Xin Li, Longyu Pan, Dongling Bi, Xudan Tian, Lihua Li, Zhaomeng Xu, Lanlan Wang, Xiaowei Zou, Xiaoqing Gao, Haihe Yang, Haiyan Qu, Xiang qian Zhao, Zheng jie Yuan, Haiyan He, Shaohong Qu

Front Plant Sci. 2021 Apr 20;12:644437.  doi: 10.3389/fpls.2021.644437. 

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 (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.

See: https://pubmed.ncbi.nlm.nih.gov/33959140/

Figure 1: Double-fluorescent protein-expressing Ac/Ds transposon vectors for the generation of marker-free transgenic plants. (A) pBDL11 is a double-fluorescent protein-expressing Ac/Ds universal vector for transgene reintegration. pLJ26 (Gao et al., 2015) is a single-fluorescent protein-expressing Ac/Ds universal vector used to compare the efficacy of screening transgenic progeny with pBDL11. The AscI restriction sites in pBDL11 and pLJ26 were used for insertion of the gene of interest (GOI). (B) A diagram of Ds transposition-mediated transgene reintegration. The working pattern of the double fluorescent protein-expressing Ac/Ds vector is as follows: (i) T-DNA is integrated into the plant genome during transformation; (ii) by the action of AcTPase, Ds is excised from T-DNA and reintegrated at another site in the plant genome; (iii) T1 progeny carrying reintegrated Ds elements, from which T-DNA sequences including HPT and AcTPase are removed, are generated by genetic recombination. (C) The Ac/Ds vectors pBDL23 and pBDL22, in which Ds elements harbor an RNAi cassette of the rice gene Pi21 conferring rice blast disease resistance, were constructed to generate marker-free disease-resistant transgenic rice plants.