Identification And RNA Editing Of Sua-CMS And Tab1-CMS In Nicotiana Tabacum

Male sterile cytoplasm used in tobacco breeding and production in China is mainly one type of sua-CMS.Long-term use of single sterile cytoplasm has a weak genetic basis and has a high potential risk in production.This study obtained the molecular characteristics of tobacco sua-CMS type by bioinformatics method and specific ORFs PCR amplification.Through the research methods of semi-quantitative,fluorescence quantitative expression,enzyme activity analysis and RNA editing,the sterility characteristics of sua-CMS were discussed,laying a foundation for the study on the sterility mechanism of sua-CMS.Tab1-CMS was derived from K326,the sterile lines were completely aborted.By comparing the differences between the agronomic characters and seedling resistance of the sterile lines and the maintainer lines,the sterile characteristics of tab1-CMS were evaluated,laying a foundation for the acquisition and utilization of new sterile cytoplasm types.The aims are as follows:(1)By ORF-finder,BLASTN and BLASTX in the mitochondrial genome of tobacco Zhongyan 100sterile line and maintainer line.By BLAST comparison,thirty-one ORFs specific primers specific to sterility lines of sua-CMS were amplified in seven tobacco cytoplasmic male sterile types(sua-,glu-,rep-,rus-,tab1-,tab2-,tab3-)and maintainer lines.It was found that six ORFs(ORF82,ORF103,ORF115~a,ORF100,ORF115~b,ORF91)were located in three regions(122845-123313,372705-343743 and 414430-415454)of the mitochondrial genome of the sterile line sua-CMS,and were unique to male sterile line of sua-CMS.Fuither use three viable tobacco Speight G-28,K326,NC82 respectively with seven types of cytoplasmic male sterility cross and backcross BC1F1 generations,as well as three sua-CMS sterile cytoplasmic backcross generations of ten or more years of sterile lines and three sua-CMS sterile cytoplasmic hybrid,PCR amplification in the three sections,only in containing sua-CMS fertility extranuclear tobacco can amplify the corresponding size stripe,other types of sterile cytoplasm and tobacco in the fertile no amplification,shows that the three sections specific to sua-CMS line.(2)Semi-quantitative PCR and Quantitative Reverse Transcription PCR analysis of the tobacco sua-CMS and keep ORF82,ORF103,ORF115~a,ORF100,ORF115~b,ORF91 six ORFs in different tissue(leaf,bud,anther and root)of the relative expression quantity,The results showed that all the six ORFs were highly expressed in the buds and anthers of sterile lines,but not in the maintainer lines.(3)After the PCR amplification was carried out on the twenty-four of mitochondrial genes encoding sequence alignment,RNA editing site analysis,analysis of the characteristics of RNA editing,and the genes(COX1,COX2,COX3,COB),the quantitative Reverse Transcription PCR analysis found that RNA editing of sua-CMS sterile line and maintainer line has a widespread influence,The results showed that RNA editing had extensive influence in sua-CMS male sterile lines and maintainer lines.There were many RNA editing sites in maintainer lines,and the relative expression levels of nine genes were high in flower buds of sua-CMS male sterile lines and maintainer lines,and the relative expression levels of sua-CMS male sterile lines were higher.(4)The difference between tab1-CMS sterile cytoplasm and maintainer lines in the main agronomic characters of flowering and seedling resistance(TMV,CMV,PVY,black shank disease,bacterial wilt)was compared and analyzed.The results showed that the sterile cytoplasm of tab1-CMS had no significant difference in fertile cytoplasm of anti-TMV,PVY,CMV,black shank and bacterial wilt disease.There were significant differences in plant height,and were no significant differences in stem circumference,leaf width,leaf length and effective leaf number between the two cytoplasms.