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An optimized double T-DNA binary vector system for improved production of marker-free transgenic tobacco plants

文献类型： 外文期刊

作者： Leng, Chunxu 1 ; Sun, Bing 1 ; Liu, Zheming 1 ; Zhang, Lei 1 ; Wei, Xiaoli 1 ; Zhou, Yun 1 ; Meng, Ying 4 ; Lai, Yongcai; 1 ;

作者机构： 1.Chinese Acad Sci, Innovat Acad Seed Design, Inst Genet & Dev Biol, State Key Lab Plant Genom, Beijing 100101, Peoples R China

2.Chinese Acad Sci, Innovat Acad Seed Design, Inst Genet & Dev Biol, Natl Ctr Plant Gene Res Beijing, Beijing 100101, Peoples R China

3.Heilongjiang Acad Agr Sci, Biotechnol Res Inst, Harbin 150086, Peoples R China

4.Heilongjiang Acad Agr Sci, Inst Crop Cultivat & Tillage, Harbin 150086, Peoples R China

5.Univ Chinese Acad Sci, Beijing 100049, Peoples R China

6.Heilongjiang Acad Agr Sci, Harbin 150086, Peoples R China

关键词： Transgene; Co-transformation; Double T-DNA; Transcriptional read-through; Linkage transfer; Marker-free

期刊名称：BIOTECHNOLOGY LETTERS （ 影响因子：2.461； 五年影响因子：2.457 ）

ISSN： 0141-5492

年卷期： 2020 年 42 卷 4 期

页码：

收录情况： SCI

摘要： Objectives In the plant transformation process, marker genes play a vital role in identifying transformed cells from non-transformed cells. However, once transgenic plants have been obtained, the presence of marker genes may provoke public concern about environmental or biosafety issues. In our previous study, a double T-DNA vector system has been developed to obtain marker-free transgenic plants, but the T-DNA left border (LB) and right border (RB) of the vector showed an RB-LB-RB-LB pattern and led to high linkage integration between the selectable marker gene (SMG) and the gene of interest (GOI). To improve this double T-DNA vector system, we inverted the first T-DNA direction such that a LB-RB-RB-LB pattern resulted to avoid transcriptional read-through at the LB and the subsequent linkage transfer of the SMG and GOI. Results We separately inserted the green fluorescent protein (GFP) gene as the GOI and the neomycin phosphotransferase II (NPTII) gene as the SMG in both optimized and original vectors and carried out Agrobacterium-mediated tobacco transformation. Statistical analysis revealed that the linkage frequency was 25.6% in T-0 plants transformed with the optimized vector, which is a 42.1% decrease compared with that of the original vector (44.2%). The frequency of obtaining marker-free transgenic plants was 66.7% in T-1 plants transformed with the optimized vector, showing a 33.4% increase compared with that of the original vector (50.0%). Conclusion Our results demonstrate that the optimized double T-DNA binary vector system is a more effective, economical and time-saving approach for obtaining marker-free transgenic plants.

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