Research On The Character Of Proteins Expressed By Genes Which Associated With S Type Cytoplasmic Male-sterility In Maize

In maize,CMS was originally classified into three groups:S,T and C.S type CMS in maize is gametophyte male sterity and it is generally recognized that S-CMS is associated with orf77 and orf355 in mitochondria(Zabala,1997).The two transcripts of orf77 and orf355 in the sterile microspore is accumulated in large amount,but in the fertility restored microspores the abundance of these two transcripts is reduced(Xiao H L, 2006)the stop editing occurs in the orf77 region,resulting in the premature stop codon, the orf77 will be translated into a peptide have 17 amino acids due to the teminaton edit(Gallagher et al.,2002),which shares high identity with the C-terminal transmembrane domian of ATP9,so some scholares presumed that the produce of orf77 can disturb the normal function of mitochonadria through competion with ATP9 or binding directly with mitochondrial inner member and disordering the membrane permeability,these interference factors may lead to the initiation of programmed cell death(PCD) in the dexeloping microspores,and then cause the sterility character.In this research,we selected the genes(orf77 and orf355)which were associated with S type CMS and used some techniques to research character of ORF77 and ORF355, such as predicting the protein structure,prokaryotic expression,sub-cellular localization in onion epidermal cells,proteome.Main results are showm below:1.We uesd software TMHMM Server v.2.0 to predict the protein secondary structure of ORF77 and ORF355,known that proteins had trans-membrane structure and high hydrophobicity.2.0rf77 mutating to orf77 depend on DpnI mediated site-directed Mutagenesis, with that we constructed three expression vector(pET-15b)carrying orf77,orf60,orf77 genes respectively,and transformated E.Coli.Inducing the proteins expression and making growth curve,we observed that cell lysis leading to a rapid decrease in cell density with orf77 and cell density had no change with orf17 or orf60.These observations indicated that complete orf77 encodes acytotoxic peptide.3.Inserting the aim genes(orf77 and orf355) to different prokaryotic expression vectors:pET-15b,pET-28a,pET-32a,pEGX-KG,pTYB-12,but not obtaining the aim proteins;synthesizing a peptides ORF17 was not successful;using the TNT SP6 High-Yield Protein Expression System to translate the protein was also unsuccessful.4.Inserting the aim genes EGFP to the prokaryotic expression vectors pET-28a and inducing the protein expression,we can observed E.coli produced strong fluorescence by confocalmicroscope;if we inserted genes orf77 or orf355,the fluorescence were very weak.It demonstrated that orf77 or orf355 can block the process of translating the EGFP.5.Transformated the pET-28a-EGFP-orf77 and pET-28a-EGFP-orf355 to the BL21(DE3) respectively,and extraction of total RNA to amplification of the aim genes.The result shown that the orf77 and orf355 had its transcripts in different stages and the amount of transcripts increased if inducing the proteins expression.So we could consider the transcription process was all right.6.Extraction of total proteins of pET-28a-EGFP and pET-28a- EGFP-orf77,and separated the proteins used dielectrophoresis.If we insert the orf77,the amount of the protein spots would decrease.Nine spots had significance various were selected to be detected by the Mass-spectrometric,and nine of them were successfully identified finally.Among which,eight proteins was involved in biosynthesis and metabolic activity,one protein was involved in forming cytoskeletonic system.7.Subcellular localization of ORF77 protein and ORF355 protein in onion epidermal cells.It demonstrated that ORF77 and ORF355 were located at the plasma membrane,and it proved the orf77 and orf355 can translate into proteins in the Eukaryotic Expression Systems.