| Objective:The Piggy Bac(PB)transposon was derived from the TTAA-type transposon of the Trichoplusia ni.It can be use as an insertion type mutation tool and can be applied to various biological cell.Common mutation tools include μltraviolet mutagenesis,chemical mutagenesis,transposition,etc.PB transposon insertion mutation can use the common sequence on the transposon to quickly detect the mutation site by PCR technology.At present,PB transposon systems have been used to mutate in mammals,plants,insects,and S.pombe species.However,the PB transposon system in S.pombe has not been studied in related aspects of ribosomal synthesis.In this study,we constructed a screening system based on PB transposon in S.pombe.PB transposon was applied to factors related to ribosomal synthesis for the first time,and gene screening was achieved by insertion mutation.The system constructed in this study coμld quickly and efficiently screen target genes related to ribosomal synthesis in S.pombe and further study their functions in the ribosomal synthesis process.Methods:In this study,the PB transposon screening system was used to investigate factors related to ribosomal synthesis,such as Nog2,Tif6,Cid4,Pol5,and Rrp14.First,build a screening system.Constructing a rescue plasmid corresponding to the target gene and knocking out a synthetic fragment,and then sequentially transferring the rescue plasmid and the target gene knockout fragment into a wild type strain by adopting a homologous recombination technology to obtain the strain containing the rescue plasmid and the target gene knockout,and integrating PB transposase to complete the construction of a screening system.Transposase PBase is controlled by the thioamine-inhibited nmt1 promoter.When cells grown in thiamine-free medium induced PBase expression,PB [ura4 +] was excised from the arg6 donor site and the cells were converted to arginine pro-cμlture(Arg+).Excision and reduction of PB[ura4 +] can be performed simμltaneously,selected by cμlturing cells in a medium lacking arginine and uracil.Through the transposition phenomenon,we can explore the importance and function of genes related to ribosome factors,which also plays a role in promoting the research on the biosynthesis of ribosome.Resμlts:With the known pst1 gene,its knockout lethality can be inhibited by the knockout of pst2 gene,so that the pst1-knockout strain can survive.Through the PB transposon system,it is verified that the PB transposon system can work normally,and pst2 gene is a candidate inhibitor of pst1 gene.The nog2,tif6 and pol5 genes are lethal genes,and their knock-out will lead to death.The rrp14 and cid14 genes are pathogenic genes.After PB transposon screening and a lot of repeated experiments on ribosome-related factors Nog2,Pol5,etc.,it was found that Nog2,Tif6,Pol5,Rrp14 and other factors may not have candidate inhibitors,indicating that these factors play a very important role in ribosome synthesis and cannot be replaced.Conclusion:1.A mutagenesis and screening system for candidate target genes by using the Piggy Bac(PB)transposon was successfμlly constructed.2.This study found that Nog2,Tif6,Pol5,Rrp14 and other factors may not have candidate inhibitors,indicating that these factors play a very important role in ribosome synthesis and cannot be replaced. |
