Construction of cDNA library of Magnaporthe grisea with magneticbead

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.