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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front Agric Chin    0, Vol. Issue () : 450-455    https://doi.org/10.1007/s11703-011-1123-1
RESEARCH ARTICLE
Construction of yeast two-hybrid cDNA libraries for wheat near-isogenic line TcLr19 under the stress of Puccinia recondita and its preliminary appreciation
Lifeng ZHANG, Hui ZHOU, Fengju WEI, Ziyi CHENG, Aihua YAN, Dongmei WANG()
College of Life Science, Agricultural University of Hebei, Baoding 071001, China
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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     
Corresponding Author(s): WANG Dongmei,Email:dongmeiwang63@hotmail.com   
Issue Date: 05 December 2011
 Cite this article:   
Lifeng ZHANG,Hui ZHOU,Fengju WEI, et al. Construction of yeast two-hybrid cDNA libraries for wheat near-isogenic line TcLr19 under the stress of Puccinia recondita and its preliminary appreciation[J]. Front Agric Chin, 0, (): 450-455.
 URL:  
https://academic.hep.com.cn/fag/EN/10.1007/s11703-011-1123-1
https://academic.hep.com.cn/fag/EN/Y0/V/I/450
Fig.1  Integrity detection of total RNA of wheat leaves. 1-3 are total RNA extracted from wheat leaf inoculated with leaf rust race 366 in 4, 8, and 12 h, respectively.
Fig.2  Range of ds cDNA. A is ds cDNA PCR product using oligo(dT) primer to reverse transcription into the first chain, and B is ds cDNA PCR product using random primer to reverse transcription into the first chain.
Fig.3  Range of inserts randomly selected from library. A: PCR detection of insert fragments in the library using oligo(dT) primer. B: PCR detection of insert fragments in the library using random primer.
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