Analysis Of Differentially Expressed Protein Of Anthers Between The Male Sterile Line K932S And The Storing Line In Maize

Male sterility exist widely in plants.Using male sterile line to produce hybrids can improve the purity of seeds and reduce the cost of artificial detasseling.The breeders have created a large number of male sterile materials through a variety of genetic pathways,but because of the abotion mechanism is still not very clear,limiting its application.In this study,the protein expression spectrums of dfferent development stage anther of male sterile line K932 S and the restorer line K932 R were constructed by iTRAQ technology,and screened differentially expressed proteins(DEPs).Through bioinformatics analysis,qRT-PCR and QC-MS verification,to clarify DEPs and metabolic pathways relatde to abotion,and to explore the relationship between theier pathways and the abotion process of K932 S,providing the basis for further research and application.The main results are as follows:1.A total of 32307 peptides and 7440 proteins were identified by iTRAQ.GO annotation showed that the proteins mainly concentrated in cell structure components and organelles,mainly in binding activity and catalytic activity,involved in oxidation reduction process and metabolism process.COG fanctions included translation,ribosomal structural components and biosynthesis,and post translational modification,protein conversion and molecular chaperones.IPR annotation mainly included kinase domain and RNA recognition domain.350 KEGG pathways were found including carbon metabolism,ribosome,amino acid metabolism,sugar metabolism and phenylpropanoid biosynthesis.2.A total of 1648 significant DEPs were screened from four anther development stages.There were 370,591,526 and 858 DEPs at pollen mother cell stage,diploid stage,tetrad stage and early mononuclear microspore stage respectively,and 210,288,163 and 530 exclusive DEPs respectively,and 22 DEPs in all stages.The number of DEPs at diploid stage and early mononuclear microspore stage was high,which may be the important period of abotion.GO enrichments of DEPs were mainly distributed in cell part and intracellular part,mainly in catalytic activity and hydrolase activity,participated in metabolic process and organic substance metabolic process.COG annotation mainly included metabolism of carbohydrate,lipid and amino acid transfer and metabolism.KEGG enrichments mainly distributed in phenylalanine biosynthesis,sugar metabolism,amino acid metabolism and fatty acid metabolism pathways,which may related to the abortive process of K932 S.3.Three DEPs,Histone H3,HSP17.7 and ubiquitin-like protein 5 were selected to perform western blot.By using beta-actin as internal reference,and the results of western blot verification were basically consistant with iTRAQ results.Six related genes in long chain fatty acid metabolic pathway were selected for qRT-PCR expression analysis,the results were basically consistent with the corresponding protein expression trend.K932 S compared to K932 R,genes related to biosynthesis and elongation of long chain fatty acids were down-regulated from diploid stage to early mononuclear microspore stage,and genes related to degradation of long chain fatty acids were up-regulated at early mononuclear microspore stage,which may lead to insufficient content of long chain fatty acids,affecting the anther cuticle and microspores development.4.Determination by gas chromatography-mass spectrometry found that,the contents of five free long chain fatty acids including palmitic acid,stearic acid and arachidic acid in anthers of K932 S was lower than that of K932 R at all of foure anther developing stages.Of which,all of them decreased significantly at diploid stage,and part of them decreased significantly at tetrad stage and early mononuclear microspore stage.It was further confirmed that the abnormal of fatty acid metabolism was closely related to abortion of K932 S.5.In conclusion,it is speculated that the differential expression of proteins relaed to apoptosis pathway and fatty acid metabolism in K932 S from diploid stage to early mononuclear microspore stage,leading to tapetum programmed cell death(PCD)delayed and cuticle degenerated,resulting in anther shriveled and degenerated.The differential expression of proteins relaed to sugar metabolisms,amino acids metabolisms and fatty acids metabolisms,resulted in insufficient substance and energy required for the microspores development,leading to small microspores and deformed,and the autolysis disappeared.Anther degradation and microspore disappear finally resulted in the male sterility of K932 S with pollen-free.