Transformation of Coniothyrium minitans, a parasite of Sclerotinia sclerotiorum, with Agrobacterium tumefaciens
Transformation of Coniothyrium minitans, a parasite of Sclerotinia sclerotiorum, with Agrobacterium tumefaciens
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DOI：
10.1016/j.femsle.2004.12.033
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发表时间：
2005-02-15
影响因子：
2.1
通讯作者：
Hou, MS
中科院分区：
文献类型：
--
作者：
Li, MX;Gong, XY;Hou, MS
小锥虫是一种潜在的植物病原菌菌核病生物防治剂。本研究采用农杆菌介导的方法，对C - minitans菌株ZS-1进行了T-DNA插入转化，并优化了转化的孢子成熟度。经PCR确认后，对转化子进行Southern blot分析，结果显示超过82.7%的转化子有单个T-DNA插入，12.1%的转化子有两个拷贝T-DNA插入。利用TAIL-PCR可以从两侧扩增出T-DNA两侧的转化子基因组DNA片段。从T-DNA插入文库中筛选出4种突变体，包括产孢缺陷突变体、致病性缺陷突变体、色素变化突变体和抗生素缺陷突变体，并对部分突变体进行了描述；计算文库中各类型突变体的数量和频率，各类型突变体的频率分别为3.27 × 10（-3）、1.0 × 10（-4）、1.4 × 10（-4）、2.5 × 10（-4）。T-DNA插入转化文库的成功建立，将有助于我们在分子水平上揭示寄生物与寄主的相互作用，阐明寄主真菌的分化和发育，以及分析和克隆三方关联的生物防治微生物的功能基因。(C) 2005年欧洲微生物学会联合会。Elsevier B.V.版权所有。
Coniothyrium minitans is a potential biological control agent of the plant pathogenic fungus Sclerotinia sclerotiorum. In this research, T-DNA insertional transformation of strain ZS-1 of C minitans mediated by Agrobacterium tumefaciens was obtained, with optimization of spore maturity for transformation. After confirmation by PCR, transformants were subjected to Southern blot analysis, and results showed that more than 82.7% of transformants had single T-DNA insertions, and 12.1% of transformants had two copies T-DNA insertions. The genomic DNA segments of transformants flanking the T-DNA could be amplified from both borders with TAIL-PCR. Four types of mutants were screened and identified from the T-DNA insertional library, which comprised sporulation deficient mutants, pathogenicity deficient mutants, pigment change mutants and antibiotic deficient mutant, and some of the mutants were described; the number and frequency of each type of mutant from the library were calculated, and the frequency of each type is 3.27 x 10(-3), 1.0 x 10(-4), 1.4 x 10(-4), 2.5 x 10(-)4, respectively. The successful creation of the T-DNA insertional transformation library may help us to unravel the interaction between a parasite and its host at a molecular level, to clarify the differentiation and development of this fungus, and to analyze and clone functional genes from the biocontrol microorganism in tripartite associations. (C) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.