Functional Analysis Of CpMMS19 Gene In Papaya

Papaya(Carica papaya L.)belongs to the genus Carica in the family Caricaceae.It is cultivated in tropical and subtropical regions worldwide.Papaya is ranked as one of the most nutritious fruits and has medicinal properties as well.It is a diploid dicotyledonous plant specices with 9 pairs of chromosomes and a genome size of 372 Mb.Due to its short generation time and characterized genome,papaya became a good model system for studying flower and fruit development in fruit trees.Therefore,it is necessary to study gene functions in papaya.MMS19(Methyl Methanesulfonate Sensivity 19)encodes a highly conserved HEAT repeat protein and plays an important role in DNA replication and DNA repair.Recent studies in human revealed that MMS19 serves as a key player in iron-sulfur protein assembly(CIA)and envoys iron-sulfur cluster to target proteins that are critical for DNA replication and repair,and telomere maintenance.At present,the study of MMS19 in plants is rare,and it is only reported in Arabidopsis haiana.Therefore,it is important to study the function of MMS19 gene in fruit tree model papaya.Papaya diminutive mutant,an undesirable mutant that was characterized by small stature of the tree with small leaves,and misshapen flowers and fruits,is caused by a 36.8 kb deletion in CpMMS19 gene.In this study,we studied gene functions of CpMMS19 gene and its diminutive allele,cpdim gene,by transformation and protein-protein interactions between CPMMS19 protein and other members of CIA complex in order to unravel the molecular mechanism underlying the diminutive phenotype.Ourmajor discoveries are listed as follows:1.There is a single copy of CpMMS19 gene in papaya genome.CpMMS19 gene contains all four conserved domains of the MMS19 gene,MMS19_N,Motif 1(amino acid sequence:G-E-K/R-D-P-R-N-L),Motif 2(amino acid sequence:F-xxxx-C-Y-F-P-x-F)and the HEAT repeat domain,while the cpdim gene misses 26 amino acids in the C-terminal conserved HEAT repeat domain although it compeletly retains all the other three conserved domains.2.The gene expression analysis showed that CpMMS19 gene was expressed in leaf,flower,fruit,pistil and stamen tissues.CpMMS19 gene exhibited a relatively lower expression in leaf compared with the rest four tissues.3.The CPMMS19 protein exhibited self-activation of transcription,while the cpdim protein did not show the same activity.The cpdim protein displayed clear interactions with CPNBP35 and CPAE7 protein,while other members of the CIA complex didn’t show direct interactions between each other,suggesting the cpdim gene has remained its function in CIA complex assembly and CPMMS19 may act as a key player in CIA complex assembly.4.No significant phenotypic change was observed in overexpression of the CpMMS19 gene in the Arabidopsis thaliana mutant met 18.However,similar phenotype as observed in papaya diminutive mutant,such as diminutive stature,embryo abortion,was found in overexpression of the cpdim gene in the met 18 mutant,indicating the cpdim gene lost the function in assisting iron-sulfur cluster to target proteins.5.Subcellular localization assay revealed that both the CpMMS19 gene and cpdim gene were localized in both the cytoplasm and nucleus.6.The constructs of CpMMS19-RNAi and CpMMS19-CRISPR/Cas9,and overexpression constructs of PMDC202-CpMMS19 and PMDC202-cpdim were made and transformed into wild-type papaya.Up to date,we have obtained transgenic plants of CpMMS19-CRISPR/Cas9 and positive callus at regeneration stage for the other constructs.In a conclusion,CPMMS19 is a highly conserved HEAT repeat protein localized in both the cytoplasm and nucleus and expressed widely in leaf,flower,fruit,pistil,and stamen tissues.It is one of the CIA pathway member in cytoplasmic,and may play the key role in CIA complex assembly.Besides,CPMMS19 may have overlapping functions with CPAE7.Our results laid a foundation for further study of the biological functions of the CpMMS19 gene.