番茄SlNAC1基因启动子低温、高温和ABA应答顺式元件分析

Analysis of Cold-,Heat-,and ABA-responsive cis Elements in Tomato SlNAC1 Promoter

番茄转录因子SlNAC1调控多种生物和非生物胁迫应答,但其上游转录调控因子至今不明,限制了对其胁迫应答机制的理解。构建了系列5'-缺失的SlNAC1启动子(起始密码子上游2039 bp、1508 bp、1373 bp和777 bp)驱动的GUS转基因烟草,并定量分析了其在低温、高温和ABA诱导下的GUS酶活,以鉴定SlNAC1启动子中的低温、高温和ABA应答顺式元件。结果显示,低温和高温诱导后,2039 bp启动子转基因烟草GUS酶活的增长显著高于其他转基因烟草和野生型烟草;而ABA诱导后,1508 bp启动子转基因烟草GUS酶活的增长显著高于其他转基因烟草和野生型烟草。这些结果说明低温和高温应答顺式元件位于-2039~-1508 bp区间,而ABA应答顺式元件位于-1508~-1373 bp区间。启动子顺式元件预测分析显示,-2039 bp~-1508 bp区间只有1个低温/高温/干旱/盐应答顺式元件DRE/CRT,而-1508 bp~-1373 bp区间只有1个ABA应答顺式元件ABRE。因此,这两个顺式元件将作为候选元件用于后续SlNAC1上游调控转录因子的筛选。

Tomato transcription factor SlNAC1 has been reported to regulate several biotic and abiotic stress responses,but its upstream regulatory transcription factors remain unknown,which limits our understanding of its molecular mechanism of stress responses.We constructed series of 5'-deleted SlNAC1 promoters(2039 bp,1508 bp,1373 bp and 777 bp upstream of start codon)-driven GUS transgenic Nicotiana benthamiana and analyzed quantitatively their GUS activity under cold,heat and ABA treatment conditions in order to identify cold-,heat-,and ABA-responsive cis elements.Our results show that the GUS activity of 2039 bp promoter-driven transgenic Nicotiana benthamiana increased much higher than that of any other transgenic Nicotiana benthamiana and wild-type Nicotiana benthamiana after cold and heat treatment,while the GUS activity of 1508 bp promoter-driven transgenic Nicotiana benthamiana increased much higher than that of any other transgenic Nicotiana benthamiana and wild-type Nicotiana benthamiana after ABA treatment.These results indicate that both cold-responsive and heat-responsive cis elements were located in the region from-2039 bp to-1508 bp,and ABA-responsive cis element(s)was(were)located in the region from-1508 bp to-777 bp.According to cis elements'prediction of SlNAC1 promoter,there were only one cold/heat/drought/salt-responsive cis element DRE/CRT in the region from-2039 bp to-1508 bp and only one ABA-responsive cis element ABRE in the region from-1508 bp to-777 bp.Therefore,these two cis elements will be used as candidates for subsequent site-directed mutagenesis validation and screening of the upstream regulatory transcription factor of SlNAC1.