稻曲病菌UV-2菌株细菌人工染色体文库构建及分析

Construction of a bacterial artificial chromosome library of Villosiclava virens UV-2 genome

摘要:

【目的】稻曲病(Rice false smut)是由稻曲病菌[Villosiclava virens (Cooke) Tak.]引起的严重危害水稻的真菌病害。构建稻曲病菌UV-2的大片段DNA细菌人工染色体(Bacterial artificial chromosome, BAC)文库, 为致病相关基因的鉴定及在图位克隆、比较基因组学等方面的研究奠定基础。【方法】以幼嫩菌丝为材料制备大分子基因组DNA包埋块, 用Hind III部分酶解后经脉冲凝胶电泳筛选, 回收大片段DNA并与pIndigoBAC536-S 载体连接, 连接产物转化大肠杆菌菌株DH10B T1 Phage-Resistant 细胞后进行蓝白斑筛选, 白色菌落捡入384孔板置于?80 °C低温保存。【结果】成功构建UV-2菌株的高质量、高覆盖度的BAC文库, 该文库共含10 368个克隆, 平均插入片段为124.4 kb, 空载率小于1%, 约覆盖该菌基因组的36.8倍。【结论】克服了真菌大分子基因组DNA制备难控制的技术难题, 建立了首个稻曲病菌的BAC文库。该文库已作为一种公共基因组资源向研究者开放(http://GResource.hzau.edu.cn)。

Abstract:

[Objective] Rice false smut caused by Villosiclava virens (Cooke) Tak. is a serious fungal disease of rice. This study aimed to construct a high molecular weight bacterial artificial chromosome (BAC) library of UV-2 genome. The BAC library will help identify patho-genicity-associated genes and provide genome resource for map-based cloning and compara-tive genomic analysis. [Methods] High molecular weight genomic DNA was isolated from young mycelia of UV-2 strain, digested with restriction enzyme Hind III and size selected by PFGE. Large genomic DNA fragments were recovered and ligated to the pIndigoBAC536-S vector. The ligation product was used to transform the Escherichia coli strain DH10B T1 Phage-Resistant cells and the transformants were selected on a blue-white selection medium. White colonies were individually picked into 384-well microtiter plates and stored at ?80 °C. [Results] We constructed a high quality deep coverage BAC library of UV-2 strain. The BAC library consisted of 10 368 clones with an average insert size of 124.4 kb and an empty clone rate of lower than 1%, covering 36.8-fold of the UV-2 genome. [Conclusion] We established a method for preparation of fungal high molecular weight genomic DNA, and constructed the first BAC library of V. virens genome successfully. The BAC library has been opened to re-searchers as a public genomic resource (http://GResource.hzau.edu.cn).