Screening Of Gene Transcriptional Regulators Of Brassica Napus L. Ribosomal Protein RPL32-2

Fission yeast (Schizosaccharomyces pombe) ribosomal protein L32 has two paralogs, RPL32-1 and RPL32-2, which have high protein sequence identy and one paralog can rescue the deficency of the other one. But their functions have divergence and may play different parts in different cellular circumstance. Our lab discovered When the amount of total protein RPL32 remains unchanged, competing restrain another homologous gene expression; When cells enter a state of silence RPL32-1 highly expression inhibition of cell division; While cells in logarithmic phase RPL32-2 high expression to promote cell division. But, why they have different expression and they have different functions? So We reserch on rpl32-1 and rpl32-2 gene transcriptional regulation.To study the funtion of RPL32-1 and RPL32-2 further, we screen the transcription factor of the ribosomal protein RPL32-2 promoter by yeast one-hybrid.Our lab has construct the bait strain which contain the promoter rpl32-2,and this resurch expect to screen the transcription factor from the high quality cDNA library we made.We got 109 positive clones which include 43 possible binding protein.6 binding protein which include five ribosomal proteins RPS6-1, RPL5-2,RPS10-1,RPL27-1, RPL5-1,and a translation elongation factor EF-1B have high frequency by analyzing.The frequency of RPS6-1, RPL5-2,RPS10-1,RPL27-1, RPL5-1,EF-1B are 11.93%,11.93%,8.26%,6.42%,5.50%,11.01%.We speculate that these proteins could be the potential transcription factor of rpl32-2.In view of the above conjecture, we construct the recombinant vector pGADT7+ rps6-1, pGADT7+rps10-1, pGADT7+rpl5-1, pGADT7+rpl5-2, pGADT7+rpl27-1, pGADT7+ef1b and transform the recombinant vector into yeast.We find that RPL5-1, RPL5-2, RPS6-1 could have interaction with rpl32-2. That means RPL5-1, RPL5-2, RPS6-1 can be transcription factor of rpl32-2. For the limitations of the one-hybrid system itself,so we have to make further verification.In this thsis,we make further verification of the interaction between the promoter of ribosomal protein RPL32-2 and ribosomal protein RPL5-1 in vivo by Chromatin Immunoprecipitation(ChIP).Before performing Chromatin Immunoprecipitation, we need to construct a yeast strain with RPL5-1-HA tag. According to the results of Chromatin Immunoprecipitation, we find that RPL5-1-HA can not be co-precipitated by the promoter of rpl32-2 fragment. We can not amplify the promoter rpl32-2 fragment by ChIP-PCR,which means the ribosomal protein RPL5-1 have no interaction on the promoter of RPL32-2. The result of the one-hybridization experiment was a false positive.But lots of possible binding protein which got from one-hybrid are still not confirmed yet.so we should reserch on them furthrely.