摘要
【目的】利用农杆菌(Agrobacterium tumefaciens)介导的遗传转化技术,对玉米大斑病菌(Setosphaeria turcica)进行转化,构建ATMT突变体库,为从分子水平上揭示病菌的致病机理奠定基础。【方法】以带有重组双元载体的农杆菌对玉米大斑病菌进行转化,利用潮霉素B进行筛选,对抗性稳定的转化子进行PCR检测,构建玉米大斑病菌ATMT突变体库;从突变体库中随机选取一定数量的突变体,对其菌落形态、菌丝及分生孢子发育、致病性等进行分析。【结果】获得了1 265株玉米大斑病菌T-DNA插入突变体;从中随机选取36株突变菌株,对其进行抗性筛选和PCR检测,发现潮霉素磷酸转移酶基因已整合进入野生型菌株的基因组中,且能够稳定遗传;与野生型菌株相比,在供试的菌株中,大部分菌株菌落形态和生长速率没有发生明显改变。生长速率明显减慢的菌株占总数的13.8%,明显加快的菌株占16.7%;发现了2株分生孢子形态发生明显改变的菌株,占菌株总数的5.6%;产孢量明显增大的菌株占5.6%,产孢量减少的菌株占13.5%;分生孢子萌发率发生明显改变的菌株占16.6%;发现了1株致病性明显增强的菌株,占菌株总数的2.8%。【结论】构建了玉米大斑病菌ATMT突变体库,并对突变体库进行了初步分析,将为克隆玉米大斑病菌生长发育和致病性相关基因奠定基础。
【Objective】 The objective of this study is to use Agrobacterium tumefaciens-mediated transformation（ATMT） to mutate the genes of Setosphaeria turcica and construct ATMT mutant library,which will lay a foundation for illuminating the pathogenesis of S.turcica at molecular level.【Method】A.tumefacien with binary recombination vector was transformed into S.turcica,and the mutants were screened by hygromycin B and PCR technology.Based on the methods,a preliminary ATMT mutant library was constructed.Some mutants were selected randomly and analysed about the colony morphologies,mycelium and conidium development,and pathogenicity.【Result】A total of 1 265 T-DNA insertion mutants were obtained in this experiment.Hygromycin B resistance screening and PCR technology were used to confirm 36 mutants,which were selected randomly from the mutant library.The results showed that hygromycin phosphotransferase genes had been integrated into wild-type genome of S.turcica and the mutants were stable in their characteristics of genetics.Compared with wild-type strains,most of the mutants did not changed their colony morphologies and growth rates,only minorities of the mutants varied in their characteristics,in which 13.8% strains slowed down their growth rate obviously,16.7% strains accelerated their growth rate distinctly,about 5.6% strains altered their shapes of conidia,13.5% strains reduced the number of conidia,16.6% strains changed their germination rate evidently,and 1 strain reinforced its pathogenicity,accounting 2.8% in all selected strains.【Conclusion】 ATMT mutant library of S.turcica was constructed and some mutants were analysed primarily.The study will lay a foundation for cloning genes related to growth,development and pathogenicity of S.turcica in the future.
出处
《中国农业科学》
CAS
CSCD
北大核心
2012年第12期2384-2392,共9页
Scientia Agricultura Sinica
基金
国家自然科学基金项目(30471126
31171805)
河北省自然科学基金项目(C2009000622
C2010001854)
