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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

作者： Li, Xin 1 ; Pan, Longyu 1 ; Bi, Dongling 1 ; Tian, Xudan 1 ; Li, Lihua 1 ; Xu, Zhaomeng 1 ; Wang, Lanlan 1 ; Zou, Xiaowei 1 ;

作者机构： 1.Zhejiang Acad Agr Sci, Inst Virol & Biotechnol, State Key Lab Managing Biot & Chem Threats Qual &, Hangzhou, Peoples R China

2.Zhejiang Normal Univ, Coll Chem & Life Sci, Jinhua, Zhejiang, Peoples R China

3.Hunan Agr Univ, Coll Plant Protect, Changsha, Peoples R China

4.Zhejiang Acad Agr Sci, Inst Crop Sci & Nucl Technol Utilizat, Hangzhou, Peoples R China

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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