Comparative Proteomic Analysis Of Anthers From Genetic Male-sterile And Fertile Cotton

Protein is the final product of gene expression and the executors of functions of life,the use of proteomics to study the phenomenon and essence of life,with irreplaceable advantages.The cotton?Gossypium hirsutum L.?21A GMS line was identified as a new single recessive nuclear male sterile lineIn this experiment,we harvested the buds in 21 A male sterile and ferlie plants,during sporogenous cells and pollen mother cells undergo meiosis stages.We performed an iTRAQ-based quantitative proteomics technology to identify differentially expressed protein?DEPs?in male sterile and fertile anther,and discuss functions and pathways enriched of DEPs.The profiling of differentially expressed proteins may provide important clues on elucidating the mechanism of genic male sterile cotton.The main results were as follows:1.In this project,there were four Gossypium samples,F1,F2,S1 and S2?F?S represents respectively fertile and sterile plants;1?2 represents respectively sporogenous cells and pollen mother cell meiosis stage?.iTRAQ technology in combination with liquid chromatographyelectrospray ionization-tandem mass spectrometry?LC-ESI-MS/MS?was applied.After Mascot 2.3.02 program searching,a total of 8,123 proteins were identified.Then,3941 proteins were mapped with at least 2 peptides.2.We performed respectively,GO,COG,Pathway function notes of 8123 proteins identified based on Gene Ontology,Clusters of Orthologous Groups of proteins,and KEGG Ortholog databases:?1?GO function annotation: In the process of biological classification,"metabolic process"?3535,62.97%?,"the cell"?3187,56.77%?occupies the largest proportion;in cellular components,proteins in the distributions of "cell"?3189,56.80%?,"part of the cell"?3189,56.80%?are significantly more;in molecular function,the largest function proportion of proteins are catalytic activity "?3362,59.89%?,the" combination "?2738,48.77%?;?2?By COG function analysis,the identified proteins were assigned to 24 categories.The main functional categories were general function prediction only?1244,25.90%?,posttranslational modification,protein turnover,and chaperones?710,14.78%?,translation,and ribosomal structure and biogenesis?533,11.10%?,energy production and conversion?459,9.56%?,carbohydrate transport and metabolism?445,9.27%?,amino acid transport and metabolism?426,8.87%?;in function classification of nuclear structure?2,0.04%?and cell motility?10,0.21%?,the number of proteins is minimum;?3?In the Pathway analyses,5743 proteins were successfully annotated,and they were involved in 128 metabolic pathways.The most ways involved in were the metabolic pathways?1768?,biosynthesis of secondary metabolites?1035?,ribosome?278?,protein processing in endoplasmic reticulum?221?,RNA transport?207?,plant-pathogen interaction?183?.3.According to the level of protein abundance,Any proteins with 1.2-fold changes and a Q-value less than 0.05 were determined to be DEPs.The results of identification of DEPs are as follows:?1?Floral buds in ferlie plants at sporogenous cells stage and pollen mother cells undergo meiosis to tetrad formation stage?F2-VS-F1?: There were 81 DEPs;floral buds in sterlie plants at sporogenous cells stage and pollen mother cells undergo meiosis to tetrad formation stage?S2-VS-S1?: A total of 181 proteins were differentially expressed.In the two stages of anther development,the proteins with different expression were different in sterile and fertile plants.?2?In the two stages of pollen development,there were 1150 differentially expressed proteins.Compared with fertile plants,at sporogenous cells stage,there were 295 up-regulated proteins and 375 down-regulated proteins in sterile plants.And,340 proteins were significantly differentially up-regulated while 442 proteins were significantly down-regulated during pollen mother cells undergo meiosis to tetrad formation stage.The number of DEPs that were up-or down-regulated during sporogenous cells stage and pollen mother cells undergo meiosis to tetrad formation stage indicated that more DEPs were identified at pollen mother cells undergo meiosis to tetrad formation stage than at sporogenous cells stage,which was consistent with the more serious male sterile phenotype observed by microscopic examination in AS anthers.4.For 1150 differentially expressed proteins during two stages of anthers development,further to have GO enrichment analysis.In the biological process,a large proportion of DEPs enriched in "metabolic process"?502?,"cellular process"?449?.In the cellular component,the most abundant differential expressed proteins were in "cell"?485?,"cell part"?485?,"organelle"?364?.In terms of molecular function,“catalytic activity”?410?,"binding"?380?were enriched by the largest differential protein.During the process of anther development,compared with the fertile anther,the differentially expressed proteins in the sterile plants were mainly enriched in the catalytic activity and binding of the molecular function.5.To analyze further the physiological and biochemical properties of DEPs,1,150 DEPs were assigned to 24 categories using the cluster of orthologous groups?COG?database.The percentages of proteins involved in O?13.4%;posttranslational modification,protein turnover,and chaperones?,J?10.6%;translation,and ribosomal structure and biogenesis?,C?9.3%;energy production and conversion?were dominant,indicating that post-transcriptional regulation,translation,and energy production and conversion play important roles during anthers development.6 The pathway enrichment analysis showed that the major pathways were involved in metabolic pathway?ko01100?,ribosome?ko03010?,protein processing in endoplasmic reticulum?ko04141?,and RNA transport?ko03013?,carbohydrate synthesis and metabolism,energy production.According to scatter plot of pathway enrichment,in the sporogenous cells stage,473 DEPs involved in 110 metabolic pathways;at the pollen mother cells meiosis period,the 576 DEPs were involved in 111 metabolic ways.Pathway enrichment analysis was carried out on specific expressed proteins during the sporogenous cells stage and pollen mother cells undergo meiosis to tetrad formation stage,and there were a total of 35 pathways.These DEPs-enriched pathways might be important for elucidating the complex mechanisms of GMS.7.To validate whether the differences in protein and transcriptional levels were consistent,and to confirm the reliability of the proteomic analysis,12 DEP candidate genes selected based on their expression abundance,were analyzed by real-time quantitative PCR?qPCR?at sporogenous cells stage and pollen mother cells undergo meiosis to tetrad formation stage in AF and AS plants.The expression patterns determined by qPCR for the genes were consistent between qPCR and iTRAQ results,except for CotAD₇2197,indicating that the proteomics analysis results in this study were relatively reliable.