Research Article

RNA interference-mediated silencing of the starch branching enzyme gene improves amylose content in rice

Published: January 04, 2013
Genet. Mol. Res. 12 (3) : 2800-2808 DOI: 10.4238/2013.January.4.19

Abstract

Amylose and amylopectin are the 2 major components of plant storage starch. The rice starch branching enzyme (RBE) plays an important role in the starch components of rice. In the present study, we selected a specific 195-bp segment from the RBE3 gene to construct hairpin DNA, which was driven by an endosperm-specific high molecular weight glutenin promoter to regulate the biosynthesis of starch. An RNA interference plasmid for the RBE3 gene was constructed to form double-stranded RNA. Following Agrobacterium-mediated rice transformation (in the cultivar Zhonghua 11), 41 transgenic plants were identified using PCR and Southern blot analysis. Semi-quantitative real-time PCR revealed that RBE3 gene expression was significantly reduced in immature transgenic seeds. Transgenic rice amylose content had an average increase of 140%. The highest rice amylose content was 47.61% and the growth rate increased 238% compared to the non-transgenic controls. Branching enzyme II activity was notably reduced, and ADP-glucose pyrophosphorylase, soluble starch synthase, isoamylase, and pullulanase enzyme activity was markedly reduced in T3 seeds. Relative enzyme activity change explained the reduction in thousand-grain weight in transgenic plants. The present study indicated that amylose content was negatively correlated with branching enzyme II activity, spike size, and thousand-grain weight.

Amylose and amylopectin are the 2 major components of plant storage starch. The rice starch branching enzyme (RBE) plays an important role in the starch components of rice. In the present study, we selected a specific 195-bp segment from the RBE3 gene to construct hairpin DNA, which was driven by an endosperm-specific high molecular weight glutenin promoter to regulate the biosynthesis of starch. An RNA interference plasmid for the RBE3 gene was constructed to form double-stranded RNA. Following Agrobacterium-mediated rice transformation (in the cultivar Zhonghua 11), 41 transgenic plants were identified using PCR and Southern blot analysis. Semi-quantitative real-time PCR revealed that RBE3 gene expression was significantly reduced in immature transgenic seeds. Transgenic rice amylose content had an average increase of 140%. The highest rice amylose content was 47.61% and the growth rate increased 238% compared to the non-transgenic controls. Branching enzyme II activity was notably reduced, and ADP-glucose pyrophosphorylase, soluble starch synthase, isoamylase, and pullulanase enzyme activity was markedly reduced in T3 seeds. Relative enzyme activity change explained the reduction in thousand-grain weight in transgenic plants. The present study indicated that amylose content was negatively correlated with branching enzyme II activity, spike size, and thousand-grain weight.