[Construction of T7 phage display library from the anther of Honglian hybrid line of rice].
Phage display is a powerful method to study protein-protein interactions. In order to study the molecular mechanism of cytoplasmic male sterility and fertility restoration in Honglian rice, the mRNA was isolated with PolyA Tract mRNA Isolation Kit from the anther of F1 hybrid rice and the double strand (ds) cDNA was synthesized by reverse transcription. Then the directional EcoRI /Hind III linkers were ligated into the ends of ds cDNA and the ds cDNA was further digested with EcoR I and Hind, which resulted in ds cDNA with EcoR I and Hind III ends. The digested ds cDNA fragments longer than 300 bp in length were fractionated with Mini Column, then ligated into the T7 Select 10-3b vertor with EcoR I and Hind III ends. After packaging in vitro, the T7 Select 10-3b vertor was transformed into BL T5403 to construct the T7 phage display library. Analysis showed that the library contained 1.03 x 106 clones per microliter, and approximately 100% of the clones in library was recombinant. The titer of the amplied library was 2.14 x 1012 pfu/mL, and the insert length of the recombinants over 300 bp was about 97%.
Key Laboratory of MOE for Plant Development Biology, College of Life Science, Wuhan University, Wuhan 430072, China. phenixfeng@yahoo.com.cn
Chao-Feng
CF
Hu
Xiao-Jue
XJ
Peng
Yang-Yong
YY
Zhou
Yan-Ping
YP
Tan
Shao-Qing
SQ
Li
Ying-Guo
YG
Zhu
D017123:Bacteriophage T7 / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D035264:Flowers / Q000235:genetics*; D006824:Hybridization, Genetic / Q000235:genetics*; D012275:Oryza / Q000235:genetics*; D019151:Peptide Library; D012333:RNA, Messenger / Q000235:genetics
D018076:DNA, Complementary; D018744:DNA, Plant; D019151:Peptide Library; D012333:RNA, Messenger