The Molecular Mechanism Of PPR-SMR Protein PTAC2 Involved In Higher Plant Chloroplast Gene Expression

Chloroplast originate from endosymbiosis bacteria,which contains its own genome,and is a specific organelle in higher plant.Chloroplast genome is transcribed at least by the two types of plastid RNA polymerase: Plastid-encoded RNA polymerase(PEP)and Nucleous-encoded RNA polymerase(NEP).PEP is a major plastid RNA polymerase,which consists of four core subunits and numerous accessory proteins.The regulatory mechanism of PEP complexes is very complicated,and most of the components' functions remain unclear.In this dissertation,the function of the p TAC2 were investigated.The phenotype of ptac2 mutant displays albino lethal,and the morphology of chloroplast was severely impaired by transmission electron microscopy.q RT-PCR results showed that the expression levels of psb A and rbc L in the ptac2 mutant were 50% and 75% of the wild-type,respectively;the expression of atp B and 16 s r RNAs co-transcribed by PEP and NEP were 90% and 75% of wild-type respectively.In contrast,the expression abundance of acc D and rpo A,were 4 times and 2 times of that wild type,respectively.These results confirmed that p TAC2 is involved in the regulation of PEP-related gene expression.p TAC2 encoded a PPR-SMR family protein.The p TAC2 gene is strongly expressed in green photosynthetic tissues,which expression was regulated by light at the gene and protein level.p TAC2 localized in chloroplast,and is associated with thylakoid membranes.BN-PAGE results showed that p TAC2 was present in 669 KDa PEP complex,co-migrated with the core subunit,Rpo B,which demonstrated that p TAC2 is one component of PEP complex.Yeast-two hybrid results showed that p TAC2 interacts with PAPs,including FLN1 and TRX z Investigation of the 34 known chloroplast RNA editing sites revealed that these RNA editing sites in the ptac2 mutant were similar to those in the wild type,suggesting that p TAC2 does not affect the chloroplast transcripts RNA editing.RT-PCR analysis revealed that there are defects in these transcripts' splicing,including trn L,ndh A,atp F,pet D,ycf3 and rps12.Thus,p TAC2 is involved in the splicing of these PEP-dependent chloroplast transcript splicing.Biochemical analysis showed that the SMR domain of the p TAC2 protein could digest the DNA and RNA.These data facilitate the knownledge of the p TAC2 in PEP complex,which extended our understanding the mechanism of chloroplast PEP complex in higher plant.