Study On Efficient Assembly Of Designer PPR Protein And Its Specific Recognition Of Targeting RNA

Pentatricopeptide repeat(PPR)proteins are sequence specific RNA-binding proteins which involve in various kinds of RNA metabolisms.An overwhelming majority of them is a large number of proteins widely spread in higher plants,which are mainly located in chloroplasts and mitochondria.Canonically,PPR proteins contain 2-30 tandem repeat sequences,with each repeat haboring 35 amino acids.PPR proteins tract RNA base by means of one-repeat : one-nucleotide binding mode.This modular architecture endows PPR proteins the ability to serve as designable module for gene manipulation.Biochemical and structural studies have revealed that amino acids at position 5,35 of each repeat which recognized as PPR base code determine the RNA base recognition specificity.In theory,the combinations of such amino acid code are as many as 400.However,up to date,only few have been uncovered by experiments,while most code combinations and the the structural basis for nucleotide-specific recognition between PPR proteins and RNA base remains to be elucidated.And this is very detrimental to the study of PPR protein function and its further application.Thus,a variety of PPR proteins with different code are needed for the biochemical validation.However,synthesis of such genes which contain a large number of PPR tandem repeats domain is costly and time consuming.Here we exploited an efficient,handy and sparing method for the assembly of designer PPR(dPPR)sequence with variable codes in this study.Based on the characteristics within the Type IIs restriction endonuclease and the basic principles of Golden Gate cloning,we designed a smart method for dPPR repeats assembly which includes three steps.Taking the construct of 10 repeats as an example,the first step is to construct 3 repeats(3R)primary elements by ?restriction cut-ligation? one step method.And the second step is to build 9 repeats(9R)secondary element using the 3R fragments.The last step is to ligate 9R element into the received expression vector which contains 1 ?repeat?(1R)to obtain a complete sequence of dPPR gene with 10 repeats.We have successfully expressed the targeted protein using the assembly dPPR sequence.And the correspondence between several codes and RNA base was verified by native polyacrylamide gel electrophoresis and electrophoretic mobility assay and electrophoretic mobility shift assay.In summary,this study provides an efficient method for assembling designer PPR sequences which can identify specific RNA bases for the first time.We successfully performed protein expression and purification,and further validated the activity of the recombinant protein.The application of dPPR repeats assembly will not only accelerates the functional study of PPR proteins,but also provides a framework for the potential tools for gene manipulation.