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Based on the gene-protein-reaction (GPR) model of E.coli_iAF1260 and the method of constraint-based analysis, we first calculated the metabolic flux distribution of E.coli_iAF1260 and determined its high-flux-backbone. Then we calculated the deletion impact of calculable 896 genes, one by one, on the metabolic flux redistribution of E.coli_iAF1260. Next we analyzed the relativity between v (describing deletion impact of one gene) and d (connection degree of one gene) and the relativity between v and vgene (flux sum controlled by one gene), and found that both of them were not of linear relativity. Furthermore, we seek out 32 important genes that most greatly affected the metabolic flux distribution, determined their functional subsystems and found that many of 32 key genes were related to "Alternate Carbon Metabolism" and "Transport Inner Membrane" subsystems. At the same time, by computation, we found these key genes affected high flux backbone(HFB) not greatly. Because the in silico model of E.coli_iAF1260 was tested by many experiments, thus is credible, we can conclude that the result we got has biological significances.