根癌农杆菌介导的新暗色柱节孢菌遗传转化

Agrobacterium tumefaciens-mediated Transformation of Neoscytalidium dimidiatumcausing Stem Canker on Pitaya

火龙果是一种新兴的热带水果,随着种植面积的不断增大,病害问题日趋严重,其中危害最大的是火龙果溃疡病,导致火龙果溃疡病的病原菌为新暗色柱节孢菌(Neoscytalidium dimidiatum)。为了研究新暗色柱节孢菌的遗传变异与基因功能,必须建立有效的遗传转化体系,但目前国内外未见相关报道。本研究通过构建含有gpdA启动子、绿色荧光蛋白(green fluorescent protein)和潮霉素(Hygromycin)抗性基因为筛选标记的双元表达载体,对新暗色柱节孢菌的孢子进行根癌农杆菌介导的遗传转化,成功获得了阳性转化子。通过荧光显微镜观察表明,阳性转化子菌丝能够产生绿色荧光,野生型菌丝不能产生绿色荧光;PCR检测证明了转化子基因组中整合了潮霉素抗性基因。因此,本研究成功实现了根癌农杆菌介导下绿色荧光蛋白基因在新暗色柱节孢菌中的稳定遗传表达,为研究新暗色柱节孢菌对火龙果溃疡病的致病机制奠定了技术基础。

In recent years, pitaya(Hylocereus spp.) has gradually become a newly-emerging tropical fruit. With the continuous increase of planting area, diseases infecting pitaya have become more serious. Among them stem canker is the most important disease, which is caused by Neoscytalidium dimidiatum. To explore the genetic variation and gene function of N. dimidiatum, an effective genetic transformation system must be established. However, there have been no reports documented at home or abroad in this aspect. A binary expression vector containing gpdA promoter, green fluorescent protein(mGFP) and hygromycin resistance gene as screening markers was constructed to establish a genetic transformation system for N. dimidiatum through Agrobacterium tumefacien-mediation, and positive transformants from the spores of N.dimidiatumwere successfully generated. Fluorescence microscope observations showed that the positive transformant hyphae could produce green fluorescence, while the wild-type hyphae could not produce green fluorescence. The PCR test confirmed the integration of hygromycin resistance gene in the transformant genome. Therefore, this Agrobacterium tumefaciens mediated transformation system produced stable genetic expression of m GFP gene in N. dimidiatum, which lays a technical foundation for the further study of the pathogenicity mechanism of dragon fruit canker disease.