用λZAPExpress作载体构建金针菇子实体表达型cDNA文库

CONSTRUCTION OF CDNA EXPRESSION LIBRARY OF FLAMMULINA VELUTIPES

采用QuickPrepmicromRNAPurification试剂盒从新鲜金针菇子实体中快速提取、纯化mRNA,TimeSavercDNASynthesis试剂盒逆转录成cDNA分子后,通过在cDNA分子两端加上EcoRⅠ/NotⅠ接头,在T4多核苷酸激酶的作用下磷酸化,与表达载体λZAPExpressPredigestedVector连接,然后用包装蛋白在体外包装连接产物,感染E.coliXL1-BlueMRF捁菇ǔ杀泶颿DNA文库。经检测:文库滴度为4.0×106pfu/ml,文库扩增后,滴度达2.0×1010pfu/ml,重组率为90%,cDNA分子长度在0.3-3.5Kb范围。应用原位免疫杂交,初步筛选出火菇素相关基因的阳性克隆。

mRNA of Flammulina velutipes was extracted with Quick Prep mRNA Purification Kit, after synthesis of double-strand cDNA molecules using Time Saver cDNA Synthesis Kit, EcoRⅠ/NotⅠadaptors were added to the end of ds cDNA, phosphorylated by T4 polynucleoted kinase. Then the cDNA was ligated with λZAP Express Predigested Vector and packaged to transfect E.Coli XL1-Blue MRF? The results revealed that the tilter of the cDNA library was 4.0×106 pfu/ml, the amplified cDNA library is 2×1010 pfu/ml, the recombinant rate reached to 90%, and the length of inserts were between 0.3-3.5 kb. A candidate cDNA related with flammulin gene was screened by western blotting in situ.