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

文献类型： 外文期刊

第一作者： Li, Xin

作者： Li, Xin;Pan, Longyu;Bi, Dongling;Tian, Xudan;Li, Lihua;Xu, Zhaomeng;Wang, Lanlan;Zou, Xiaowei;Gao, Xiaoqing;Yang, Haihe;Qu, Haiyan;Yuan, Zhengjie;He, Haiyan;Qu, Shaohong;Pan, Longyu;Tian, Xudan;Xu, Zhaomeng;Li, Lihua;Zhao, Xiangqian

作者机构：

关键词： rice; rice blast (Magnaporthe oryzae); disease resistance gene; selection marker; Ac; Ds transposable element; marker-free transgenic plant

期刊名称：FRONTIERS IN PLANT SCIENCE （ 影响因子：4.402； 五年影响因子：5.207 ）

ISSN： 1664-462X

年卷期： 2021 年 12 卷

页码：

收录情况： SCI

摘要： 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 (Zea mays) Activator (Ac)/Dissociation (Ds) 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 Pi21 gene that negatively regulates resistance to rice blast as a GOI into the Ds element in the Ac/Ds vector and obtained marker-free T1 rice plants from 13 independent transgenic lines. Marker-free and Ds/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 Pi21 expression levels and increased resistance to rice blast. TAIL-PCR results showed that the Ds (Pi21-RNAi) transgenes in two rice lines were reintegrated in intergenic regions in the rice genome. The Ac/Ds 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.

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