植物果实特异性启动子E8基因的克隆

采用高盐低pH值法、分步离心法、SDS法、CTAB法、改良CTAB法提取毛粉802番茄幼苗基因组DNA,其中改良CTAB法提取的DNA效果最好,以此DNA为模板,用特异性引物进行PCR扩增,得到了预期大小的片段,将目的片段回收,克隆进pMD18-T Simple Vector载体,经PCR及酶切检测具有与目标片段长度相符的插入片段,构建的重组pMD18-E8载体经测序结果分析显示,番茄果实特异性E8启动子序列具有高度保守性,与GenBank上发表的E81.1启动子同源性为99.1%,说明成功获得了果实特异性E8启动子基因,为实现外源基因在转基因桃果实中特异性表达做准备。

Clong of Plant Fruit-Specific E8 Promoter

The DNA was extracted from cotyledons of tomato Maofen 802 by high salt and low pH,step by step acentric,SDS,CTAB and improved CTAB method.The improved CTAB method was the best method in the DNA extract.We use this DNA as stencil and amplified by PCR,the product of which was reclaimed and subcloned into pMD18-T Simple Vector.After identification by PCR and restriction enzymes,the recombinant pMD18-E8 vectors were subjected to sequence analysis.As indicated by homology analysis,the resultant tomato fruit-specific E81.1 promoter was highly conservation,with GenBank submission,proved to share 99.1% homology.Therefore,tomato fruit-specific E8 promoter has been successfully cloned,thus making possible the subsequent research in transgenic peach.