Research On Cytoplasmic Male Sterility-Related Mitochondrial Protein In S-Type CMS Maize

Chimeric gene created by rearrangements of coding and noncoding segments of mtDNA have been found to be associated with cms in a number of species.It could either interfere with the normal fuction of mitochondria through expression products, or affect the adjacent genes'mRNA transcription and translation through co-transcription, and then the development of pollen or floral organ abolished. In maize CMS-S,orf355 and orf77 resulted from recombination located downstream coxâ… and coxâ…¡, We use the male sterile line S-Mo17rf3rf3 in this research, maize CMS-S associated gene orf77 and orf355 was the object we foucs on. We discuss biochemical and functional characterization of ORF77 and ORF355 through structure prediction, prokaryotic expression, onion transient expression system and obtain the following results:1.We used ConPredâ…¡and the ExPASy Proteomics Server to predict polarity and secondary structure of ORF77 and ORF355.ORF77 had one transmembrane domain, ORF355 had three transmembrane domain, they both had high hydrophobicity.2.Clone orf17, orf60, orf77, orf355 into pET-28a and use atp9 as hydrophobic control, then, we induced vector to express protein and make bacterial growth curve until E.coli grown to mid-log(OD6oo=0.5).We could not detect any specific proteins on SDS-PAGE gel. We observed that E.coli contained pET-28a-orf355 had a decrease in cell density while cell density of E.coli contained other plasmids had no obvious change.It indicated that pET-28a vector could express hydrophobic protein and expression of ORF355 could suppress bacterial growth for its'potential toxicity instead of its'hydrophobicity.3.Clone orfl7, orf60, orf77, orf355 into pTYB11,and then we induced vector to express protein and make bacterial growth curve until E.coli grown to mid-log(OD6oo=0.5).we could not detect ORF355 on SDS-PAGE gel. We observed that E.coli contained pTYB11-orf355 had a decrease in cell density while cell density of E.coli contained other plasmids had no obvious change.Expression of ORF355 would inhibit bacterial growth. ORF355 may be a toxic protein.4. Based on former vector pET-28a-EGFP, we clone orf17, orf60, orf77, orf355 into C-terminal of EGFP, and then we induced vector to express protein and make bacterial growth curve until E.coli grown to mid-log(OD6oo=0.5).we could not detect ORF355 on SDS-PAGE gel. we observed E.coli contained pET-28a-EGFP-orf355 had weaker fluorescence compared with control plasmid pET-28a-EGFP and uninducined control by Laser Scanning Confocal Microscope. Expression of ORF355 also inhibited bacterial growth.It demonstrated that ORF355 might be a toxic protein.5.We constructed pET-28a-egfp by cloning egfp into pET-28a, and then cloned orf77, orf355 into N-terminal of egfp.we induced vector to express protein and make bacterial growth curve until E.coli grown to mid-log(OD600=0.5).Compared with control plasmid pET-28a-egfp and uninducined control, only E.coli contained pET-28a-orf355-egfp was inhibited, E.coli contained pET-28a-orf77-egfp was unaffected. E.coli contained pET-28a-orf77-egfp had weaker fluorescence, E.coli contained pET-28a-orf355-egfp had no fluorescence through Laser Scanning Confocal Microscope. As the result 4, ORF355 might be a toxic protein.6. Result of onion epidermal cells demonstrated that ORF77 and ORF355 could be expressed in eukaryotic cells.ORF77 appeared in the cytoplasm and cell membrane, ORF355 appeared in cell membrane.It indicated location that the protein might play a role in.7.Conclusion:The expression of mitochondrial gene had the same pattern with prokaryote, so in this paper we analyze maize cms-associated gene through prokaryotic expression. The expression of ORF355 strongly inhibited bacterial growth, but too little protein was produced for direct test by SDS-PAGE gel. our find in the experiment may provide a clue to get some information about the mechanism on CMS of S-type maize using the prokaryote model system, we knew the role orf355 might play through onion transient expression system. So, the expression of orf355 may be responsible for S-type CMS maize.