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

Abstract

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 efficiently assembled in a binary vector simultaneously, and is compatible with the Cre enzyme-mediated 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 transgenic 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.

Prospects of microgreens as budding living functional food: Breeding and biofortification through OMICS and other approaches for nutritional security

“…The impact of endogenous genes could be regulated by modifying biological processes and metabolic pathways to boost carotenoids and flavanoids using various abiotic stimuli such as drought, salinity, mineral toxins, cold, temperature and RNA interference technologies ( Eapen, 2008 ). Interestingly, desirable multi-trait transgenic plant can be developed through in vitro gene stacking system: GuanNan Stacking ( Qin et al, 2022 ). However, transgenic rice have been developed by construction of binary vector and insertion of five desirable foreign genes that would be helpful for regulation of metabolic engineering and trait improvements through breeding and multiomics approaches in future ( Qin et al, 2022 ).…”

“…Interestingly, desirable multi-trait transgenic plant can be developed through in vitro gene stacking system: GuanNan Stacking ( Qin et al, 2022 ). However, transgenic rice have been developed by construction of binary vector and insertion of five desirable foreign genes that would be helpful for regulation of metabolic engineering and trait improvements through breeding and multiomics approaches in future ( Qin et al, 2022 ). Moreover, improvement of chickpea and pigeonpea have been explored through transgenic and molecular approaches ( Arya and Mishra 2022 ).…”

Harnessing Knowledge from Plant Functional Genomics and Multi-Omics for Genetic Improvement

“…Kirov et al sequenced the full-length glutenin genes ( Glu-1Ax , Glu-1Bx , and Glu-1By ) and their promoters in hexaploid triticale using cas9-targeted nanopore sequencing (nCATS) technology, disclosing the potential of the nCATS approach for sequencing target genes in large genome size plants [ 50 ]. A new in vitro gene stacking system, GuanNan Stacking (GNS), was developed by Qin et al [ 51 ]. This system allows multiple expression cassettes to be efficiently assembled in a binary vector simultaneously and is compatible with the Cre enzyme-mediated marker deletion mechanism, emphasizing this technique as a powerful tool for plant metabolic engineering and transgenic breeding of complex traits.…”

Pyramiding stacking of multigenes (PSM): a simple, flexible and efficient multigene stacking system based on Gibson assembly and gateway cloning

“…The joint systems combine the advantages of multiple stacking systems. The recently developed TGSII-UNiE system (combined nickase-based cloning and Cre/loxP recombination) and GNS system (combined Golden Gate cloning and Gateway cloning) demonstrated higher stacking efficiency and flexibility than a single stacking system (Qin et al, 2022;Zhao et al, 2022). In this study, we developed a multigene vector construction system named Pyramiding Stacking of Multigenes (PSM) with the combination of Gibson assembly and Gateway cloning.…”