[1]张林平,叶建仁*,李月阳,等.根癌农杆菌介导的异角状拟盘多毛孢菌转化系统研究[J].南京林业大学学报(自然科学版),2012,36(01):028-32.[doi:10.3969/j.jssn.1000-2006.2012.01.006]
 ZHANG Linping,YE Jianren*,LI Yueyang,et al.Agrobacterium tumefaciens mediated transformation system for Pestalotiopsis heterocornis[J].Journal of Nanjing Forestry University(Natural Science Edition),2012,36(01):028-32.[doi:10.3969/j.jssn.1000-2006.2012.01.006]
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根癌农杆菌介导的异角状拟盘多毛孢菌转化系统研究
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《南京林业大学学报(自然科学版)》[ISSN:1000-2006/CN:32-1161/S]

卷:
36
期数:
2012年01期
页码:
028-32
栏目:
研究论文
出版日期:
2012-01-30

文章信息/Info

Title:
Agrobacterium tumefaciens mediated transformation system for Pestalotiopsis heterocornis
作者:
张林平12叶建仁1*李月阳1黄麟1许剑涛1张扬2
1.南京林业大学,江苏省有害生物入侵预防与控制重点实验室,江苏南京210037;2.江西农业大学园林与艺术学院,江西南昌330045
Author(s):
ZHANG Linping12YE Jianren1*LI Yueyang1HUANG Lin1XU Jiantao1ZHANG Yang2
1.Jiangsu Key Laboratory for Prevention and Management of Invasive Specie, Nanjing Forestry University,Nanjing 210037,China;2.College of Landscape Architecture and Art, Jiangxi Agricultural University, Nanchang 330045,China
关键词:
异角状拟盘多毛孢菌根癌农杆菌遗传转化转化效率
Keywords:
Pestalotiopsis heterocornisAgrobacterium tumefacienstransformationtransfer efficiency
分类号:
S764
DOI:
10.3969/j.jssn.1000-2006.2012.01.006
文献标志码:
A
摘要:
以携带潮霉素B磷酸转移酶抗性基因(hph)的pBHt1作为转化载体,根癌农杆菌AGL-1作为转化介体,实施转化异角状拟盘多毛孢菌。研究发现,异角状拟盘多毛孢菌的最优转化体系为:异角状拟盘多毛孢菌分生孢子悬浮液浓度为1×106个/mL,根癌农杆菌OD600为0.3,共培养时间48 h,共培养温度为25 ℃,诱导培养基中乙酰丁香酮(AS)浓度为200 μmol/L,选择培养基添加250 μg/mL潮霉素B、250 μg/mL头孢噻肟钠。1×106个异角状拟盘多毛孢菌分生孢子可以产生200~300个转化子,随机挑选10个转化子进行PCR检测,均能扩增出预期条带,且在不含潮霉素B的PDA培养基平板上转化子连续培养5代后,hph基因仍能稳定遗传,这表明T-DNA已经插入到异角状拟盘多毛孢菌的基因组中。此次建立的异角状拟盘多毛孢菌的转化体系可为该病菌的功能基因研究和寄主与病原菌的互作研究提供有效的工具。
Abstract:
In this study, we developed an Agrobacterium tumefaciensmediated transformation system for Pestalotiopsis heterocornis by using A. tumefaciens strain AGL1 which carried plasmid pBHt1 harboring the hygromycin B phosphotransferase gene(hph). The results showed a total of 200-300 transformants were obtained per 1×106 spores when the spore suspension were cocultivated with A. tumefaciens cells,the optimal conditions were OD600=0.3, 25 ℃,and 48 h cultivation in the presence of induction medium containing acetosyringone at 200 μg/mL. Selection medium performed best with concentrations of 250 μg/mL of hygromycin B and 250 μg/mL of cefotaxime sodium in PDA. 〖JP2〗To detect the heredity stability, ten random transformants were cultivated on the PDA medium for five generations, colony PCR of each generation obtained expected amplification bands, which showed that the transformants exhibit high heredity stability. The transformation system will be useful for further studies of functional genes and hostpathogen interaction in P. heterocornis.

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备注/Memo

备注/Memo:
收稿日期:2011-10-28修回日期:2011-11-15基金项目:国家重点基础研究发展计划(2009CB119205);国家自然科学基金项目(31000303);江苏省自然科学基金项目(BK2010566);国家林业局林业公益性行业科研专项项目(200704026);江苏省高校优势学科建设工程资助项目第一作者:张林平,讲师,博士生。*通信作者:叶建仁,教授。Email: jrye@njfu.com.cn。
更新日期/Last Update: 2012-01-30