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目的构建灭蚊真菌-贵阳腐霉Pythiumsp.GY1938菌株cDNA文库,为进一步筛选和克隆GY1938菌株特异表达基因奠定基础。方法以GY1938菌丝体为材料,液氮研磨法制备总RNA并分离纯化mRNA,应用SMARTer技术将LD-PCR法合成并分级纯化的ds cDNA连接至pSMART2IFD载体,电穿孔法转化至大肠埃希菌HST08,构建GY1938菌株cDNA文库,进行滴度、重组率和重组片段大小等文库质量检测。结果检测GY1938菌株cDNA文库滴度达1.36×106 cfu/ml,重组率为98%,插入片段长度约为300~2 500bp。结论成功构建了较为理想的贵阳腐霉GY1938菌株cDNA文库,该文库可用于新基因的筛选、克隆和功能研究。
Objective To construct a cDNA library of Pythium sp.GY1938, a mosquito-killing fungus, which laid the foundation for further screening and cloning of the gene specifically expressed in GY1938 strain. Methods GY1938 mycelium was used as the material and the total RNA was prepared by liquid nitrogen grinding method. The mRNA was isolated and purified. The ds cDNA synthesized and purified by LD-PCR method was linked to pSMART2IFD vector by SMARTer technology and transformed into Escherichia coli HST08, the GY1938 strain cDNA library was constructed and the titers, recombination rates and the size of the recombinant fragments were tested. Results The titer of cDNA library of GY1938 strain was 1.36 × 106 cfu / ml, the recombination rate was 98%, and the inserted fragment length was about 300-2 500 bp. Conclusion The ideal cDNA library of Pythium guiyangense GY1938 strain was constructed successfully. This library can be used for screening, cloning and functional studies of novel genes.