玉米S型CMS线粒体DNA R区及其orf77的表达研究

Expression Analysis of orf77 and R Region in Mitochondrial DNA of S-type CMS Maize

玉米S型CMS胞质育性基因被认为与其线粒体DNA高频重组R区相关。在多种S胞质不育材料中都观察到R区DNA的重组及其转录产物的变化。R区含orf35 5和orf77两个开放阅读框 ,其中orf77有 3段与atp9同源。选用R区全长 1 6 5kbDNA探针、2 5 4bp的orf77DNA探针以及另外 3个线粒体基因atp9、atp6和coxⅡ为探针 ,进行了Northern分析。研究发现 :orf77与R区共转录 ;在不同核背景的S rf3rf3基因型试材的雄穗小花中 ,这两个探针均检测到 2 8,1 6 ,1 1,0 9,0 7及 0 4kb共 6个转录本 ,而在恢复基因杂合的S Rf3rf3基因型植株的雄穗小花中 ,2 8,1 6kb 2个转录本表达量减少或消失 ,1 1,0 9,0 7及 0 4kb转录本则没有明显变化 ;另外T、C胞质材料的雄穗小花中则只检测到 1 1,0 9,0 7及 0 4kb 4个转录本 ,没有 2 8,1 6kb 2个转录本。以atp9为探针的Northern分析证明 ,所有以R区和orf77为探针所检测到的 1 1,0 9,0 7及 0 4kb转录本实际上为atp9基因的转录产物 ,只有 2 8,1 6kb的转录本为R区 orf77所特有。另外 ,atp6探针和coxⅡ探针在所有实验材料中均只检测到 1种转录本 ,未发现RNA表达的差异。研究结果说明atp9,atp6和coxⅡ基因与玉米S CMS的形成没有关系 ,而R区或其中orf77是玉米S型CMS胞质育性基因的重要候?

It is generally recognized that S type of CMS in maize associated with the recombination region R in mitochondria.Complicated DNA recombination and changes of transcripts of R are observed in several subgroups of S cytoplasm.R region includes two open reading frames (orf355 and orf77) and they are all chemeric.Among the sequence of orf77,there are three stretches similar to atp9 of mitochondria. Many different S cytoplasms are found in maize in China and researches on them show that similar R region and sequence are also found in the mitochondrial DNA in Tangxu,Shuang and J (cytoplasm.Northern analysis of Tangxu,J cytoplasm with Probe R and orf77 indicates that orf77 co-transcribes with R region and the nuclear restorer gene Rf3 affects their expression.In all tested S cytoplasm plants with the genotype of s-rf3rf3,both probe orf77 and R can detect six transcripts of 2.8,1.6,1.1,0.9,0.7 and 0.4 kb.In the presence of the nuclear restorer-of-fertility gene Rf3,previous transcripts of 2.8 kb and 1.6 kb are disappeared,but the other four transcripts are not changed.In T and C cytoplasm of CMS,only four transcripts of 1.1,0.9,0.7 and 0.4 kb appear when hybrid to probe R.Further Northern analysis of probe atp9 proved that all the four transcripts of 1.1,0.9,0.7 and 0.4 kb were actually transcripts of gene atp9,so only the transcripts of 2.8 kb and 1.6 kb are particular to R region of S.In addition,atp9,atp6 and coxⅡ each has the same transcription pattern in tassel with different genotypes respectively.It can be included that R region and orf77 are the most important candidate gene for S-CMS.