Chen Liang, Sun Gengwu, Wu Shujing, Liu Huixiang, Wang Hongkai
State Key Laboratory for Rice Biology, Biotechnology Institute, Zhejiang University, Hangzhou, 310058, People's Republic of China.
World J Microbiol Biotechnol. 2015 Jan;31(1):227-35. doi: 10.1007/s11274-014-1780-3. Epub 2014 Nov 26.
Valsa mali var. mali, the causal agent of valsa canker of apple, causes great loss of apple production in apple producing regions. The pathogenic mechanism of the pathogen has not been studied extensively, thus a suitable gene marker for pathogenic invasion analysis and a random insertion of T-DNA for mutants are desirable. In this paper, we reported the construction of a binary vector pKO1-HPH containing a positive selective gene hygromycin phosphotransferase (hph), a reporter gene gfp conferring green fluorescent protein, and an efficient protocol for V. mali var. mali transformation mediated by Agrobacterium tumefaciens. A transformation efficiency up to about 75 transformants per 10(5) conidia was achieved when co-cultivation of V. mali var. mali and A. tumefaciens for 48 h in A. tumefaciens inductive medium agar plates. The insertions of hph gene and gfp gene into V. mali var. mali genome verified by polymerase chain reaction and southern blot analysis showed that 10 randomly-selected transformants exhibited a single, unique hybridization pattern. This is the first report of A. tumefaciens-mediated transformation of V. mali var mali carrying a 'reporter' gfp gene that stably and efficiently expressed in the transformed V. mali var. mali species.
苹果腐烂病菌苹果变种（Valsa mali var. mali）是苹果腐烂病的病原菌，在苹果产区导致苹果产量大幅损失。该病原菌的致病机制尚未得到广泛研究，因此需要一种适用于致病侵袭分析的基因标记以及用于突变体的T-DNA随机插入。在本文中，我们报道了一种二元载体pKO1-HPH的构建，该载体包含阳性选择基因潮霉素磷酸转移酶（hph）、赋予绿色荧光蛋白的报告基因gfp，以及一种由根癌农杆菌介导的苹果腐烂病菌苹果变种转化的有效方法。当苹果腐烂病菌苹果变种与根癌农杆菌在根癌农杆菌诱导培养基平板中共培养48小时时，转化效率达到每10⁵个分生孢子约75个转化体。通过聚合酶链反应和Southern杂交分析验证hph基因和gfp基因插入苹果腐烂病菌苹果变种基因组，结果表明随机选择的10个转化体呈现单一、独特的杂交模式。这是关于携带“报告”gfp基因的根癌农杆菌介导苹果腐烂病菌苹果变种转化的首次报道，该基因在转化的苹果腐烂病菌苹果变种中稳定高效表达。
