冬虫夏草cDNA文库的构建和鉴定

Cordyceps Sinensis cDNA Library Construction and Indentification

目的分别构建冬虫夏草子座和虫体部分的cDNA文库。方法裂解法提取总RNA,磁珠法分离mRNA,反转录合成双链cDNA,末端补平,连接EcoRI并磷酸化,胶回收目的片断,连接载体pBluescriptⅡSK(+)XR并电转化感受态细胞DH10B,测定文库的克隆数、重组率,PCR测定插入片断大小。结果所建文库库容量分别为:子座部分1.0×106克隆、虫体部分1.28×106克隆;重组率分别为:子座部分87.5%、虫体部分68.75%,片段的大小范围均在0.5～2kb之间。结论分别构建了野生冬虫夏草子座和虫体部分的cDNA文库。

Objective To construct a cDNA library from Hepialus armoricanus parasitized by Cordyceps sinensi. Methods Total RNA was extracted from Hepialus armoricanus parasitized by Cordyceps sinensi and mRNA was purified, cDNA was synthesized by reverse transcription, and cDNA fragments larger than lkb were ligated with dephosphorylated arms of pBluescriptⅡ SK（＋） XR vectors. The recombinant vectors were eletroporated into DH10B.The size of cDNA inserts and the diversity of library were detected by PCR. Results The cDNA library of stroma contained 1.28×10^6 recombine clone,that of mycelium was 1.0×10^6 recombine clone. The rate of recombination of stroma was 87.5%, that of mycelium was 68.75%. The insert size ranged from 0.5 to 2 kb. Conclusion The cDNA library Hepialus armoricanus parasitized by Cordyceps sinensi has been constructed successfully.