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| 苜蓿轮枝菌的生物学特性分析及ATMT遗传转化体系的建立 | |
| 引用本文： | 贾瑞芳,王乐,于飞,张园园,林克剑.苜蓿轮枝菌的生物学特性分析及ATMT遗传转化体系的建立[J].植物病理学报,2022,52(5):727-737. |
| 作者姓名： | 贾瑞芳 王乐 于飞 张园园 林克剑 |
| 作者单位： | 中国农业科学院草原研究所,呼和浩特 010010; 敖汉旗植保植检站,敖汉 024300 |
| 基金项目： | 中央级公益性科研院所基本科研业务费专项(1610332020013);内蒙古自治区自然科学基金 ( 2021BS03025) |
| 摘 要： | 为了揭示苜蓿轮枝菌Verticillium alfalfae的生物学特性及其致病机理,在室内条件下测定不同温度、pH值、碳源和氮源对菌株Ms198的生长速率和产孢量的影响,同时利用农杆菌转化法(Agrobactirium tumfacience-mediated transformant,ATMT)将带有潮霉素(Hygromycin,Hyg)抗性标记和绿色荧光蛋白(green fluorescent protein,GFP)报告基因的双元载体转入苜蓿轮枝菌的分生孢子,获得147株阳性转化子,以野生型菌株为对照,对挑取的15株阳性转化子的菌落形态、生长速率、产孢量、孢子萌发率、粗毒素分泌量和致病力进行研究。结果表明,苜蓿轮枝菌具有较宽的温度和酸碱度适应范围,最适生长和产孢温度分别为25 ℃和20 ℃,最适生长pH值为6 ~ 9,最适产孢pH值为9。可以利用多种碳、氮源,最适生长的碳、氮源分别为可溶性淀粉和牛肉膏,最适产孢的碳、氮源分别为D-牛乳糖和胰蛋白胨。与野生型菌株相比,15株供试转化子中有66.67%的转化子在菌落形态方面与野生型菌株无明显差别,而其生长速率、产孢量和孢子萌发率均有不同程度的降低。在产毒能力和致病力方面,1株转化子的粗毒素分泌量显著高于野生型菌株,9株显著低于野生型菌株,其余5株与野生型菌株无显著差异;4株转化子的致病力显著低于野生型菌株,其余11株转化子的致病力与野生型菌株均无显著差异。研究结果表明,转化子的生物学特性以及产毒能力和致病力,随外源基因插入位点的随机性而有所变化。 |
| 关 键 词： | 苜蓿轮枝菌 生物学特性 农杆菌转化法 转化子鉴定 |
| 收稿时间： | 2021-07-07 |
Analysis of biological characteristics of Verticillium alfalfae and establishment of ATMT genetic transformation system |
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| JIA Ruifang,WANG Le,YU Fei,ZHANG Yuanyuan,LIN Kejian.Analysis of biological characteristics of Verticillium alfalfae and establishment of ATMT genetic transformation system[J].Acta Phytopathologica Sinica,2022,52(5):727-737. | |
| Authors: | JIA Ruifang WANG Le YU Fei ZHANG Yuanyuan LIN Kejian |
| Institution: | Institute of Grassland Research of Chinese Academy of Agricultural Sciences, Hohhot 010010, China; Aohan Plant Protection and Plant Inspection Station, Aohan 024300, China |
| Abstract: | To unravel the biological characteristics and pathogenic mechanism of alfalfa Verticillium alfalfae. The effects of different temperatures, pH values, carbon sources and nitrogen sources on the growth rate and sporulation of strain Ms198 under in vitro conditions were studied. A binary vector containing hygromycin resis-tant gene and green fluorescent protein gene (GFP) was transformed into conidia of V. alfalfae using Agrobacterium tumefaciens-mediated transformation system (ATMT) resulting in 147 positive transformants. Fifteen out of the 147 transformants were chosen for observation and comparison of colony morphology, growth rate, conidial production, spore germination rate, toxin production and pathogenicity. The results showed that V. alfalfae had a wide range of temperature and pH adaptation. The optimum growth and sporulation temperature were 25 ℃ and 20 ℃, respectively. The optimum pH range for growth was 6-9, and the optimum spore production pH was 9. We observed that a variety of carbon and nitrogen sources could be used but the most suitable carbon and nitrogen sources for growth were soluble starch and beef extract, respectively; and the most suitable for sporulation were D-nougat and tryptone. Compared with the wild-type strain, 66.67% of the 15 tested transformants had no significant difference in colony morphology, but their growth rate, conidia production and spore germination rate was reduced to various degrees. In terms of toxin production and pathogenicity, only one transformant had a crude toxin secretion that was significantly higher than the wild-type strain. Whereas, 9 transformants had a significantly lower secretion than the wild-type strain, and the remaining 5 transformants were not significantly different from that of the wild-type strain. The pathogenicity of the 4 transformants was significantly lower than the wild-type strain, and the remaining 11 transformants were not significantly different from that of the wild-type strain. The above research results revealed that the biological characteristics, toxin production and pathogenicity of the transformants could vary depending on the insertion site of the foreign gene. |
| Keywords: | Verticillium alfalfae biological characteristics Agrobacterium tumefaciens-mediated transformant transformants characterization |
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