Construction Of Yeast Two Hybrid CDNA Library Of Wild-type Drosophila W¹¹¹⁸ And BD-vector

Drosophila have many characteristics such as small in size,very short life cycle,a large number of offsprings,and the complete genome of Drosophila has been sequenced,the gene sequences are high homology with other organisms?especially mammalian?.Meanwhile,some complex biological phenomena of Drosophila also have been researched and understood deeply.Thus,Drosophila becomes one of the most important model organisms,it plays a very crucial role in research area of genetics,neuroscience and human diseases.The most widely used species is Drosophila melanogaster,the wild-type Drosophila w¹¹¹⁸ is one of them.It is convenient to have a Drosophila w¹¹¹⁸ cDNA library with the yeast two-hybrid system for studying the interaction between protein-RNA,protein-small molecule and protein-protein.The prerequisite for these studies is the construction of a high quality cDNA library.In this study,a full-length cDNA library in yeast Y187 have been constructed with the SMART?Switching Mechanism At 5'end of RNA Transcript?technology.The first chain of cDNA was synthesized by reverse transcriptase using the total RNA of wild-type Drosophila w¹¹¹⁸ as template.The double strands cDNA was amplified by long distance-PCR in the existence of DNA polymerase.The purified ds cDNA and the linearized vector pGADT7-Rec were co-transformed into competent yeast Y187.Through homologous recombination the recombinant plasmid was formed in yeast cells.All clones were selected and collected from SD/-Leu plates,the conversion efficiency and the library titer were calculated by dilution method.The recombination rate and the range of inserted fragments were calculated by colony PCR.Meanwhile,according to the CDS sequence of CG2056 on Flybase,two primers were designed.The gene was amplified by RT-PCR,and the BD-vector pGBKT7-CG2056 was constructed and transformed into competent yeast AH 109,after that the self-activated reaction was detected.The total RNA of wild-type Drosophila w¹¹¹⁸ was examined by agarose gel electrophoresis:28S and 18S was very clear.So the concentration and purity of the total RNA was enough to construct a cDNA library.The fragment of ds cDNA was 500-2000 bp,they were able to transform into the competent yeast Y187.As a result,the conversion efficiency of the cDNA library was 5.5×105/3ug pGADT7-Rec,and the titer was 1.42×107cfu/ml.The recombination rate was 100%,and the fragments of inserted cDNA were 300-2000bp.All the results indicated that the quality of the cDNA library was very good,and it provided the important materials and foundation for selecting the receptor protein through the yeast two-hybrid.Meanwhile,the BD-vector pGBKT7-CG2056 which was transformed into competent yeast AH 109 had no self-activated reaction.