| Early stages are the foundation for kernel development.And embryo plays an important role in kernel development.To provide reference for the research on early development of kernel and embryo development respectively,on the basis of current research,we choose two inbreds with different grain size meterials,and analyze differential expressed proteins in different periods through 2-DE to find key proteins in early development of kernel and embryo,respectively.In addition,we analyzed the expression levels of targeted genes,cloned the cDNA sequences of two protein peptide with important functions and align sequences.This result could lay the fundamental basis for further research in the mechanism of kernel development and cloning key genes.The main results were as follows:1.In early development,63 differential expressed proteins were found.Through GO annotation,these proteins were involved in 15 biological processes,including metabolic process(23.08%),cellular process(22.44%),response to stimulus(14.74%),regulation of biological process(6.41%)and so on.In molecular function,these proteins were involved in 9 functions,including binding(42.86%),catalytic activity(40.26%)and so on.In cellular component,these proteins were involved in 9 components,including cell(25.16%),cell part(24.53%),organelle(16.35%),membrane(9.43%)and so on.Through COG function classification,these proteins were involved in 15 functions,including posttranslational modification,protein turnover,chaperones(33.33%),energy production and conversion(14.04%)and so on.Compared among different stages for Dan232 in D4/D0,D6/D4 and D6/D0,the numbers of increasingly expressed proteins were 1,6 and 5,respectively,and the numbers of decreasingly expressed proteins were 7,1 and 5,respectively.Compared among different stages for N04 in N4/N0,N6/N4 and N6/N0,the numbers of increasingly expressed proteins were 0,5 and 12,respectively,and the numbers of decreasingly expressed proteins were 1,10 and 10,respectively.Compared between inbreds Dan232 and N04 in the same stages in D0/N0,D4/N4 and D6/N6,the numbers of increasingly expressed proteins were 12,17 and 19,respectively,and the numbers of decreasingly expressed proteins were 10,7 and 10,respectively.Further analysis to differently expressed proteins showed that signal transducer,heat shock protein,translation initiation factor,elongation factor and RNA helicase family protein may play important roles in early developmental kernels.Compared with N04,GDP-mannose 3,5-epimerase 1,malate dehydrogenase2,triosephosphate isomerase involved in carbohydrate transport and metabolism expressed up in Dan232 in three stages,which may participated in regulation of kernel development.2.In embryo development process,74 differential expressed proteins were found in the same periods of different inbreds and in different periods of the same inbreds.Through GO annotation,these proteins were involved in 11 biological processes,including metabolic process(28.26%),response to stimulus(25.00%),cellular process(20.65%),multi-organism process(7.61%)and so on.To molecular function,these proteins were involved in 7 functions,including catalytic activity(35.44%),binding(31.65%),nutrient reservoir activity(22.78%)and so on.To cellular component,these proteins were involved in 11 components,including cell(24.70%),cell part(24.70%),organelle(21.08%)and so on.Through COG function classification,these proteins were involved in 9 functions,including posttranslational modification,protein turnover,chaperones(27.78%),carbohydrate transport and metabolism(25.00%),energy production and conversion(13.89%)and so on.Compared among different stages for Dan232 in D33/D20,D46/D33 and D46/D20,the numbers of increasingly expressed proteins were 15,14 and 13,respectively,and the numbers of decreasingly expressed proteins were 19,24 and 27,respectively.Compared N33/N20 for N04,10 proteins were increasingly expressed,and 16 proteins were decreasingly expressed.Compared between inbreds Dan232 and N04 in the same stages in D0/N0,D4/N4 and D6/N6,the numbers of increasingly expressed proteins were 12,17 and 19,respectively,and the numbers of decreasingly expressed proteins were 10,7 and 10,respectively.Further analysis in differently expressed proteins showed that cyclophilin,glyoxalase,late embryogenesis abundant protein and heat protein may play important roles in early developmental kernels.3.Five proteins related with carbohydrate transport and metabolism,energy production and conversion and transcription in early developmental kernels and 4 proteins related with carbohydrate transport and metabolism and response to stimulus in embryo were chosen to analyze relative abundance of gene expression.The results showed that there were no consistent correlations between RNA and the cognate protein expression levels,indicating that there were significant effects for posttranscriptional modification to protein levels.4.Two protein peptides with important functions of GME and PGAM were chosen to clone their cDNA sequences of the two inbreds.Through analysis of CDD in NCBI,the results showed that 2 proteins represent a member of NADB_Rossmann superfamily and iPGAM_N superfamily.Through cDNA cloning and alignment of gene ZmGME and gene ZmPGAM in Dan232 and N04,the results showed that there were 7 bases difference in gene ZmGME between the two inbreds among which two amino acids changed.There were 3 bases difference in gene ZmPGAM between the two inbreds among which 1 amino acid changed.Their functions in early kernel and embryo development needed to be studied in further research. |
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