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

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

doi:10.3390/plants11040488

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.

Highlights

In contrast to traditional breeding techniques such as mutagenesis and hybridization, transgenic technology can accurately transfer advantageous traits to recipient plants regardless of reproductive isolation
In the GuanNan Stacking (GNS) system, elements that are commonly used in plant genetic engineering, including promoters, coding sequences (CDSs), terminators and expression cassettes, are provided in the form to assemble them into donor vector backbones, producing entry vectors in a modular manner (Figure 1A)
A destination vector is a binary vector used for Agrobacterium-mediated plant transformation and accepts target “CARGO” sequences carried by entry vectors

Summary

Introduction

In contrast to traditional breeding techniques such as mutagenesis and hybridization, transgenic technology can accurately transfer advantageous traits to recipient plants regardless of reproductive isolation. Transgene stacking is the method of combining two or more foreign genes in the same plant. Gene stacking can be achieved by methods such as hybridization, cotransformation, retransformation, or the transformation of a single vector expressing multiple genes [2,3,4]. Single-vector transformation with multigene expression cassettes (ECs) presents advantages of timesaving and cosegregation, so it is favored by researchers [5,6]. Some technical obstacles to the implementation of this strategy remain, such as the construction, propagation, and plant transformation of large vectors carrying multiple foreign genes. A number of strategies for realizing the construction of multigene stacking vectors have been reported. Goderis et al [11] reported a modular transgene stacking system based on homing endonucleases

Methods

Results

Conclusion