| 农杆菌介导油菜黑胫病菌遗传转化 |
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| 引用本文： | 宋培玲,皇甫海燕,史志丹,燕孟娇,郝丽芬,皇甫九茹,贾晓清,郭晨,杨永青,李子钦.农杆菌介导油菜黑胫病菌遗传转化[J].中国油料作物学报,2021,43(2):293-299. |
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| 作者姓名： | 宋培玲 皇甫海燕 史志丹 燕孟娇 郝丽芬 皇甫九茹 贾晓清 郭晨 杨永青 李子钦 |
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| 作者单位： | 1. 内蒙古自治区农牧业科学院植物保护研究所，内蒙古呼和浩特，010031；
2. 内蒙古大学生命科学学院，内蒙古呼和浩特，010021 |
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| 基金项目： | 内蒙古自治区农牧业创新基金项目(2017CXJJN15);国家重点研发计划(2018YFD0200907);内蒙古自治区农牧业科学院院青年创新基金(2017QNJJNO2)。 |
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| 摘 要： | 为优化建立农杆菌介导的油菜黑胫病菌遗传转化技术体系，以携带潮霉素磷酸转移酶基因的pCH-sGFP
为载体，农杆菌LBA4404为介体，对油菜黑胫病菌的分生孢子进行转化。油菜黑胫病菌的最佳转化体系为:分生孢
子浓度106个/mL，农杆菌OD600值0.6，乙酰丁香酮浓度200 μmol/mL，pH 5.8，25℃，共培养4 d，获得120～161个转化
子。随机选取26个转化子连续培养5代能够稳定遗传，绿色荧光蛋白基因gfp 表达的菌丝和孢子可见绿色荧光。
PCR鉴定发现T-DNA 已整合进油菜黑胫病菌基因组中，Southern blot鉴定发现T-DNA以单拷贝的形式插入黑胫病
菌中,转化子均能够稳定遗传。建立油菜黑胫病菌遗传转化体系为该病菌的致病机制研究和功能基因分析奠定了
基础。
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| 关 键 词： | 油菜黑胫病菌 遗传转化 转化子 |
Agrobacterium tumefaciens-mediated transformation of Leptosphaeria biglobosa |
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| SONG Pei-ling,HUANGFU Hai-yan,SHI Zhi-dan,YAN Meng-jiao,HAO Li-fen,HUANGFU Jiu-ru,JIA Xiao-qing,GUO Chen,YANG Yong-qing,LI Zi-qin.Agrobacterium tumefaciens-mediated transformation of Leptosphaeria biglobosa[J].Chinese Journal of Oil Crop Sciences,2021,43(2):293-299. |
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| Authors: | SONG Pei-ling HUANGFU Hai-yan SHI Zhi-dan YAN Meng-jiao HAO Li-fen HUANGFU Jiu-ru JIA Xiao-qing GUO Chen YANG Yong-qing LI Zi-qin |
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| Institution: | 1. Plant Protection Institute, Inner Mongolia Academy of Agricultural & Animal Husbandry Sciences, Hohhot 010031, China;
2. College of Life Science, Inner Mongolia University, Hohhot 010021, China |
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| Abstract: | To optimize and establish Agrobacterium tumefaciens-mediated transformation system of Leptosphaeria biglobosa, A. tumefaciens strain LBA4404 carrying plasmid pCH-sGFP was used as transforming carrier, and conidia of L. biglobosa as transformation recipients. Hygromycin B phosphotransferase gene(hyg) and green fluorescent protain gene(gfp) were transformed into L. biglobosa. The optimal transformation system of L. biglobosa was established. Under the optimal condition of 106 spores/mL, A. tumefaciens OD600=0.6, 200 μmol/mL acetosyringone,pH 5.8,co-cultivation at 25℃ for 4 d, the transformation efficiency could get 120-161 transformants. When selecting 26 transformants randomly for successive cultures, all of them were still resisitant to hygromycin after 5 generations grown on PDA without hygromycin. Green fluorescence was visible in hypha and conidia of the transformants.PCR amplification and Southern blot showed T-DNA insertion in L. biglobosa. |
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| Keywords: | Leptosphaeria biglobosa genetictransformation transformant |
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