烟草PR1a基因启动子的克隆及其活性检测

Cloning of tobacco PR1a promoter and its activity evaluation

【目的】从烟草中克隆得到病程相关蛋白1a基因的启动子PR1aP,并对其进行活性检测。【方法】通过PCR方法,从烟草基因组中克隆病程相关蛋白1a基因的启动子PR1aP,构建该启动子驱动的可溶性绿色荧光蛋白基因(smGFP)的植物表达载体p3300-PR1aP-GFP,并用农杆菌介导法将其导入烟草,检测转基因烟草植株经水杨酸(SA)诱导前后GFP的表达水平,通过SDS-PAGE和ELISA对其在不同诱导条件下GFP的表达情况进行分析。【结果】序列分析表明,PR1aP的长度为1 320 bp,含已报道序列的全部顺式作用元件,如水杨酸相关的W-box、ACGT序列等,也有增强子as-a-like元件及CAAT、TATA等常见应答元件。PCR检测结果显示,成功地获得了转基因植株;荧光检测表明,启动子PR1aP在正常条件下不表现活性,而在水杨酸诱导下有GFP表达。SDS-PAGE和ELISA分析结果表明,当水杨酸诱导时间为72 h、质量浓度为0.25 mg/L时,GFP表达量达到最大值,GFP最高占到总可溶性蛋白的0.083%,产率最高可达15μg/g。【结论】烟草病程相关蛋白1a的启动子PR1aP具有水杨酸诱导性。

【Objective】 This paper aimed to clone and identify the pathogenesis-related protein 1a gene promoter（PR1aP） from tobacco.【Method】 In this study,the promoter,PR1aP,was isolated from the genomic DNA of tobacco by PCR amplification.The cloned promoter PR1aP was used to drive the soluble modified fluorescent protein（smGFP） reporter gene to construct plant expression vector p3300-PR1aP-GFP,which was introduced into tobacco via agrobacterium mediated transformation.Analysis of the smGFP activities,induced by salicylic acid（SA） and before,was employed to detect the induced expression.SDS-PAGE and ELISA analysis were performed to investigate the expression status under different inducible conditions.【Result】 Sequencing analysis showed that PR1aP was 1 320 bp in length,with all cis-elements appeared in the reported sequence,such as SA repeat element W-box,ACGT-box,and enhancer as-a-like,as well as the normal elements CAAT-box,TATA-box etc.PCR results showed that the transgenic plants were obtained successfully.Fluorescence activity assays indicated that PR1aP performed no activity under normal condition,but expression of GFP was actively induced by salicylic acid.Results from SDS-PAGE and ELISA analysis indicated that the GFP reached the max level after being induced by 0.25 mg/L of SA for 72 h,which resulted in 0.083% of total soluble protein and a yield of 15 μg/g.【Conclusion】 The pathogenesis-related protein 1a gene promoter（PR1aP） was inducible using SA as revulsant.