Development and Event-specific Detection of Transgenic Glyphosate-resistant Rice Expressing the G2-EPSPS Gene

Abstract

Glyphosate is a widely used herbicide, due to its broad spectrum, low cost, low toxicity, high efficiency, and non-selective characteristics. Rice farmers rarely use glyphosate as a herbicide, because the crop is sensitive to this chemical. The development of transgenic glyphosate-tolerant rice could greatly improve the economics of rice production. Here, we transformed the Pseudomonas fluorescens G2 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene G2-EPSPS, which conferred tolerance to glyphosate herbicide into a widely used japonica rice cultivar, Zhonghua 11 (ZH11), to develop two highly glyphosate-tolerant transgenic rice lines, G2-6 and G2-7, with one exogenous gene integration. Seed germination tests and glyphosate-tolerance assays of plants grown in a greenhouse showed that the two transgenic lines could greatly improve glyphosate-tolerance compared with the wild-type; The glyphosate-tolerance field test indicated that both transgenic lines could grow at concentrations of 20,000 ppm glyphosate, which is more than 20-times the recommended concentration in the field. Isolation of the flanking sequence of transgenic rice G2-6 indicated that the 5′-terminal of T-DNA was inserted into chromosome 8 of the rice genome. An event-specific PCR test system was established and the limit of detection of the primers reached five copies. Overall, the G2-EPSPS gene significantly improved glyphosate-tolerance in transgenic rice; furthermore, it is a useful candidate gene for the future development of commercial transgenic rice.

Whole-Genome Sequencing: An Effective Strategy for Insertion Information Analysis of Foreign Genes in Transgenic Plants

“…Three samples, transgenic rice G2-6, conventional rice cultivar Zhonghua 11 (ZH11), and spike-in control (ZH11-P), were used for WGS analysis. Transgenic rice G2-6 containing the G2-aroA gene was generated by Agrobacteriummediated transformation (Dong et al, 2017). ZH11 was the recipient of G2-6.…”

“…Southern blotting analysis revealed one copy of the foreign gene in the rice genome. The 3end flanking sequence of the foreign DNA fragment was not obtained by the PCR-mediated genome-walking method (Dong et al, 2017). In this study, we determined the copy number, integrity, insertion site, and flanking sequence of foreign DNA fragments and identified the presence or absence of vector backbone sequences in transgenic plants by combining WGS data and bioinformatics analysis.…”

Development and identification of transgenic rapeseed with a novel gene for glyphosate resistance

“…利用反向 PCR 的方法分离转基因的 T-DNA 插 入位点。具体程序见李杰华 [22] 的方法。第 1 轮 PCR 效果 [15,22] 。本研究使用的 I. variabilis EPSPS 是从细 菌 I. variabilis 中克隆到的一种新型的抗草甘膦基 因 [11] , 与 已 获 得 转 基 因 批 准 的 CP4-epsps 、 玉 米 EPSPS 突变体以及 GRG23 突变体中的 EPSPS 基因 的 氨 基 酸 序 列 同 源 性 分 别 只 有 27.1% 、 33.5% 和 31.5% [24] 。因此, I. variabilis EPSPS 是一种具有完全 自主知识产权的新型抗草甘膦基因 [19]…”

Construction and Transformation of OsERA1 Gene into Expression Vector and Response of Nipponbare-OsERA1 Transgenic Rice to Drought Stress

“…Penggunaan primer spesifik dapat menjamin bahwa tanaman transforman yang diperoleh benar-benar merupakan tanaman transgenik (true transgenic plant). Deteksi transgen dengan primer spesifik ini merupakan tahapan yang selalu ada di dalam perakitan tanaman transgenik (Melloul et al 2014;Lu et al 2016;Dong et al 2017).…”