拟南芥中人工启动子GWSF转录特性及乙烯诱导活性分析

Transcriptional properties and transcriptional activities to ethylene of a synthetic pathogen-inducible promoter GWSF in Arabidopsis thaliana

理想的病原诱导型启动子可用来精确调控抗病基因的时空表达,消除转基因中的副作用。选用Gst1-box、W-box、S-box、F-box元件和CaMV minimal 35S启动子设计长度为374 bp的人工启动子GWSF。用GWSF替代p BI121中调控gus基因的CaMV 35S启动子后,将重组质粒导入农杆菌GV3101,并通过花序浸泡法得到转GWSF:gus拟南芥。以T_4代植株为材料,GUS染色发现GWSF本底表达低,受疫霉菌、青枯菌及抗病信号分子水杨酸的诱导,但不受非致病菌大肠杆菌和逆境激素脱落酸(ABA)的诱导。序列分析发现,GWSF含有乙烯应答元件(ERE)。用乙烯处理T_4代转基因植株,通过实时荧光定量PCR(qPCR)检测GWSF的乙烯诱导活性。当乙烯浓度为0.2 mmol·L^(-1)时,GWSF转录活性最高,达到CaMV 35S的11.33倍。结果表明,GWSF是较为理想的植物病原和乙烯诱导型启动子,具有本底表达低、诱导因子广、启动表达快、诱导效率高等优点,可用于植物转基因抗病育种。

Ideal pathogen-inducible promoters meet the demands for desired temporal and spatial regulation of transgenes with minimal side effects. GWSF, a synthetic pathogen-inducible promoter（SPIP）, was designed with Gst1-box, W-box, S-box, F-box and CaMV 35 S minimal promoter. GWSF was used to replace the wildtype CaMV 35 S promoter in the plasmid pBI121 in order to control the expression of the β-glucuronidase（gus） gene. The transcriptional properties and transcriptional activities in response to ethylene of GWSF were evaluated by histochemical staining and real-time quantitative PCR（qPCR） in homozygous T_4 lines of transgenic Arabidopsis thaliana. GWSF had a low basal expression and responded to Ralstonia solanacearum, Phytophthora capsici and salicylic acid, but did not respond to abscisic acid and Escherichia coli. GWSF could be induced by ethylene for an ethylene response element in its sequence. At a concentration of 0.2 mmol·L^（-1） ethylene, the transcriptional activities reached to the peak that was 11.33 times as high as the wild-type CaMV 35 S promoter. The results indicate that GWSF is an ideal SPIP with the advantages of a low background expression, a wide range of inducing pathogens, rapid responses and efficient transcriptional activities. It can be potentially improved further to apply to plant genetic engineering for disease resistance.