紫花苜蓿高温诱导启动子pMsMBF1c的克隆与功能分析

Cloning and function analysis of the high temperature inducible promoter pMsMBF1c in alfalfa (Medicago sativa)

植物耐热性受复杂而精细的调控。热诱导启动子能经济、高效的激活或关闭耐热调节途径关键基因的表达,在植物功能基因组研究与现代分子育种技术中起十分重要的作用。以紫花苜蓿耐热候选基因MsMBF1c的编码序列为基础,采用酶切连接的方法分离获得了其上游1748bp序列,生物信息学分析发现该区域具有HSE与GATA结合位点等2个与植物耐热调节相关的保守模体,此外还有5个ABA应答元件(ABRE、MYB2、MIC2、CBF与DPBF)和2个MYB蛋白结合位点,说明MsMBF1c除了参与植物耐热性调节外,还可能参与其他抗逆性调节。构建pBI121-MsMBF1c::GUS双元载体转化野生型拟南芥,荧光定量分析热诱导后的转基因植物中GUS与AtMBF1c基因的表达发现其分别上调了5.4与4.8倍,并且高温诱导下转基因植株的组织化学染色分析同样证明MsMBF1c启动子显著受高温诱导。分离获得紫花苜蓿MsMBF1c启动子序列并转化拟南芥,并且从生物信息学、组织化学染色与基因表达等方面验证该序列能显著被高温诱导,为探讨紫花苜蓿耐热调控机制及通过分子生物技术改善紫花苜蓿耐热性提供理论支撑,最终为培育适应南方高温气候条件的紫花苜蓿新品种提供技术储备。

Plant thermotolerance is controlled by a complex and finely regulated network of transcription processes.High temperature inducible promoters can economically and efficiently activate or shut down the expression of key genes involved in regulating thermotolerance.Knowledge of these promoters is therefore important in plant functional genomics research and the molecular breeding industry.In the present study,a 1748 bp sequence upstream of MsMBF1c,which is a candidate gene involved in thermotolerance in alfalfa,was isolated using a digestion ligation PCR method,based on its coding sequence.Bioinformatics analysis showed that two conserved motifs representing the HSE and GATA binding sites involved in thermotolerance regulation existed in the MsMBF1c promoter region.There were 5 abscisic acid response cis-elements(ABRE,MYB2,MIC2,CBF and DPBF)and 2 MYB protein binding motifs in the MsMBF1c promoter region,which suggested MsMBF1c might regulate other abiotic stress response in addition to thermotolerance.A vector of pBI121-MsMBF1c::GUS was constructed and transformed into wild type Arabidopsis.The expression of GUS and AtMBF1c genes,was examined by qRT-PCR,and found to be up-regulated 5.4 and 4.8 fold,respectively,after heat stress.At the same time,histochemical staining analysis also showed the MsMBF1c promoter to be markedly induced by high temperature.To summarize,the MsMBF1c promoter was isolated and transformed into Arabidopsis,and was proved to be heat inducible by bioinformatics and gene expression data,and by histochemical staining analysis.This information provides technical and theoretical support for further investigation of heat regulation mechanisms and breeding of heat resistant alfalfa varieties.