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用棉花生殖器官优势表达启动子替代CaMV35S启动子是解决目前转基因抗虫棉存在“前期抗虫性强,后期抗虫性弱”这一生产实践问题的关键.运用反向PCR技术从陆地棉中分离到一个棉花生殖器官优势表达基因——腺苷酸核糖基化作用因子-1(Arf1)基因的启动子序列.该启动子具有独特的结构特征,不仅同时包含有转录起始子(initiator),TATA box,CAAT box和GC box这四种特异元件,而且还包含有一个5’非翻译区的内含子.通过构建四个启动子缺失突变体, 定向替换植物表达载体pBI121上的CaMV35S启动子,并与GUS基因融合,构建了4个植物表达载体.用花粉管通道技术将它们导入到棉花中,转基因棉花后代的GUS染色和荧光定量分析结果表明:Arf1启动子是一个典型的棉花生殖器官优势表达启动子,它为棉花生殖器官性状的分子设计和抗虫棉的再创新提供了有用的工具.
Replacing the CaMV35S promoter with the dominant promoter of cotton genitalia is the key to solve the production practice problem of transgenic cotton with “strong prophase of pest resistance and weak pest resistance in the late stage.” By using reverse PCR technique, A promoter of Arf1 gene, which is the dominant gene of cotton reproductive organ, was isolated and characterized by its unique structural features, including not only the initiator of transcription, , TATA box, CAAT box and GC box, but also contained an intron of 5 ’untranslated region. By constructing four promoter deletion mutants, directional replacement of CaMV35S on plant expression vector pBI121 was initiated Four plant expression vectors were constructed by fusion with GUS gene.The GUS staining and fluorescence quantitative analysis of transgenic cotton progeny showed that the Arf1 promoter is a typical cotton reproduction The organ-superior expression promoter provides a useful tool for the molecular design of cotton reproductive organ traits and the reinvention of insect-resistant cotton.