Generation of Marker-Free Transgenic Rice Resistant to Rice Blast Disease Using Ac/Ds Transposon-Mediated Transgene Reintegration System

Abstract

Rice blast is one of the most serious diseases of rice and a major threat to rice production. Breeding disease-resistant rice is one of the most economical, safe, and effective measures for the control of rice blast. As a complement to traditional crop breeding, the transgenic method can avoid the time-consuming process of crosses and multi-generation selection. In this study, maize (<i>Zea mays</i>) Activator (<i>Ac</i>)/Dissociation (<i>Ds</i>) transposon vectors carrying green fluorescent protein (GFP) and red fluorescent protein (mCherry) genetic markers were used for generating marker-free transgenic rice. Double fluorescent protein-aided counterselection against the presence of T-DNA was performed together with polymerase chain reaction (PCR)-based positive selection for the gene of interest (GOI) to screen marker-free progeny. We cloned an RNAi expression cassette of the rice <i>Pi21</i> gene that negatively regulates resistance to rice blast as a GOI into the <i>Ds</i> element in the <i>Ac/Ds</i> vector and obtained marker-free T1 rice plants from 13 independent transgenic lines. Marker-free and <i>Ds</i>/GOI-homozygous rice lines were verified by PCR and Southern hybridization analysis to be completely free of transgenic markers and T-DNA sequences. qRT-PCR analysis and rice blast disease inoculation confirmed that the marker-free transgenic rice lines exhibited decreased <i>Pi21</i> expression levels and increased resistance to rice blast. TAIL-PCR results showed that the <i>Ds</i> (<i>Pi21</i>-RNAi) transgenes in two rice lines were reintegrated in intergenic regions in the rice genome. The <i>Ac/Ds</i> vector with dual fluorescent protein markers offers more reliable screening of marker-free transgenic progeny and can be utilized in the transgenic breeding of rice disease resistance and other agronomic traits.