灭蚊真菌-贵阳腐霉Pythium sp.GY1938菌株cDNA文库的构建及鉴定

发布时间：2018-05-14 08:45

本文选题：cDNA + 文库　；参考：《中国病原生物学杂志》2017年03期

【摘要】：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。
[Abstract]:Objective to construct the cDNA library of Pythiumsp.GY1938 strain of Pythium Guiyang, which is a fungicidal fungus, and to establish the foundation for further screening and cloning the specific expression gene of GY1938 strain. Methods using GY1938 mycelium as material, total RNA was prepared by liquid nitrogen grinding method and purified mRNAs were isolated and purified. DS cDNA synthesized by LD-PCR method was ligated to pSMART2IFD vector by SMARTer technique, and transformed into Escherichia coli HST08 by electroporation. CDNA library of GY1938 strain was constructed. The quality of library such as titer, recombination rate and size of recombinant fragment were detected. Results the titer of cDNA library of GY1938 strain was 1.36 脳 10 ~ 6 cfur / ml, the recombination rate was 98 and the length of inserted fragment was about 300 ~ 2 500 BP. Conclusion the cDNA library of GY1938 strain of Pythium Guiyang was successfully constructed, which could be used for screening, cloning and functional study of new genes.
【作者单位】：成都医学院基础医学院;成都医学院生物医学系;成都医学院检验医学院;成都医学院科研实验中心;
【基金】：四川省科技厅应用基础研究项目(No.2015JY0205) 国家级大学生创新项目(No.201313705003,201313705007,201413705005) 成都医学院大学生创新实验计划项目(No.CXJS201309) 四川省教育厅项目(No.13ZB0220) 四川省卫生厅项目(No.130302,130298)
【分类号】：Q78;R184.31

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