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本研究以莲瓣兰‘玉兔彩蝶’花瓣为材料,根据GenBank中已经登录的春兰DEF基因序列(HM106982.1)设计引物,克隆DEF基因开放阅读框,构建表达载体,转化‘拟南芥’,并验证基因功能。研究结果表明:所扩增的DEF基因开放阅读框大小为669bp,编码222个氨基酸,预测蛋白质分子质量为25.560kD。该基因具有典型的植物MADS-box基因结构域,与春兰开放阅读框同源性达99%,与其它兰花同源性都在90%以上。将DEF基因构建到植物真核表达载体pCAMBIA2300中,利用农杆菌花序法转化拟南芥,经PCR分子鉴定,获得含DEF转基因拟南芥植株。
In this study, primers were designed according to the sequence of Chunlan DEF gene (HM106982.1) registered in GenBank. The ORF of the DEF gene was cloned and the expression vector was constructed and transformed into Arabidopsis thaliana , And verify gene function. The results showed that the size of open reading frame of DEF gene amplified was 669bp, encoding 222 amino acids, and predicted molecular mass of 25.560kD. The gene has the typical plant MADS-box gene domain, which has 99% homology with Chunlan open reading frame and 90% homology with other orchids. The DEF gene was constructed into the plant eukaryotic expression vector pCAMBIA2300. The Arabidopsis thaliana was transformed by Agrobacterium tumefaciens and identified by PCR. The DEF transgenic Arabidopsis plants were obtained.