陳明杰; 趙紹惠; 張樹庭
上海市農(nóng)業(yè)科學(xué)院食用菌研究所; 香港中文大學(xué)生物系; 香港中文大學(xué)生物系 上海 201106 ; 香港 ; 香港
【中文摘要】 應(yīng)用基因重細(xì)技術(shù),我們建立丁一個草菇總DNA的部分基因文庫。通過限制性內(nèi)切酶Sau 3AI部分酶切純化的草菇總DNA,得到小于4kb的DNA片段。這些DNA片段插入到經(jīng)Bam HI酶切及堿性磷酶酯酶處理的pUC18質(zhì)粒,轉(zhuǎn)化感受態(tài)細(xì)胞DH5α。在此研究中。我們比較了兩種轉(zhuǎn)化方法(氯化鈣轉(zhuǎn)化法和電轉(zhuǎn)化法)的轉(zhuǎn)化效率,電轉(zhuǎn)化法的轉(zhuǎn)化效率高于氯化鈣轉(zhuǎn)化法數(shù)千倍,達(dá)到2×10(2)CFU/μg DNA。在此實驗中DNA重組率是64%,這個草菇總DNA的部分基因文庫由8000個克隆組成,平均每個克隆含有1.5kb的草菇DNA。此基因文庫含有15%的草菇總DNA。可作為分子標(biāo)記用于DNA指紋圖譜分析及作為遺傳學(xué)標(biāo)記用于RFLPs的遺傳圖譜分析。
【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. 還原
【中文關(guān)鍵詞】 草菇; 基因文庫; 轉(zhuǎn)化
【英文關(guān)鍵詞】 Straw mushroom(Volvariella volvacea); Genomic library; Transformation
【文獻(xiàn)出處】 食用菌學(xué)報,Acta Edulis Fungi,編輯部郵箱,1994年02期 【DOI】CNKI:SUN:SYJB.0.1994-02-004