陈明杰; 赵绍惠; 张树庭
上海市农业科学院食用菌研究所; 香港中文大学生物系; 香港中文大学生物系 上海 201106 ; 香港 ; 香港
【中文摘要】 应用基因重细技术,我们建立丁一个草菇总DNA的部分基因文库。通过限制性内切酶Sau 3AI部分酶切纯化的草菇总DNA,得到小于4kb的DNA片段。这些DNA片段插入到经Bam HI酶切及碱性磷酶酯酶处理的pUC18质粒,转化感受态细胞DH5α。在此研究中。我们比较了两种转化方法(氯化钙转化法和电转化法)的转化效率,电转化法的转化效率高于氯化钙转化法数千倍,达到2×10(2)CFU/μg DNA。在此实验中DNA重组率是64%,这个草菇总DNA的部分基因文库由8000个克隆组成,平均每个克隆含有1.5kb的草菇DNA。此基因文库含有15%的草菇总DNA。可作为分子标记用于DNA指纹图谱分析及作为遗传学标记用于RFLPs的遗传图谱分析。
【英文摘要】 A partial genomie. library was constructed for straw mushroom (Volvariella volvacea) using the shotgun approach. Purified genomic DNA was partially digested into small fragments of less than 4 kb in size using restriction enzyine, Sau 3AI. Extracted plasmid pUC18 was digested with an isoehizmerous enzyme, Bam HI and treated with alkaline phosphatase to prevent self-ligation. Then the two DNAs were iigated. The transformation effieieney into the host Escherichia coli with the ligation mixture was compared between calcium chloride treatment and electroporation. The latter yielded several thousand-fold more transfor.mants reaching 2×10 CFU/μg DNA. The reeombinant frequency was 64%. Up to now, 8000 clones, each .carrying an average size of 1.5 kb DNA insert were obtained. This is estimated to represent 15% of the total genome of V. voluacea. These clones in the constructed genoinic library can serve as molecular markers for DNA fingerprinting or genetic markers as restriction fragment leng...更多th polymorohisms for elaborating the inheritance pattern. 还原
【中文关键词】 草菇; 基因文库; 转化
【英文关键词】 Straw mushroom(Volvariella volvacea); Genomic library; Transformation
【文献出处】 食用菌学报,Acta Edulis Fungi,编辑部邮箱,1994年02期 【DOI】CNKI:SUN:SYJB.0.1994-02-004