Preliminary Study On Function Of A New Cytoplasmic Sterility Related Protein ORF290 In Rice

Cytoplasmic male sterility-related gene,HL-sp1,was previously cloned from Honglian-type cytoplasmic male sterility(HL-CMS)rice Yuetai A(YTA)in this laboratory.An independent exon encoded 290 amino acids,orf290,was predicted by bioinformatics analysis.The sterile lines containing only orf290 were successfully obtained from AA wild rice and 824 cultivated varieties through molecular markeraided selection.In this study,ORF290 was expressed with both prokaryotic and eukaryotic expression systems,respectively,and the ORF290-interacted proteins were screened from yeast membrane protein library.The main results are as following:1.The inducible E.coli prokaryotic expression system was used for ORF290 expression,and its growth curve was measured.It was showed that the growth of E.coli was inhibited by ORF290 accumulation.2.The inducible Pichia pastoris eukaryotic expression system was used to express ORF290 protein,and the growth curve and the concentrations of ATP and reactive oxygen species(ROS)were detected.it was showed that the growth of Pichia pastoris was obviously inhibited by ORF290 accumulation,accompanied with the decrease of ATP concentration and the accumulation of ROS.It was suggested that ORF290 was toxic to Pichia pastoris through the accumulation of ROS and the decrease of ATP energy.3.A transmembrane domain in the N-terminal of ORF290 was predicted by bioinformatics analysis,and to screen the ORF290 interacted proteins,the full-length and C-terminal bait protein vectors were constructed.Two ORF290 interacted proteins,OsAPx8 and E37,were finally obtained from 39 primarily selected potential proteins.Through WoLF PSORT prediction,both OsAPx8 and E37 are located in mitochondria and chloroplasts.Furthmore,OsAPX8 can be combined with both FAD/NAD(P)binding domain and/or glutathione transferase.And E37 is involved in vitamin E biosynthesis,being interacted strongly with those related proteins TMT,VTE1,HPT1 and HPT2.