Investigation On The Sterile Molecular Mechanism Of The Cytoplasmic Male-sterile Line KTM3315A In Wheat By Proteomic Thchnology

A thermo-sensitive cytoplasmic male-sterile(TCMS)line KTM3315 A was developed by Northwest A&F University using tiller regeneration.According to our study,KTM3315 A,a K-type thermo-sensitive cytoplasmic male-sterile wheat line,is completely male sterile at lower-temperatures conditions(<18?)during binucleate stage and it is capable of producing self-pollinated seeds when the temperature exceeds 20? during this growth period.Therefore,it has great potential in the two-line hybrid breeding of wheat.Some scholars have studied the physiology,phenotype and transcriptome of KTM3315 A.However,the molecular mechanism of temperature-sensitive male sterility remains to be studied.In this study,the phenotype and cytology of the male sterile line KTM3315 A and its homotypic maintainer TM3315 B were studied.The key period and possible mechanism of abortion were identified through cytological studies.Then,we compared the anther protein in wheat between sterile line KTM3315 A its maintainer line TM3315 B using isotope-labeled relative to absolute quantification(iTRAQ).To find out the pathways of significant enrichment of differential expressed proteins,we performed the enrichment analysis of differentially abundant proteins,and to determine fertility-related metabolic pathways and fertility candidate genes.At the same time,this study is expected to provide new ideas for the research of cytoplasmic male sterility in wheat.The main findings are as follows:1.According to the results of DAPI staining,the peak of abortion occurred at the late uninucleate stage.From the results of semi-thin and ultra-thin sections,the tapetum of the sterile anther began to degrade in the early uninucleate stage,and the fertile tapetum was complete at this time.From those can estimated that the early degradation of tapetum may be an important cause of KTM3315 A wheat infertility.2.Proteomics sequencing was performed on six samples from three phases of KTM3315 A and its maintainer line TM3315 B,matching 23,277 peptides through 73,688 spectra,eventually identifying 5,570 proteins containing 1,450 differential abundance proteins(DAPs).The results of functional annotation and metabolic pathway enrichment analysis in each period showed that the differentially expressed proteins of three stages were significantly enriched in carbohydrate metabolism and energy metabolism pathways,indicating that carbohydrate metabolism and energy metabolism pathway are vital for wheat fertility.Through the analysis of carbohydrate metabolism and energy metabolism,it was found that the mainly enriched KEGG pathways of differentially abundant proteins include glycolysis pathway,citric acid cycle and oxidative phosphorylation pathway,among which many key proteins,such as glyceraldehyde-3-phosphate dehydrogenase,pyruvate kinase and 6-phosphofructokinase 1,isocitrate dehydrogenase,citrate synthase,NADH-dehydrogenase,and ATP synthase are all significantly down-regulated in sterile line,indicating male sterility in wheat may be related to down-regulation of key proteins in these key pathways.3.Through the analysis of the relationship between the significantly enriched pathways of differentially abundant proteins,a protein regulatory network that may cause male sterility was obtained.The network regulation relationship was verified to some extent from the measured ATP and soluble sugar content determination results,thereby further proving the credibility of the relationship between protein regulatory networks and male infertility.4.The results of qRT-PCR showed that the quantitative results of differentially abundant proteins were significantly different from the results of protein sequencing.This shows that there is no good correlation between protein level and mRNA level,indicating that messenger RNA(mRNA)is not a reliable indicator of protein levels.In this study,the molecular mechanism of sterility of KTM3315 A was revealed through proteomics analysis and the verification of cytological and physiological indicators.At the same time,these results of this experiment provided theoretical support for the study of male sterility.