数字农科院2.0

An Efficient Modular Gateway Recombinase-Based Gene Stacking System for Generating Multi-Trait Transgenic Plants

文献类型： 外文期刊

作者： Guannan Qin;Suting Wu;Liying Zhang;Yanyao Li;Chunmei Liu;Jianghui Yu;Lihua Deng;Guoying Xiao;Zhiguo Zhang

作者机构：

关键词： Gateway recombination;Golden gate cloning;Modular;Multi-transgene stacking;Plants

期刊名称： Plants

ISSN： 2223-7747

年卷期： 2022 年 11 卷 4 期

页码：

收录情况： JCR（2021版）

摘要： Transgenic technology can transfer favorable traits regardless of reproductive isolation and is an important method in plant synthetic biology and genetic improvement. Complex metabolic pathway modification and pyramiding breeding strategies often require the introduction of multiple genes at once, but the current vector assembly systems for constructing multigene expression cassettes are not completely satisfactory. In this study, a new in vitro gene stacking system, GuanNan Stacking (GNS), was developed. Through the introduction of Type IIS restriction enzyme-mediated Golden Gate cloning, GNS allows the modular, standardized assembly of target gene expression cassettes. Because of the introduction of Gateway recombination, GNS facilitates the cloning of superlarge transgene expression cassettes, allows multiple expression cassettes to be effi-ciently assembled in a binary vector simultaneously, and is compatible with the Cre enzyme-medi-ated marker deletion mechanism. The linked dual positive-negative marker selection strategy ensures the efficient acquisition of target recombinant plasmids without prokaryotic selection markers in the T-DNA region. The host-independent negative selection marker combined with the TAC backbone ensures the cloning and transfer of large T-DNAs (>100 kb). Using the GNS system, we constructed a binary vector containing five foreign gene expression cassettes and obtained trans-genic rice carrying the target traits, proving that the method developed in this research is a powerful tool for plant metabolic engineering and compound trait transgenic breeding.

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