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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
发表时间：2012-10-15 浏览量：1807 下载量：690
| 全部作者： | 孔令广，韩宽德，巨延虎，丁新华，储昭辉 |
| 作者单位： | 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 |
| 摘 要： | 利用根癌农杆菌（Agrobacterium tumefaciens）介导的转化法把报告基因绿色荧光蛋白（green fluorescent protein, GFP）转入香蕉枯萎病菌(Fusariumoxysporum f.sp.cubense, FOC)1号生理小种。首先，利用PCR扩增GFP基因，通过引入ClaⅠ和SacⅡ酶切位点，连接置换pUCATPH载体的潮霉素B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（Aspevgrillus inidulans）启动子（Ptrpc）和GFP的片段，并连入pBHT载体中，成功构建了携带GFP报告基因的适合真菌转化的表达载体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FOC 1号（FOC1）生理小种。转化子在含潮霉素B平板上经多次单孢纯化后，在无选择压下仍能发出稳定且强烈的绿色荧光，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FOC1生理小种的基因组中，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： | 植物病理学；载体构建；根癌农杆菌介导的转化；香蕉枯萎病菌；绿色荧光蛋白� |
| Title： | Transformation of green fluorescent protein gene in Fusarium oxysporum f. sp. cubense race 1 mediated by Agrobacterium tumefaciens |
| Author： | KONG Lingguang, HAN Kuande, JU Yanhu, DING Xinhua, CHU Zhaohui |
| Organization： | Shandong Province Key Laboratory of Agricultural Microbiology, College of Plant Protection, Shandong Agricultural University |
| Abstract： | In this study, the Agrobacterium tumefaciens-mediated transformation system of Fusarium oxysporum f.sp. cubense (FOC) race 1 isolate was established successfully. Firstly, the DNA fragment of green fluorescent protein (GFP) was amplified by polymerase chain reaction (PCR). After digested with Cla〖KG-*2〗Ⅰ and SacⅡ, it was ligated into helper vector pUCATPH to replace the hygB 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 LB4044 after several turns of proving with PCR, size filtration and sequencing. Furthermore, it was transformed into the race 1 isolate of F. oxysporum f. sp. cubense (FOC1) with Agrobacterium-mediated method. Transformants were regenerated and selected on hygromycin B containing medium. Strong green fluorescence could be observed in single-spore isolates after several successive generations on the medium without hygromycin B. The presence of the green fluorescent protein DNA in the fungal cells was also confirmed by PCR using a GFP-specific primer pair. The results showed that the ectogenous DNA had been stably integrated into the strain FOC1, without any observed difference on growth ability as wild isolate. They may lay a good foundation for studying the infection process and pathogenic mechanism of this pathogen.� |
| Key words： | phytopathology; vector construction; Agrobacterium tumefaciens-mediated transformation; Fusarium oxysporum f. sp. cubense; green fluorescent protein |
| 发表期数： | 2012年10月第19期 |
| 引用格式： | 孔令广，韩宽德，巨延虎，等. 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[J]. 中国科技论文在线精品论文，2012，5（19）：1833-1840. |
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