| Rice cytoplasmic male sterility is closely related to mitochondrial genes in cells.An important way to understand rice CMS(Cytoplasmic male sterility)is studying mitochondrial genes and proteins.In our previous research on HL-CMS rice YTA,a new CMS related sequence HL-sp1 was cloned,which there is no homology with the known HL-CMS related gene orfH79.There is an open reading frame(ORF)encoding 290 amino acids,which was named as orf290,in HL-sp1 through bioinformatics analysis.The analysis of ORF290 showed that it is a mitochondrial membrane protein with two transmembrane domains in N terminal.Two potential interacting proteins APX8 and E37 of ORF290 were screened by membrane-based yeast two-hybrid.On this basis,In this experiment,the interaction relationship between ORF290 and APX8,E37 were further verified through yeast point-to-point,BiFC,GST pull-down and other experiments.The mainly results are as following:1.Both of APX8 and E37 were located in mitochondria and chloroplasts predicted by Plant-m PLoc.STRING results show that APX8 encodes a 478 amino acid ascorbate peroxidase,which is involved in the homeostasis of ascorbic acid and the removal of reactive oxygen species in cells.E37 encodes 37 KDa MPBQ/MSBQ methyltransferase,which is mainly involved in the methylation of tocopherol(vitamin E)and plastoquinone synthesis,and is involved in protecting the body’s redox homeostasis.2.It is proved that ORF290 and ORF290 C interact with APX8 and E37 in eukaryotic yeast cells by yeast point-to-point and Lac Z coloration reaction.The ORF290/ORF290 C and APX8/E37 expression vectors were constructed by the Gateway method,and the interactions between ORF290/ORF290 C and APX8/E37 were verified by BiFC in tobacco leaf epidermal cells.The prokaryotic expression vectors of pET-32a-orf290/orf290 C and p GEX6p-1-APX8/E37 were constructed,and the fusion protein was successfully induced in vitro.The Pull-down experiment showed that there were interactions between ORF290/ORF290 C and APX8/E37.The three methods all proved that ORF290 interacted with APX8 and E37 through its C-terminal.3.The APX8 and E37 recombinant plasmid vectors containing His tags were constructed and co-transformed yeast strains with the ORF290 bait protein vector.The expression at the RNA and protein levels has been validated,and the interacting proteins are located in the membrane system.results of the growth curve showed that when ORF290 interacted with APX8 and E37 in yeast,the growth of yeast was inhibited and the ATP level in the strain was significantly lower than that of the control group.4.The APX8/ E37 RNAi interference expression vector p DS1301-APX8/E37 was constructed and genetic transformed into NIP and YTB,the callus are currently in the differentiation stage. |
