Studies On Mitochondrial Genes And Differential Gene Expression In Microspore Development Of Tobacco Cytoplasmic Male Sterile Lines

Tobacco is an important economic crop, cytoplasmic male sterile (CMS) lines and hybrid varietieshave becoming the main development trend and direction in tobacco agricultural production. In thisstudy, mitochondrial DNA from3male sterile lines and their maintain lines of tobacco cultivars wereextracted, specific primers were designed according to the complete nucleotide sequence of the tobaccomitochondrial genome in GenBank to amplify purpose genes from tobacco mitochondrial DNA. PCRproducts were cloned and sequenced. Bioinformatics method was applied to analyze the differencebetween male sterile lines and maintainer lines on nucleoside acid and speculate protein structure ofseveral levels in tobacco.At the same time, the floral organ total RNA of microspore development frommale sterile lines MSG28and its maintain line G28of tobacco were extracted, cDNA-AFLP wasanalyzed at the stage of microspore development between cytoplasmic male sterile line and maintainerline in tobacco. The results are as follows:(1) Tobacco mitochondrial DNA extractionSix selected materials such as MS Zhongyan100, MSG28, MSK326, Zhongyan100, G28and K326cultivate at weak light conditions in greenhouse. The mitochondrial DNA were extracted using repeateddifferential centrifugation, electrophoretic bands were clear and complete testing by the agarose gelelectrophoresis, it can meet the needs of the follow-up experiment of molecular biology.(2) The contrast of target genes genetic coding sequences and amino acid sequences38Electron transport and oxidative phosphorylation genes and exons from tobacco mitochondrialDNA were amplified by using Auele specific primers. The differences of nucleotide and predictedprotein structure were analysed between male sterile lines and maintain lines. The results showed that25genes and exons had no difference on mtDNA level,6genes and exons had difference ontranscription level,7genes and exons had difference on translation level.(3) Three-dimensional structure prediction for speculate proteinsIn this study, ten bases lost in the sdh3gene of male sterile lines, and led to the speculate proteinlack a helix structure, two bases mutated in the atp4gene of male sterile lines, and the speculate proteinshortened significantly, three bases mutated in the atp6gene of male sterile lines, and the speculateprotein had two more strands. The mutation of mitochondrial gene sdh3, atp4and atp6is likely to beone of the root causes of tobacco CMS.(4) The analysis of differential gene expression in Microspore developmentRich polymorphism was observed by using same or different primer combinations to amplifydifferential gene in the stage of microspore development between cytoplasmic male sterile line andmaintainer line in tobacco.15different fragments were recovered, of which10fragments were notfound in NCBI, we speculate they are unknown gene. The other5fragments had high similarity withSTY-L, phyB and L-allo-threonine aldolase etc., those fragments involved in anther development andenergy metabolic pathway.