根癌农杆菌介导的绿色荧光蛋白基因在香蕉枯萎病菌1号生理小种中的转化.doc

  根癌农杆菌介导的绿色荧光蛋白基因在香 蕉枯萎病菌 1 号生理小种中的转化# 孔令广，韩宽德，丁新华，储昭辉** 5 10 15 （山东农业大学植物保护学院，山东省农业微生物重点实验室，山东 泰安 271018） 摘要：香蕉枯萎病是世界范围内香蕉种植区最为严重的病害之一，严重威胁着香蕉产业的发 展。本文利用根癌农杆菌介导的转化法把报告基因绿色荧光蛋白（Green fluorescent protein ， GFP）转入香蕉枯萎病病原菌 1 号生理小种。首先，利用 PCR 扩增 GFP 基因, 通过引入 Cla I 和 Sac酶切位点，连接置换 pUCATPH 载体的潮霉素选择基因,获得中间载体 pUC-GFP。 然后利用 XbaI 从该载体切下含有构巢曲霉启动子(Ptrpc)和 GFP 的片段，并连入 pBHT 载体 中，成功构建了携带 GFP 报告基因适合真菌转化表达载体 pBHT-GFP。该载体经过 PCR、 酶切和测序验证后通过电转化方法转入根癌农杆菌菌株 LB4404，利用农杆菌介导的遗传转 化体系转入香蕉枯萎病菌 1 号生理小种。转化子在含潮霉素平板上经多次单孢纯化后，在无 选择压下仍能发出稳定而且强烈的绿色荧光，用 GFP 特异性引物扩增转化子菌株基因组 DNA 获得预期大小的片段，表明 GFP 基因已成功转入香蕉枯萎病菌 1 号生理小种基因组中， 且后代能够稳定遗传；GFP 标记的转化子生长正常，生长情况和野生型菌株无明显差别， 这为进一步研究该病菌在自然界的侵染过程及致病机制提供了基础性研究材料。 关键词：载体构建；ATMT 转化；香蕉枯萎病菌；绿色荧光蛋白 中图分类号：S432.1 20 Agrobacterium-mediated transformation of green fluorescent protein gene into race 1 isolate of Fusarium oxysporum f.sp. cubense KONG Lingguang, HAN Kuande, DING Xinhua, CHU Zhaohui 25 30 35 40 45 (Shandong Province Key Laborotory of Agricultural Microbiology,College of Plant Protection,Shandong Agricultural University, ShanDong Tai’an 271018) Abstract: Wilt disease of banana is one of the most devastating diseases in the banana-growing regions around the world, which caused seriously threaten and influence on the development of banana industry. In this stduy, we reported to successfully establish the Agrobacterium-mediated transformation system of Fusarium oxysporum f.sp. cubense(FOC) race 1 isolate. Firstly, the DNA fragment of GFP was amplified by ploymerase chain reaction (PCR). After digested with ClaI and SacⅡ, it was ligated into helper vector pUCATPH to replace the hgt gene , which results to construct the intermediate vector pUC-GFP. Then the pUC-GFP was digested with XbaI. And the DNA fragment carried GFP and promoter Ptrpc, was inserted into fungi expression vector pBHT to form the expression construct pBHT-GFP. It was mobilized into LBA4044 after several turns of proved with PCR, size filtration and sequenced. Furthermore, it was transformed int