Dual Reproductive Cell-Specific Promoter-Mediated Split-Cre/LoxP System Suitable for Exogenous Gene Deletion in Hybrid Progeny of Transgenic Arabidopsis.

Chen Yang,Changzheng Xu,Keming Luo,Jia Ge,Xiaokang Fu

doi:10.3390/ijms22105080

Chen Yang, Changzheng Xu + Show 3 more

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https://doi.org/10.3390/ijms22105080

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Abstract

Genetically modified (GM) crops possess some superior characteristics, such as high yield and insect resistance, but their biosafety has aroused broad public concern. Some genetic engineering technologies have recently been proposed to remove exogenous genes from GM crops. Few approaches have been applied to maintain advantageous traits, but excising exogenous genes in seeds or fruits from these hybrid crops has led to the generation of harvested food without exogenous genes. In a previous study, split-Cre mediated by split intein could recombine its structure and restore recombination activity in hybrid plants. In the current study, the recombination efficiency of split-Cre under the control of ovule-specific or pollen-specific promoters was validated by hybridization of transgenic Arabidopsis containing the improved expression vectors. In these vectors, all exogenous genes were flanked by two loxP sites, including promoters, resistance genes, reporter genes, and split-Cre genes linked to the reporter genes via LP4/2A. A gene deletion system was designed in which NCre was driven by proDD45, and CCre was driven by proACA9 and proDLL. Transgenic lines containing NCre were used as paternal lines to hybridize with transgenic lines containing CCre. Because this hybridization method results in no co-expression of the NCre and CCre genes controlled by reproduction-specific promoters in the F1 progeny, the desirable characteristics could be retained. After self-crossing in F1 progeny, the expression level and protein activity of reporter genes were detected, and confirmed that recombination of split-Cre had occurred and the exogenous genes were partially deleted. The gene deletion efficiency represented by the quantitative measurements of GUS enzyme activity was over 59%, with the highest efficiency of 73% among variable hybrid combinations. Thus, in the present study a novel dual reproductive cell-specific promoter-mediated gene deletion system was developed that has the potential to take advantage of the merits of GM crops while alleviating biosafety concerns.

Highlights

To improve the characteristics of agricultural products, new plant breeding techniques have been developed to produce genetically modified (GM) crops with advantageous traits, such as high yield, insect resistance and abiotic stress resistance [1,2]
The recombination activity of split-Cre could be obtained when both NCre and CCre were supplied [31], and the recombination activity of split-Cre linked to split-intein was higher in the hybrid progeny of transgenic Arabidopsis when Cre recombinase was split on Asp232/Asp233 (866 bp) cleavage points [32]
All the components, including promoters, reporter genes, split-Cre genes, and other exogenous genes, were flanked by two loxP sites in the same direction, and the C-terminal (Ic) fragments of intein linked to CCre were fused with the GUS reporter gene via a linker LP4/2A [36] (Figure 1A)

Summary

Introduction

To improve the characteristics of agricultural products, new plant breeding techniques have been developed to produce genetically modified (GM) crops with advantageous traits, such as high yield, insect resistance and abiotic stress resistance [1,2]. Traditional genetic engineering techniques modify target genomes by inserting DNA elements stably, but the escape of exogenous genes from GM crops may cause damage to the original ecological environment [2,4]. This has raised public concern about the biosafety of GM crops [5,6], and many research laboratories have begun to resolve such issues by improving biotechnology [5,7]. Zinc-finger nuclease, and TALEN- and CRISPR-mediated gene editing technology can accurately edit the target gene sites of crops, but new exogenous genes can be introduced into crops [9,10,11,12]

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