摘要
该研究以野生型番茄(Solanum lycopersicum)为材料,采用PCR技术克隆得到了番茄SlWRKY31基因起始密码子ATG上游启动子序列,并利用该启动子驱动GUS基因在野生型番茄中表达,对获得的转基因番茄采用不同胁迫处理后进行GUS染色和定量分析。结果表明:(1)序列分析显示,该启动子全长1 849bp,含有多个与非生物胁迫和激素响应相关的顺式作用元件,主要包括热胁迫响应元件HSE、干旱诱导响应元件MBS、防卫和胁迫响应元件TC-rich repeats、创伤诱导响应元件WUN-motif、脱落酸(ABA)响应元件ABRE和水杨酸(SA)响应元件TCA-element。(2)实时荧光定量PCR结果显示,SlWRKY31基因呈组成型表达模式,且在叶和果实中表达量较高,茎中较低;在NaCl、甘露醇、SA、ABA和42℃高温的胁迫处理下,其表达量显著升高。(3)构建SlWRKY31启动子和GUS基因融合的植物表达载体,并通过农杆菌介导法将其转化野生型番茄,对获得的转基因番茄进行GUS组织化学染色分析结果显示,SlWRKY31基因在番茄的各个组织(根、茎、叶、花、果实和种子)中均有表达,表明SlWRKY31启动子是组成型表达启动子。(4)对转基因番茄在不同胁迫处理后的GUS染色和定量分析显示,SlWRKY31启动子显著受到NaCl、甘露醇、SA、ABA和42℃高温的诱导表达,说明该启动子是一个可以响应多种逆境胁迫的诱导型启动子。
In this study, the wild type tomato (Solanum lycopersicum) was used as the experimental material. The 1 849 bp promoter fragment in the upstream of the start codon of the SlWRKY31 gene from tomato was isolated by PCR cloning method. This promoter was then used to drive the GUS gene expression in wild type tomato, and the obtained transgenic tomato was analysed by GUS histochemical staining and quantitative assay of GUS activity after different stress treatments. These results showed that:(1) sequence analysis revealed that the length of this promoter was 1 849 bp, and contained a large number of abiotic stess and phytohormone responsive elements, such as heat stess responsive element (HSE), drought inducibility element (MBS), defense and stess responsive element (TC rich repeats), wound responsive element (WUN motif), abscisic acid responsive element (AREB), and salicylic acid responsive element (TGA element).(2) Quantitative real time PCR (qRT PCR) showed that the SlWRKY31 gene was expressed ubiquitously in all tissues examined, but its expression level was significantly increased under Mannitol, NaCl, SA, ABA, and heat stress (42 ℃) treatments.(3) A plant expression vector was constructed using the SlWRKY31 promoter to drive the GUS reporter gene (SlWRKY31 Pro::GUS), and was transformed into wild type tomato by Agrobacterium mediated transformation. GUS histochemical staining analysis of the transgenic tomato plants showed that the GUS gene was expressed in all tissues of tomato, indicating that the SlWRKY31 promoter is a constitutive promoter.(4) The GUS histochemical staining and the quantitative assay of transgenic plants after different stress treatments showed that the SlWRKY31 promoter was significantly induced by NaCl, Mannitol, SA, ABA and heat stress (42 ℃), indicating that the SlWRKY31 promoter is an inducible promoter that can respond to multiple stresses.
作者
高永峰
杨丰铭
李琴中
张国燕
王丹
姚银安
刘继恺
GAO Yongfeng;YANG Fengming;LI Qinzhong;ZHANG Guoyan;WANG Dan;YAO Yin an;LIU Jikai(School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang, Sichuan, 621010, China)
出处
《西北植物学报》
CAS
CSCD
北大核心
2018年第12期2155-2164,共10页
Acta Botanica Boreali-Occidentalia Sinica
基金
四川省教育厅科研项目(17ZB0456)
国家自然科学基金(31770644)
四川省国际科技合作项目(2017HH0050)
西南科技大学龙山学术人才科研支持计划(18LZX626)
