棉花器官特异病原相关启动子的克隆与分析

摘要

通过Tail PCR分离了GHNBS基因的5'侧翼序列,PLACE分析表明其含有CAAT-box、TATA-box及乙烯、茉莉酸甲酯、脱落酸应答元件,病原菌诱发子反应元件W boxes、GT-1、MYB、MYBST1和MYB1LEPR等.同时,在这段序列上还存在一些根特异表达元件.包括2个as-1和1个EECCRCAH1在内的3个增强子元件也存在于这段序列的上游区域,它们可能增强GUS基因在转基因植物中的表达.将GHNBS基因的5'调控序列以不同缺失与GUS基因融合转化拟南芥,发现GUS基因主要在根、茎的韧皮部和叶脉中表达.PGN-1559和PGN-1117表现为器官特异性表达.而PGN-476不能特异表达,同时也不能在根部表达.SA,ABA,MeJA,Ethylene,枯萎病菌和细菌DC3000处理后GUS活性均有显著上升,说明GHNBS基因的5'调控序列含有相应的应答反应因子,是一个器官特异以及与病原相关的启动子.%A promoter region was isolated from the 5' flanking sequence of GHNBS in Gossypium hirsutum by Tail PCR. CAAT-box, TATA-box as well as several pathogen, SA, MeJA and ethylene responsive elements, such as W box, GT-1, MYB and MYBST1 motifs were identified by PLACE analysis while some root specific motifs were also identified in this region. Three enhancer elements including two as-1 and one EECCRCAH1 motifs that were responsible for the strong expression of GUS gene in transgenic plants were located in the far upstream. Fusions of different 5' promoter deletion derivatives with the coding region of the GUS gene were transformed into Arabidopsis. Histochemical localization showed strong staining in roots, phloem of the stem and leaf veins. PGN-1559 and PGN-1117 showed organ specific GUS staining patterns while PGN-476 did not. The fact that GHNBS promoter could enhance the expression level of the GUS gene in transgenic Arabidopsis when treated with SA, ABA, MeJA, ethylene, Fusarium oxyporum and DC3000 showed that there were some regulatory elements in the 5' flanking sequence of GHNBS and which was most possible a pathogen responsive and organ specific promoter.