Construction And Application Of Yeast Two-hybrid System Of Maize Roots And Leaves

Maize is an important feed , industrial raw materials and food crops . With molecular biology and breeding technology means progress and update , more and more new technologies and new metheods were applied to maize breeding and production of basic research . among them , yeast two-hybrid system is a very important research methods . Yeast two-hybrid system is to study protein and protein, protein and RNA , protein and small molecules , such as the interaction of useful technology . Through the establishment of backbone inbred lines of maize roots , leaves , the development of the endosperm cDNA library , and build a suitable bait vector to screening one or a group of proteins which interaction which the bait vector is of great significance to study of maize roots , leaves and endosperm of the physiological functions and macromolecular material research function, and the same time, for the breeding of maize provide a theoretical basis .For this reason , the following text is devoted to cDNA library construction . The first chain of cDNA was synthesized by reverse transcript from maize roots and leaves mRNA as template by SMART metheods . The ds cDNA was amplified by long distance-PCR in existence of DNA polymerase . The purified ds cDNA and the linearized plasmid pGADT7-Rec were co-transformed into the competent yeast AH109 . Recombinants plasmids formed through homologous recombination in vivo . All the transformants were harvested from the SD/- Leu plates . After examntion , the library conversion of maize roots was 1.76×10~7 transfers beggar/3μg pGADT7-Rec , the capacity of the maize roots library was 1.17×10~6 pfu/mL, the reorganization rate of the maize roots library was 95% and the recombinant fragments were larger than 500bp . The conversion of maize leaves library was 1.54×10~7 transfers beggar/3μg pGADT7-Rec , the capacity of the maize leaves library was 1.03×10~6 pfu/mL , the reorganization rate of the maize leaves library was 95% and the recombinant fragments were larger than 500bp . All those showed that the yeast two-hybrid library of maize roots and leaves were successfully constructed .