biostudies-literatureQin GAgricultural Science and Technology Innovation ProgramJIEBANGGUASHUAI program of Hainan Yazhou Bay Seed LaboratoryNational Key Research and Development Program of China488https://www.ebi.ac.uk/biostudies/studies/S-EPMC8879548biostudies-literatureUnknown11(4)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 caPlants (Basel, Switzerland)An Efficient Modular Gateway Recombinase-Based Gene Stacking System for Generating Multi-Trait Transgenic Plants.PMC8879548CAAS-ZDXT2019003 and CAAS-ZDRW202109B21HJ01072020YFA0907603Li YZhang ZWu SXiao GDeng LQin GZhang LLiu CYu JfalseAn Efficient Modular Gateway Recombinase-Based Gene Stacking System for Generating Multi-Trait Transgenic Plants.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 ca2022-01-01T00:00:00Z2022 Feb2025-04-04T08:41:39.958Z2025-04-04T08:41:39.958ZS-EPMC88795483521482010.3390/plants11040488