Preliminary Study On The Mechanism Of Cytoplasmic Male Sterility In Tobacco K326

Cytoplasmic male sterility(CMS)is an important tool for hybrid production and a good system for studying nucleus-cytoplasm interactions,but its mechanism is still unclear.In this study,we used tobacco K326 CMS line and its maintainer line as materials to observe the morphological characteristics of their floral organ development,conduct genome-wide identification and expression characterization of tobacco floral organ development gene MADS-box and boundary gene SUPERMAN,conduct differential gene analysis by transcriptome sequencing,and conduct cloning and transient expression analysis of tobacco GLOBOSA gene,in order to make a preliminary investigation of its sterility mechanism and provide reference for its application in agricultural production.The main findings were as follows:(1)The CMS line K326 show a variety of abnormalities in stamen carpellation,petalization,absence,apical stigmatization,polyphyletic separation and fusion.The bases of the stamens are all fused together,showing that the boundaries of the third and fourth whorl of stamens have been disrupted.Stamen abnormalities appear already at the smalle bud stage,showing that the differences in stamen development between the CMS line K326 and its maintainer line should be at an earlier stage.Analysis of cytological observations showed that cell division in the floral meristem of the CMS line was slowed and development was abnormal.Therefore,the sterility of CMS line is due to nucleoplasmic interactions affecting the floral meristem,which in turn affects floral organ determination and thus triggers sterility.Sterility occurs at the time of the floral meristem.(2)A total of 160 MADS-box genes and five SUPERMAN genes were identified in tobacco,and seven class B genes(four PI genes and three AP3 genes)were found in the MADS-box.Covariance analysis indicated that tandem and fragment DNA duplication and ploidy were the driving forces behind the expansion of the MADS-box gene family.The expression levels of PI gene Nit MADS115 were found to be lower in small,medium,and large buds than in the keeper lines in the assay of class B MADS-box genes and SUPERMAN genes in the buds of tobacco K326 sterile lines,and cis-acting element analysis indicated that Nit MADS115 has a growth hormone response element,Aux RR,and that growth hormone may play an important role in tobacco cytoplasmic retrograde The growth hormone may play an important role in regulating the nucleus in the tobacco cytoplasm.(3)Transcriptome sequencing results showed that 11008,14273 and 12591 differentially expressed genes were identified in the CMS line at three periods.Significant changes in the important genes for floral meristem tissue,WUS,SUPERMAN,CRC,LFY3,NZZ/SPL and ARF,and significant down-regulation of class B genes indicated that floral meristem tissue development and flower development were affected in the CMS line.The expression of COX(Nita Mp068),NAD9(Novel.39047),peroxidase gene PRX40(LOC107794446)and glutathione sulfotransferase gene GSTF14(Novel.54828)were significantly down-regulated in the CMS line of tobacco K326,indicating abnormal mitochondrial function and disturbance of redox metabolism in the CMS line.The expression of calcium-binding protein gene was significantly altered.The significant change in calcium-binding protein gene expression may be related to nucleoplasmic dysregulation.The expression of the alternate oxidase gene AOX1a(LOC107763364),a mitochondrial retrograde regulatory signal,was abnormal during bud development,and the CMS line tobacco K326 may have affected floral meristem development through the alternate oxidase gene,which in turn affected floral development.(4)The tobacco K326 GLOBOSA gene was cloned and analyzed for its physicochemical properties and subcellular localization.The results showed that GLOBOSA is a hydrophilic protein without transmembrane region and signal peptide,with SRF-TF and K-box structural domains,which can dimerize to form complexes and bind to specific DNA to participate in gene regulation,and its subcellular localization is in the nucleus,which is consistent with the characteristics of transcription factors.