关键词: 文库构建, 噬菌体展示, 花药, 水稻

Abstract:

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/HindⅢlinkers were ligated into the ends of ds cDNA and the ds cDNA was further digested with EcoRⅠand HindⅢ, which resulted in ds cDNA with EcoRⅠand HindⅢends. 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 EcoRⅠand HindⅢends. After packaging in vitro, the T7 Select 10-3b vertor was tansformed into BL T5403 to construct the T7 phage display library. Analysis showed that the library contained 1.03×106 clones per microliter, and approximately 100% of the clones in library was recombinant. The titer of the amplied library was 2.14×1012 pfu/mL, and the insert length of the recombinants over 300 bp was about 97%.