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[目的]对控制辣椒辣味基因pun1的克隆及表达进行研究。[方法]以益都红(Capsicum annuum L)为材料,获得Capun1基因c DNA序列,全长1 457 bp,编码440个氨基酸;研究了其瞬时表达、原核表达和实时荧光定量表达。[结果]系统进化分析显示,CAPUN1与同属C.chinense辣椒PUN1遗传距离最近,为0.019 3。构建植物表达载体p CAM-Pun1-GFP转化烟草瞬时表达,发现融合基因Pun1::GFP编码的蛋白定位在细胞膜上。构建原核表达载体,通过SDS-PAGE和Western Blot检测,得到了一个分子量为63 ku的诱导蛋白。实时荧光定量表达发现Pun1基因在花后30 d表达量最高,而后开始下降,花后40和45 d基本不表达。[结论]该研究为研究辣椒辣味基因Pun1的调控分子机制提供了信息和参考。
[Objective] The research aimed to study the cloning and expression of the puni gene pun1. [Method] The cDNA sequence of Capun1 gene was obtained from Capsicum annuum L. The full length cDNA was 1 457 bp encoding a protein of 440 amino acids. The transient expression, prokaryotic expression and real - time fluorescence quantitative expression of Capun1 gene were studied. [Result] Phylogenetic analysis showed that the genetic distance between CAPUN1 and the same genus C. chinense pepper PUN1 was 0.019 3. The transient expression of transformed plant p CAM-Pun1-GFP was constructed and the protein encoded by the fusion gene Pun1 :: GFP was located on the cell membrane. The prokaryotic expression vector was constructed and an induced protein with molecular weight of 63 ku was obtained by SDS-PAGE and Western Blot. The expression of Pun1 gene was highest at day 30 after flowering, and then began to decline after 40 days and 45 days after flowering. [Conclusion] The study provided information and reference for studying the regulatory molecular mechanism of Pun1.