能源橡胶草GGPPS基因启动子的克隆及瞬时表达研究

以多年生宿根型草本植物橡胶草为材料,根据已获得的橡胶草牻牛儿基牻牛儿基焦磷酸合酶(geranylgeranyl pyrophosphate synthase,GGPPS)基因序列设计引物,采用TAIL-PCR扩增到GGPPS基因5'上游大小为1131 bp的序列。采用PlantCare和PLACE软件分析,表明该序列不仅具有启动子的基本元件,同时还有与多个器官特异表达元件以及与胁迫相关的顺式作用元件,命名为pTkGGPPS(GenBank:KT901796)。将该序列代替pCAMBIA1304质粒上的CaMV 35S启动子序列,以GFP基因作为报告基因,构建pCAMBIA1304-pTkGGPPS-GFP植物表达载体,利用农杆菌介导法转化洋葱表皮细胞,结果表明,该启动子能够驱动GFP基因表达,具有一定的活性。TkGGPPS基因启动子克隆及瞬时表达为橡胶草中橡胶合成及组织特异性研究奠定了基础。

Cloning and transient expression of the GGPPS gene promoter from the energy plant Taraxacum kok-saghyz

Based on the known sequence of GGPPS, which encodes geranylgeranyl pyrophosphate synthase, the GGPPS promoter sequence was amplified from the root of the perennial herb Taraxacum kok-saghyz by thermal asymmetric interlaced PCR (TAIL-PCR) using nested specific primers. Analyses of the full-length 1131-bp fragment by PlantCare and PLACE software showed that the promoter sequence contained basic cis-acting elements, multiple organ-specific expression elements, and a number of stress-related cis-acting elements. The promoter sequence was designated as pTkGGPPS (GenBank: KT901796). The plant expression vector pCAMBIA1304-pTkGGPPS-GFP was constructed using this sequence to replace the CaMV 35S promoter sequence in the plasmid of pCAMBIA1304 with GFP as the reporter gene. The construct was transformed into onion epidermal cells using Agrobacterium tumefaciens, and the promoter successfully drove expression of the GFP gene. The cloning and transient expression of the GGPPS gene promoter from T. kok-saghyz provides a reference for further studies on the mechanisms and tissue-specificity of rubber synthesis.