水稻铵转运蛋白基因OsAMT1;2的启动子分析

Promoter Analysis of Rice Ammonium Transporters OsAMT1;2

AMT是介导植物铵态氮高亲和吸收的跨膜蛋白,已报道编码AMT蛋白的大部分基因的表达依赖外界氮水平和环境的调控,但调控的机制目前尚不清楚。水稻的Os AMT1;2基因在氮缺乏条件下强烈诱导表达,因此,本研究从水稻基因组DNA中PCR克隆Os AMT1;2基因上游1 940 bp的启动子序列,采用软件和试验分析Os AMT1;2启动子存在的顺式调控元件与外界氮调控的相关性。软件预测结果表明,启动子区域内除含有TATA-box和CAAT-box外,同时含有多种非生物胁迫、生物胁迫、激素响应和光响应相关的顺式作用元件,如Box-w1、MBS、Me JA、TATC和ERE应答元件。将Os AMT1;2启动子的5′端顺序删除获得5个不同缺失片段分别与GUS报告基因融合构成表达载体,通过农杆菌介导转入水稻成熟胚诱导的愈伤组织中获得转基因水稻,T1代植株水平上的GUS活性分析表明,最短启动子片段(–211 bp)能够启动GUS表达,但是GUS活性较低,对缺氮效应也不显著;中等长度启动子片段(–763 bp)的GUS活性提高,受缺氮诱导显著,且缺氮诱导不再随启动子长度增加而显著增强,说明应答氮响应的顺式作用元件位于–211 bp和–763 bp之间,非生物胁迫和激素相关的顺式作用元件多在这个区域内,是否与氮的调控存在相关性有待后续进一步证明。

AMT is transmembrane protein mediated high-affinity ammonium uptake in plants. It has been reported that the expression of most genes encoding AMT protein depends on external nitrogen level and environmental condition, but the mechanism of expression regulation is unclear. The expression of OsAMT1;2 was greatly induced by nitrogen-deficiency. Therefore, the sequence of OsAMT1;2 promoter was cloned by using PCR from rice genome DNA and analyzed by using software and experimental analysis fusion GUS reporter gene. Software predicted results showed that the promoter region contained not only the TATA box and CAAT box, but also a variety of cis-elements of abiotic stress, biological stress, hormone and light response, such as Box-w1, MBS, MeJA, TATC and ERE. OsAMT1;2 promoter was deleted along with 5＇ end to construct five different pAMT12-x-GUS expression vector, which subsequently were transformed into rice callus to establish different transgenic rice lines. The GUS activity of T1 generation plants showed that the shortest promoter fragment （-211 bp） could start the GUS expression, but GUS activity was low and the effect of nitrogen-deficiency was not significant. The GUS activity and inductive effect of nitrogen-deficiency significantly increased when -763 bp promoter fragment fusion with GUS, additionally the inductive effect no longer significantly enhanced with the increase of the promoter length. The cis-element of abiotic stress and hormones related lies between -211 bp and -763 bp, whether correlation between nitrogen regulation and cis-elements of abiotic stress and hormones remains to be further probed.