Cloning And Functional Analysis Of Wheat TaPPR4 Gene

Pentatricopeptide repeat(PPR)genes form a large gene family in terrestrial plants.Members of the eukaryotic PPR protein family contain tandem arrays of a degenerate 35-amino acid repeat motif and have functions in RNA or DNA modification,acting through sequence-specific binding.A large majority of the PPR genes in the higher plants are localized in chloroplasts and mitochondria.The majority of characterized PPR proteins have been found to function in modulating the expression plastid and mitochondrial genes in plants.Among the PPR genes in Arabidopsis,many have additional motifs or domains.The commonest are the C-terminal E and DYW domains,but other RNA-binding or DNA-binding domains,such as small Mut S-related(SMR)domain.Although few in number,they figure prominently in the chloroplast biogenesis and retrograde signaling literature due to their striking mutant phenotypes.However,the underlying molecular regulation mechanism is not clear yet.This study cloned a TaPPR4 gene from wheat,and this gene encodes a protein with a PPR-SMR domain.The effects of TaPPR4 gene on plant growth and development were studied by biochemical and genetic methods,and we also analyzed the role of this gene in the process of sugar signaling in wheat.The main results are as follows:1.The phylogenetic analysis shown that the orthologs of TaPPR4 were identified in the bryophyte models Physcomitrella patens(moss),which suggested that the orthologs of TaPPR4 emerged early in the evolution of the PPR protein family.However,the multiple sequence alignment analysis revealed that the sequences of the orthologs of TaPPR4 only in monocots are highly conserved.2.The subcellular localization vectors p16318-TaPPR4-GFP and p16318-pTAC2-mCherry were constructed,and co-transformed into wheat protoplasts.The results revealed that TaPPR4-GFP was localized to chloroplasts.As well as we could clearly observe co-localization of the GFP and mCherry signals,indicating that TaPPR4 and pTAC2 may have similar functions.3.qRT-PCR,GUS expression experiments and in situ hybridization were performed.to analysis the expression pattern of TaPPR4.The result showed that TaPPR4 was ubiquitously expressed in various tissues throughout the various developmental stages.4.The TaPPR4-overexpressing(TaPPR4-OX)and TaPPR4-RNAi transformation vectors were constructed,and the plasmids were transformed into wheat cv by biolistic bombardment,respectively.Finally,we generated transgenic wheat plants with an overexpressing or underexpressing(RNAi)-Ta PPR4 gene.We did not observe significant phenotypic differences in the growth or morphology between the RNAi and WT.However,the various stages of TaPPR4-OX lines growth,including the tiller,jointing,heading,flowering,and mature stage,were significantly earlier than that of the RNAi and WT.The Ta PPR4-OX plants have a shorter root length,a higher plant height,a less tiller number,a longer ear length,a lower grain number per ear,and a wrinkled grain than RNAi and WT.5.We employed a qRT-PCR to gain a general overview of plastid transcripts in the RNAi and Ta PPR4-OX plants.The result suggested that the TaPPR4 may affect the accumulation of atpE transcripts in the chloroplast.6.We next sought to identify possible TaPPR4 interacting proteins using a yeast two-hybrid approach.TaPPR4,as bait protein,was used to screen the wheat cDNA library.Finally,TaSnRK1 was found,and TaPPR4 interacts physically with TaSnRK1 in the chloroplast of living,which was proved by yeast two-hybrid,BIFC,and CO-IP approachs.7.The expression pattern of TaPPR4 was monitored by q RT-PCR,which showed that the TaPPR4 gene was induced by sucrose.At the same time,the gene can affect the expression of genes involved in sugar signaling.TaPPR4-OX lines were insensitive to high sucrose concentration.TaPPR4 gene regulates the expression of TaTPS1 gene to change the content of T6 P regulating the activity of TaSnRK1.