利用磁珠构建稻瘟菌cDNA文库

Construction of cDNA library of Magnaporthe grisea with magneticbead

摘要:

本文以稻瘟菌菌丝体为材料提取RNA，【目的】改进张学敏等人的方法，通过磁珠构建稻瘟菌cDNA文库，并用于下一步研究稻瘟菌与水稻之间的互作关系。【方法】通过共价键连接的寡聚oligo（dT）磁珠纯化mRNA，并以磁珠上的oligo（dT）为引物引导第一链cDNA的合成，再利用末端转移酶加尾法合成第二链cDNA。构建过程中避免使用限制酶和连接接头。【结果】用此方法构建的文库容量为8.9×107cfu，滴度为8.9×106 cfu/mL，随机挑取的25个克隆插入片断平均大小达到1380bp。【结论】实验结果表明用改进的方法可构建高质量的cDNA文库，并且方便快捷，所用材料少，构建时间短，利于大规模的功能基因分析。

Abstract:

[Objective] We constructed cDNA library of Magnaporthe grisea. The good quality cDNA library could facilitate finding proteinaceous elicitors of M. grisea, and elucidating the mechanisms of the M. grisea--rice interaction. [Methods] The Oligo(dT) combined with the magnetic bead was used to extract mRNA from total RNA of Magnaporthe grisea and as primers to synthesize the first-strand cDNA. Terminal transferase introduced PolyA into 3’terminal of the first cDNA strand, then the PolyA was used for amplifying the second-strand cDNA. Restriction enzyme and adapter were avoided in this research, which could solve technical limitation of the traditional method. Because all reactions were done in one centrifuge tube, this process could reduce the risk of cDNA loss and cross-contamination. The primers designed in this research could clone the amplified cDNAs into expression vector in a desirable orientation. [Results]The cDNA library constructed had a high titer of 8.9×106 cfu/mL, and contained a total clones of 8.9×107 cfu, with an average inserts size of about 1380 bp. [Conclusion]Constructing cDNA library with magnetic bead was a highly efficient method useing only small amount of experimental materials within a short period.