盐生草HgNHX1基因启动子的克隆及功能验证

摘要

为了进一步探明盐生草HgNHX1基因启动子的功能,从盐生草基因组中克隆了HgNHX1基因上游5'侧翼调控区1523 bp的序列,即HgNHX1基因启动子(pHgNHX1)序列,并利用PlantCARE、PLACE等在线软件对HgNHX1基因5'端上游序列进行预测和分析,发现该启动子中除具有TATA-box、CAAT-box等核心启动子元件外,还含有多个与盐、干旱、缺水、冷、伤害等逆境胁迫诱导有关的作用元件;同时具有生长素、脱落酸、赤霉素及乙烯等植物激素诱导响应的功能元件,表明分离得到的DNA片段具有典型启动子的一般特征.构建pBI-HgNHX1启动子植物表达载体并转化拟南芥和烟草,利用组织染色法鉴定转基因烟草和拟南芥的β-葡萄糖苷酸酶基因(GUS)表达模式,发现在转基因拟南芥的各个器官均有GUS酶的活性,说明pHgNHX1具有一定的组成型启动子活性.%In order to test the function of pHgNHX1,the promoter sequence of the HgNHX1 gene from Halogeton glomeratus was cloned and a 1523 bp fragment flanking 5'-upstream of HgNHX1 was isolated and named pHgNHX1.The software PlantCARE and PLACE were used to predict and analyze the elements of pHgNHX1.The results showed that besides the TATA-box and CAAT-box,a number of potential cis-elements and transcription binding motifs related to stress responses were found,including salt,dehydration,cold and wound responsive elements.Other potential cis-elements responsive to phytohormones,including auxin,abscisic acid,gibberellin and ethylene were also found in the sequence.Sequence analysis indicated that it had the general characteristics of typical promoter.In order to evaluate the activity of pHgNHX1,we constructed pBI-pHgNHX1 expression vector and introduced it into tobacco and Arabidopsis using agrobacterium mediated transformation.The expression pattern was monitored using GUS histochemical staining.Results showed that GUS activity driven by the pHgNHX1 was detected in almost all vegetative and reproductive tissues,indicating that pHgNHX1 has a constitutive promoter activity.