Molecular Characterization of Transgenic Rice Producing Resveratrol

Abstract

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT Resveratrol, a plant phenolic compound, has potential therapeutic benefits due to its antioxidant properties. This is substantiated by previous studies that show that resveratrol derived from rice grains is an effective treatment agent for metabolic syndrome. Here, we characterized the T-DNA sequence, inserted T-DNA structure, copy number, integrity of the transgene locus, resveratrol synthase gene expression and resveratrol contents in the grains of two resveratrol transgenic rice lines, Iksan515 and Iksan526. The T-DNA transformation vector contained two expression cassettes of the resveratrol synthase gene under the control of the ubiquitin promoter and the bar selection marker gene under the control of the CaMV35S promoter. Flanking sequence analysis indicated that the T-DNAs were inserted into intergenic regions of chromosome 4 for Iksan515 and chromosome 12 for Iksan526. Two T-DNAs connected in an inverted repeat structure at a single locus of the rice genome were identified by whole genome sequencing and Southern blot hybridization in both Iksan515 and Iksan526. No novel open reading frames (ORFs) around insertion sites, sequences encoding allergenic or toxic protein, or other unintended effects by T-DNA insertion were found in either case. In addition, resveratrol synthase gene expression in leaves and resveratrol detection in brown rice grains suggested the successful expression of the inserted foreign resveratrol synthase gene in two transgenic rice lines.

Flanking Sequence and Copy-Number Analysis of Transformation Events by Integrating Next-Generation Sequencing Technology with Southern Blot Hybridization

“…1A. The resveratrolproducing rice line Iksan515 has undergone one transformation event and was molecularly characterized by Qin et al (2013) as the conventional Korean rice 'Dongjin' variety modified with an inverted-repeat insertion of two copies of a transformation vector, which we confirmed shown in Fig. 1B.…”

T-DNA Inheritance Stability of Resveratrol Rice Iksan526 over Multi-generations

“…The whole genome sequencing and southern blot hybridization analysis of Iksan526 showed two T-DNAs with head-to-head inverted insertion into the chromosome 12 of Dongjin genome in the previous studies (Qin et al 2013). To illustrate the inserted T-DNA structure through generations, a long fragment of two T-DNAs (4.4-4.5kb) was amplified by LA-PCR.…”

“…LA-PCR, southern blot hybridization and RT-PCR were performed to detect T-DNA integrity structure, flanking sequence, functional elements, backbone absence and gene expression following the description in Qin et al 2013. Event-specific primers were designed for LA-PCR and RT-PCR as shown in supplementary Table 1.…”

“…Resveratrol rice breeding shared the same breeding process to the other transgenic crops which started through RS3 gene transformation, evaluation and selection, along with complete molecular characterization. Specifically, analysis of the transgenic line event Iksan526 was initiated for commercialization (Qin et al 2013).…”

“…In the previous study, the resveratrol transgenic rice line Iksan526 was identified as one intergenic insertion locus by two copies with inverted repeats conjunction structure and expected mRNA expression and resveratrol production (Qin et al 2013). However, the complex T-DNA integrity structure of inverted repeats whether it will lead to a instable inheritance from generation to generation not only for the agronomic and yield-related traits, but also T-DNA integrity structure and gene expression, need to be proved.…”