Nucleic Acid Chain Induced Bimolecular Fluorescence Complementation

Bimolecular fluorescence complementation(BiFC)is a technique typically utilized to validate interactions between proteins,in which proteins that are postulated to interact are bond to two fluorescent parts with complementary structure.The interaction between the two proteins causes fluorescent fragments reach proximity,permitting them to form back to reporter protein and regain its inherent 3D structure,therefore,generating the florescent signal.This thesis utilized the technology to study interactions of proteins capable of specifically recognize to target nucleic acid chain.PUF protein refers to a family of highly conserved proteins that can specifically bind to RNA,which can be found in various eucaryon.All PUF protein possess sequences that particularly bind to 35 UTR of target RNA,known as RNA binding domain or PUF protein domain,which regulate the expression of target RNA.Such already-known examples are cysteine and glutamine bind to adenine;asparagine and glutamine bind to uracil;serine and glutamate bind to guanine.serine and arginine bind to cytosine.Crystal structure of complex of human PUF protein PUM1 with its target RNA demonstrated that given PUF helix correspond to particular base,such concise recognition model suggests that recognition sequence of PUM1 could be applied site-directed mutagenesis.Research results has illustrated that those mutant PUF protein can recognize cytosine and original protein bind tightly to target RNA either,which means PUF protein are capable of playing a part in other biological research.Firstly,we constructed CEYFP-PUF1 and PUF2-NYFP fusion expression vectors,and they were seperated prokaryotically expressed and purified.Upon incubation of fusion proteins of CEYFP-PUF1 and PUF2-NYFP with oligonucleotides which contains the PUF1 and PUF2 binding sequences with interval of several nucleotides between them,the successful BiFC in vitro,demonstrated that the particularly designed PUF proteins can accurately recognize their specific nucleic acid sequences.Secondly,GUS gene was selected as target of PUFgus in Arabidopsis.GUS staining showed that no GUS activity were detected in transgenic plant baring PUFgus,on the contrary,it is normal in contol plants.Further analysis with qPCR of GUS mRNA showed that the corresponding PUFgus down-regulated the expression of GUS to lower than 0.1%of the control.Interestingly,expression of other native Arabidopsis genes baring the recognition sequence of PUF gus were not affected based on qPCR analysis.whereas,coexpression of PUF-HBZ and HBZ in mammalian cells showed that PUF-HBZ did not affect the expression of HBZ gene based on qPCR analysis,which suggested that the different roles of PUF in regulating gene expression in different organisms.