Proteomic analysis of Zhangzagu3 (Setaria italica) and its parents based on iTRAQ technique

Abstract

Zhangzagu3, derived from Zhangzagu3fu and A2 (photoperiod- and thermo-sensitive genic male-sterile line) is well-known for its strong adaptability, good stability, disease, and lodging resistance. To evaluate the parental contributions to its elite traits, isobaric tags for relative and absolute quantitation (iTRAQ) and mass spectrometry (MS) were used to analyze the protein expression profile of these three lines. Enrichment analysis of differentially expressed proteins (DEPs) was performed through GO term and KEGG pathway. A total of 4015 proteins were detected, among which 504/390 DEPs between Zhangzagu3 and the female/male parent, respectively. The cluster analysis using Cluster 3.0 showed that the DEP expression profiles of Zhangzagu3 were closer to those of the male parent. GO functional classification divided the DEPs into three functional categories (cellular component, biological process and molecular function) and 71 subfamilies, among which three subfamilies had 100% difference between Zhangzagu3 and its parents. KEGG metabolic pathway exhibited significant enrichment of differentially expressed genes (DEGs) in Carbon metabolism (mainly Carbon fixation, Glycolysis and Gluconeogenesis), Photosynthesis and Oxidative phosphorylation. qRT-PCR verification confirmed the differential expression data and revealed that the expression level of 17 selected genes (such as putative receptor-like protein kinase, copper chaperone for superoxide dismutase etc.) was higher in Zhangzagu3 than that in its male parent, but significantly lower than that in A2. Thus, A2 can potentially optimize the agricultural qualities of Zhangzagu3. Taken together, we hope that this study provides valuable information for the further utilization and study on the molecular mechanisms of heterosis in hybrid millet. Abbreviations iTRAQisobaric tags for relative and absolute quantitation DEPsdifferentially expressed proteins DEGsdifferentially expressed genes PMSFphenylmethanesulfonyl fluoride HEPES4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid DTTdithiothreitol EDTAethylene diamine tetraacetic acid GOgene ontology IAMiodoacetamide MSmass spectrometry KEGGKyoto Encyclopedia of Genes and Genomes PVPPpolyvinylpolypyrrolidone HPLChigh performance liquid chromatography TCAtrichloroacetic acid TEABtetraethylammonium bicarbonate ACNacetonitrile HCDhigher collision energy dissociation FDRfalse discovery rate SDSsodium dodecyl sulphate qRT-PCRquantitative real-time polymerase chain reaction

Keywords

• proteomic analysis
• iTRAQ
• heterosis
• Zhangzagu3
• hybrid millet