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

Abstract

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 (Glu) promoter to regulate the biosynthesis of starch. An RNA interference (RNAi) 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 realtime (RT)-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 nontransgenic controls. Branching enzyme II (BEII) enzyme activity was notably reduced, and ADP-glucose pyrophosphorylase, soluble starch synthase, isoamylase, and pullulanase enzyme activity was markedly reduced in T 3 seeds. Relative enzyme activity change explained the reduction in thousand-grain weight (TGW) in transgenic plants. The present study indicated that amylose content was negatively correlated with BEII activity, spike size, and TGW.

Modulation of amylose content by structure‐based modification of OsGBSS1 activity in rice (Oryza sativa L.)

“…Unfortunately, the relative effects of gene silencing cannot be controlled to a specific degree, which limits the use of this technology in rice breeding and crop improvement. For cultivating cultivars with high AC, two main strategies were developed: (i) over-expression of Wx alleles, such as Wx a (Itoh et al, 2003), and (ii) down-regulation of enzymes involved in amylopectin synthesis, such as starch synthase IIIa (SSIIIa) (Crofts et al, 2012) and starch branching enzymes (SBEs) (Butardo et al, 2011;Jiang et al, 2013;Man et al, 2013). The strategies mentioned above for reducing AC in rice have so far been based on transcriptional or post-transcriptional regulation.…”

Regulation Mechanism of Exogenous Brassinolide on Bulbil Formation and Development in Pinellia ternata

“… Li et al (2020) reported that 0.05 mg/L BR treatment significantly increased the transcript level of BAM in guard cells, this may be related to different cellular functions, and BR treatment promoted starch degradation in guard cells, thus inducing stomatal opening. Silencing of the starch branching enzyme gene increased the amylose content in rice, which was associated with reduced ISA enzyme activity ( Jiang et al, 2013 ). Chourasia et al (2008) reported that the expression level of edg gene correlated with edg activity during mango ripening, which reduced cellulose/hemicellulose content.…”

Mining of Potential Gene Resources for Breeding Nutritionally Improved Maize

“…According to the starch synthesis process described above, SBE is the most critical factor in converting Am and Ap. Studies on rice [ 10 , 11 ], wheat [ 12 ], barley [ 13 ], potatoes [ 14 , 15 ] and cassava [ 16 ] showed the content of Am was increased when the activity of SBEs was suppressed, supporting the notion that manipulation of SBE is an effective way to increase the Am content. The effect of SSs on starch synthesis has been investigated and confirmed in rice [ 17 , 18 ] and sweet potatoes [ 19 ].…”