Construction of yeast two-hybrid cDNA libraries for wheat near-isogenic line TcLr19 under the stress of Puccinia recondita and its preliminary appreciation

Abstract

Two cDNA libraries for wheat near-isogenic line TcLr19 under Puccinia recondita stress were constructed by using SMART technique and homologous reorganization method. Wheat near-isogenic line TcLr19 was infected with leaf rust race 366, and total RNA was extracted from the leaves after infection for 4, 8, and 12 h. The total RNAs were reverse transcribed to cDNA by using oligo(dT) primer and random primer, respectively. According to the evaluation on quality, the transformation efficiency was about 1.32 × 10⁶ and 1.0 × 10⁶ transformants/3 μg pGADT7-Rec, respectively, and the library titers were up to 2.62 × 10⁸ and 3.51 × 10⁸ pfu/mL, with 93% and 100% recombinant rate, which indicated the high quality of the two libraries for next screening.

Keywords

wheat leaf rust / SMART technique / homologous reorganization / cDNA library

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Acknowledgements

This research was supported by the National Natural Science Foundation of China (Grant Nos. 30671244 and 31171472), the Key Basic Research Project of Applied Basic Research Program of Hebei Province, China (No. 08965505D), and the Natural Science Foundation of Hebei Province, China (Nos. C2007000515 and C2010000787).

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