Identification of major responding proteins of Abnormal Leaf and Flower in soybean with an integrative “omics” strategy

Abstract

Proteomics has been utilized as an effective approach to bridge the gap between phenotype and genome sequence, however, more effective strategies need to be explored to find target gene(s) from many differentially expressed proteins (DEPs). Here, we utilized an interdisciplinary approach employing a range of methodologies and software tools of genomics, proteomics and metabolomics to identify important responding proteins of the Abnormal Leaf and Flower (ALF) gene involved in soybean leaf and flower development, then to get a global insight into the relevant regulating networks underpinning the alf phenotype. The main results were as follows: (1) A pair of soybean near-isogenic lines (NILs), i.e. NJS-10H-W and NJS-10H-M, differing from ALF locus, was developed with highly consistent genetic background verified by 167 simple sequence repeats (SSR) molecular markers, and an optimized 2-DE procedure was established to separate the whole proteins of leaves of the NILs. Among more than 1000 visualized protein spots, 58 spots presented expression difference, of which 41 proteins were successfully identified by mass spectrometry. The DEPs distributed on all the twenty soybean chromosomes, indicating a complicated regulation network involved in the development of leaf and flower in soybean. (2) The ALF gene was located at the end of the short arm of linkage group C1 (Chromosome 4) by gene mapping method using an F2 population. Three DEPs were also detected in the same region. (3) Ten proteins/genes of DEPs were located in the metabolism pathway by Kyoto Encyclopedia of Genes and Genomes Application Programming Interface (KEGG API), and most of the defects occurred at intersections among carbohydrate, amino acid, energy and cofactors and vitamins metabolism. The Gene Ontology (GO) annotation results of DEPs demonstrated considerable part of proteins as DNA-binding factors, metalloproteases and oxidoreduction enzymes. The GSA (Glutamate-1-Semialdehyde2,1-Aminomutase) and PIN (Peptidyl-prolyl cis-trans isomerase) genes were selected as potential candidate genes for ALF locus based on the affluent information from different “omics” analyses, and the possible regulating profile underpinning the phenome of the mutant was also inferred. In conclusion, some important responding proteins as upstream regulated factors within ALF expression network were identified and marked to the involved pathways for further analysis of the target gene. It showed that combination of “omics” methods could accelerate the process to isolate new gene(s) and provide potential information for further study on genes and proteins regulatory network.