转录因子DREB1A基因的克隆与Gateway克隆技术构建植物表达载体

Cloning of Transcription Factor DREB1A Gene and Construction of Plant Expression Vector by Gateway Clone Technique

目的:研究转录因子DREB1A在植物抗渗透胁迫反应中的作用,并探讨利用Gateway克隆技术构建植物表达载体的方法。方法:根据GenBank中登录的DREB1A基因的全长mRNA序列设计引物,克隆了拟南芥的转录因子DREBIA基因。根据Gateway克隆技术的要求,设计含有attB接头的引物,利用高保真的PlatinumpfxDNA聚合酶,通过PCR方法在克隆基因的两端加上B序列。通过BP反应将包含有attB接头的PCR产物克隆到含有attP的donor载体上以产生Entry克隆,通过LR反应将已经重组入Entry载体的DREB1A基因再克隆到pH2GW7双元载体。结果:对重组载体pH2GW7-DREB1A的鉴定结果表明成功构建了DREB1A基因的植物表达载体。结论:利用Gateway克隆技术构建植物表达载体简便易行,该结果为遗传转化研究奠定了基础。

Objective： To research the functional the DREB1A gene in plant tolerance to osmotic stress and to develop the constructing way of the plant expression vector by Gateway technology. Methods： A pair of primer was designed according to the mRNA sequence of DREB1A gene in GenBank databases, DREB1A of Arabidopsis thaliarta was cloned. Two pair of primers containing attB adapter were designed separately by Gateway cloning technology. Platinum pfx DNA polymerase which can reduce the non-specific binding greatly was used during two PCR in which adding B sequence to the cloned gene. By the BP recombination reaction, the PCR product containing attB was transfered to an attP-containing donor vector to create an entry clone. Finally, DREB1A gene was shutted into pH2GW7 vector by LR recombination reaction. Results： The plant expression vector of pH2GW7-DREB1A was successfully constructed by identification. Conclusion： The results showed that it is easy to construct a plant expression vector by Gateway cloning technology, and it provides basic information for genetic transformation with this gene.