Preliminary Functionialanalysisof The Candidate Genes For C-type Cytoplasmic Male Sterility In Maize

Maize is one of the most widely used cropsfor heterosis.The male sterile line is an efficient and cost effective method for seeds production.However,the molecular mechanism of C-cytoplasmic male sterility in maize is very complex.In the present study,a preliminary functional analysis of the candidate genes of C-cytoplasmic male sterility in maize was carried out.The main conclusions are as follows:1.The candidate genes(atp9,atp6-C and cox2-C)of C-cytoplasmic male sterility in maize were recombinant genes while atp9 has recominataion in the non coding region.The 39 bp of the 5'end of theatp6-C gene in the male sterile line are homologous to 5'end of atp9 gene while the remaining 38 bases are identical except the fifteenth bp.;the middle portionof the atp6-C gene is composed of an unknown source region of 442 bp.The 3 'end of the gene is highly homologous to the 764 bp gene sequence of theatp6 in maintainer line with a difference of 2 SNP.cox2-Cgene is composed of three parts.The first part of the cox2-C gene containing 440 bp 5'end is homologous to the 5` end ofatp6 gene of the maintainer;the second part is composed of 118 bphaving the same homologous sequences of 111 bp to that of atp6 gene as well as to the upstream of cox2 gene sequences;the third part has exceedingly homology with the 1577 bp gene of cox2 except for a 2 bp differencein the intron.Both atp6-Cand cox2-C have RNA editing phenomenon of C to U,and both of them have transmembrane domains.2.The Circularized RT-PCR resultsfor mitochondrial gene showed thatthere was only one transcript of atp9 gene in the anther of male sterile line,while atp6-C and cox-C hadat least 4 transcripts.These results were further confirmed by Northern blot.The investigation alsorevealed that the transcription of the male sterile gene was affected by the nuclear restorer gene.The results of Real-time q PCR analysis showed that the two chimeric genes were constitutively expressed and their expressionwas significantly higher than F1.3.Transient expression vector was transferred into protoplasts of maize by PEG.The cellswere stained with a mitochondrial dye and examined underconfocal laser scanning microscope.It was found that both atp6-C and cox2-Cwere localized on the mitochondria.4.We transformed Arabidopsis thaliana with plant expression vector containing GFP tag.The transformedplants were not found to be completely aborted while the transformation of the same vector in tobacco revealed that the empty vector and mts could be expressed normally although the fluorescence signals of GFP were weak enoughto detect contained atp6-Cor cox2-C.These results suggest that GFP may affect the expression of target genes,it was also possible that pollen abortion was caused by short transcripts or other mitochondrial genes.