基础。

In order to further analyze the function of MsLEA4,we cloned MsLEA4 promoter sequence from alfalfa(Medicago Sativa)genome by genomic walking technique,constructed MsLEA4 promoter::GUS transient expression vector,and also conducted corresponding stresses on alfalfa according to the promoter sequence analysis.The results showed that:MsLEA4 promoter contained the basic elements TATA-box and CAAT-box,included abscisic acid(ABA)responsive elements(ARBE),gibberellin(GA)responsive elements(P-box),and cis-elements(GCN4,Skn-1)involved in endosperm synthesis.In addition to this,the promoter sequence also contained 6 cis-elements related to light regulation and a cis-element related to circadian rhythm.ABA,GA,continuous light and darkness all can induce MsLEA4 expression;the GUS gene was expressed only in flowers and siliques of Arabidopsis thaliana.Therefore,MsLEA4 can participate in the regulation of alfalfa stress responses,which is related to a series of cis-elements contained in the promoter.This study laid a foundation for further exploring the role of MsLEA4 in stresses,light regulation and tissue specific expression of alfalfa.