| Maize is an important food crop in the world.Cloning genes related with maize grain weight and yield has important impact on both academic and application value.However,few genes involved in the regulation of grain development have been cloned.In addition,previous studies on maize grain development are mainly focused on DNA and m RNA level,but very little on protein level.In this study,the functions of two new genes,a RING type zinc finger protein gene,Zm RFP2,and a small GTP-binding protein gene,Zm Arf2,were studied by subcellular localization,in vitro biochemical test,interaction protein screening,genetically-modified technology and so on.Meanwhile,the i TRAQ quantitative proteomics of pericarp and endosperm at 3~4 key developmental stages for two maize inbred lines with contrasting grain size,inbred line N04 with small grain and inbred line Dan232 with large grain,were studied.The differentially expressed temporal and spatial proteins were analyzed using bioinformatics tools,and the differentially expressed protein networks were constructed.These results not only laid a foundation for exploring the regulatory mechanism of maize grain development,and provided new genes for improving grain weight in transgenic breeding,but also provided reliable genes and interacting protein information for further study on the protein regulatory networks and physiological biochemical basis of grain development.The main results were as follows:1.We fused the Zm RFP2 and Zm Arf2 with the GFP,respectively,and the recombinant constructs were introduced into Arabidopsis and protoplast,respectively.The results suggested that two genes were all localized in the cytoplasm and on the plasma membrane.2.The prokaryotic expression vectors of Zm RFP2 and Zm Arf2 were constructed and the fusion proteins were induced under low temperature,respectively.Zm RFP2 and Zm Arf2 protein had E3 ubiquitin ligase and GTP-binding activity,respectively.Zm RFP2 could regulate other genes through ubiquition proteasome pathway,and Zm Arf2 could be activated by binding to GTP and take part in signal transduction through regulating downstream effector.3.The yeast two-hybrid bait vectors of Zm RFP2 and Zm Arf2 were constructed,and the bait vectors were used to screen the interacting protein of Zm RFP2 and Zm Arf2 yeast two hybrid library,which was constructed using the grain at different stages of maize inbred line N04.Totally,15 interacting proteins for Zm RFP2 and 8 interacting proteinsfor Zm Arf2 were screened through TDO/X and QDO/X/A culture media screening and sequencing.4.The overexpression vector of Zm RFP2 were constructed and transformed into maize Hi II and Arabidopsis.Two BC3F2 maize transgenic lines and 3 T3 Arabidopsis transgenic lines were selected for phenotypic observation.Compared with the nontransgenic sibling control,a significant increase in grain width(+22.4%)and 100 grain weight(+27.5%)were observed for maize transgenic lines.And the activity of 3 key enzymes for starch biosynthesis at all grain developmental stages also increased significantly.For Arabidopsis transgenic lines,we observed significant increases in leaf areas(+71.2%),plant heights(+20.3%),1000-seed weight(+55.8%),seed area(+44.3%),seed length(+17.2%)and seed width(+16.3%)compared with the wild type.We also found a significant increase in cell size by 22.9% and 39.3%,and cell number by 43.3%and 44.3%,for the leaf epidermal cells and the cotyledon cells of mature embryo.Therefore,Zm RFP2 could improve the development of grain and other organs by both cell size and cell numbers.5.The overexpression vector of Zm Arf2 were constructed and transformed into Arabidopsis.Three T3 Arabidopsis transgenic lines were selected for phenotype observation.We observed a significant increase in leaf area(+62.3%),plant height(+33.2%),1000-seed weight(+33.9%),seed area(+38.6%),seed length(+20.7%)and seed width(+13%)compared with the wild type.We also found a significant increase in cell size for the leaf epidermal cells and the cotyledon cells of mature embryo by 22.9%and 39.3%,respectively.However,the cell numbers of the leaf epidermal cells and the cotyledon cells of mature embryo were not significantly different compared with the wild type.In addition,the real time PCR results for 2 cell proliferation-associated genes,At GIF1 and At GIF5,and 3 cell expansion-associated genes,At EXP3,At EXP5 and At EXP10,suggested that Zm Arf2 could only regulate the expression of cell expansion-associated genes.6.The wild type and the Zm RFP2 overexpression transgenic Arabidopsis lines were used for stress study under different concentration of Nacl and mannitol.The seed germination rate and root length for the transgenic lines were all higher than the wild type under different stress treatments.The results suggested that Zm RFP2 could improve the capability of salt and drought resistance in Arabidopsis.7.The characteristics of dynamic quantitative proteomics of pericarp and endosperm at 3~4 key development stages were studied for two maize inbred lines Dan232 and N04 with contrasting kernel size by i TRAQ.34559 unique specras were obtained and a total of16415 proteins were identified in all samples,among which 1401 were nonredundant.Uniquely identified pericarp and endosperm proteins were 7 and 24 for inbred Dan232,104 and 4 for inbred N04.For inbred Dan232,4,0,1 and 3 pericarp proteins and 10,1,0and 2 endosperm proteins were recognized as stage-specific proteins at 10 d,20 d,33 d and 46 d,respectively.For inbred N04,114,9 and 3 pericarp proteins and 48,1 and 8endosperm proteins were recognized as stage-specific proteins at 10 d,20 d and 33 d,respectively.Most pericarp and endosperm proteins for the 2 inbreds were involved in metabolic process,cellar process and stimulus response according to gene ontology(GO),and most pericarp and endosperm proteins for the 2 inbreds were involved in posttranslational modification,general function prediction,energy production and conversion,ribosomal structure and biogenesis and carbohydrate transport and metabolism according to COG database for functional prediction.8.There were 206 significantly differently expressed proteins between any two time-points with a criterion of fold change no less than 5.The gene model sequences of these proteins were blasted against KEGG orthologs to relate proteins to the functional pathway maps from the KEGG database.Finally,we constructed 3 protein networks,each for totally 206 differentially expressed proteins,10 differentially expressed pericarp proteins and 110 differentially expressed endosperm proteins.In the pericarp network,15 proteins with first-degree interactions and 255 proteins with second-degree interactions were found.For the endosperm network,50 proteins with first-degree interactions and357 proteins with second-degree interactions were found. |
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