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The <i>opaque2</i> (<i>o2</i>) mutation in maize is associated with high lysine content in endosperm and good nutritional value. To improve the nutritional quality of waxy maize, the <i>o2</i> allele was introgressed into the <i>wxwx</i> line using marker-assisted backcrossing selection technology. The lysine content of <i>o2o2wxwx</i> lines was higher than that of the <i>wxwx</i> line. To reveal the mechanism of increasing lysine content through introgression of the <i>o2</i> in waxy maize, the transcriptome on kernels (18th day after pollination) of the <i>o2o2wxwx</i> and parent lines was analyzed using RNA-sequencing (RNA-Seq). The RNA-Seq analysis revealed 49 differentially expressed genes (DEGs). Functional analysis showed that these DEGs were mostly related to the catalytic activity and metabolic processes. The <i>O2</i> gene regulated multiple metabolic pathways related to biological processes (BP) and molecular function (MP) during waxy maize endosperm development. In particular, in the <i>o2o2wxwx</i> lines, the two genes that encode the EF-1&#945; and LHT1 were up-regulated, but the gene that encodes sulfur-rich proteins was down-regulated, raising the grain lysine content. These findings are of great importance for understanding the molecular mechanism underlying the lysine content increase due to <i>o2</i> allele introgression into waxy maize.