| Plastid-encoded polymerase(PEP)is the main polymerase in chloroplasts,responsible for the transcription of most chloroplast genes.PEP is composed of the core subunit encoded by the chloroplast and the peripheral protein encoded by the nucleus.PTAC15 / mTERF8 is a member of the PEP complex(also known as TAC complex).In this work,the function of pTAC15 in chloroplast gene expression was investigated.Sequence analysis showed that pTAC15 belongs to the member of mTERF family containing eight conserved mTERF domain.Transient expression analysis in tobacco showed that pTAC15 is localized in chloroplast.Immunblotting analysis using the pTAC15: MYC transgenic plants showed that pTAC15 associated with chloroplast thyakoid membrane.BN-PAGE,2-D and immunblotting analysis showed that pTAC15:MYC was present in a supermolecular complex with molecular weight 660 kD,and co-migrated with the core subunit,rpoB.Yeast two hybrids showed that pTAC15 was present in PEP complex and interacted with pTAC10.In order to analyze the role of pTAC15 in the process of chloroplast gene expression,mutants of ptac15 were identified.QRT-PCR analysis showed that the expression of photosynthesis genes were similar to the wild type in the ptac15,except that the transcription of psbJ was upregulated.Circularized RNA RT-PCR results showed that the transcription of psbJ terminated at the 94 th and 95 th bp after the stop codon in the wild type,while its termination was at 94 th,95th bp and many other positions in the ptac15,which suggested the knockout of the pTAC15 affects the termination of psbJ at the correct site.EMSA and cpCHIP results showed that pTAC15 specially binds to the 3' terminal of psb J DNA.Further in vitro biochemical analysis showed that pTAC15 protein has the effect of transcription termination.These results of this dissertation showed that pTAC15,a subunit of PEP complex,binds to the 3' terminal of chloroplast gene psbJ to involve in its transcription termination. |
PDF Full Text Request