In vivo Assembly in Escherichia coli of Transformation Vectors for Plastid Genome Engineering.

Yuyong Wu,Lili You,Meiqi Ma,Shengchun Li,Ralph Bock,Jiang Zhang,Ling Chang,Lixin Ma,Mengting Wu

doi:10.3389/fpls.2017.01454

Abstract

Plastid transformation for the expression of recombinant proteins and entire metabolic pathways has become a promising tool for plant biotechnology. However, large-scale application of this technology has been hindered by some technical bottlenecks, including lack of routine transformation protocols for agronomically important crop plants like rice or maize. Currently, there are no standard or commercial plastid transformation vectors available for the scientific community. Construction of a plastid transformation vector usually requires tedious and time-consuming cloning steps. In this study, we describe the adoption of an in vivo Escherichia coli cloning (iVEC) technology to quickly assemble a plastid transformation vector. The method enables simple and seamless build-up of a complete plastid transformation vector from five DNA fragments in a single step. The vector assembled for demonstration purposes contains an enhanced green fluorescent protein (GFP) expression cassette, in which the gfp transgene is driven by the tobacco plastid ribosomal RNA operon promoter fused to the 5′ untranslated region (UTR) from gene10 of bacteriophage T7 and the transcript-stabilizing 3′UTR from the E. coli ribosomal RNA operon rrnB. Successful transformation of the tobacco plastid genome was verified by Southern blot analysis and seed assays. High-level expression of the GFP reporter in the transplastomic plants was visualized by confocal microscopy and Coomassie staining, and GFP accumulation was ~9% of the total soluble protein. The iVEC method represents a simple and efficient approach for construction of plastid transformation vector, and offers great potential for the assembly of increasingly complex vectors for synthetic biology applications in plastids.

Highlights

Plastid transformation has become a promising approach for both basic research and plant biotechnology applications
We simultaneously introduced four DNA fragments and one linear vector backbone into E. coli cells via the in vivo Escherichia coli cloning (iVEC) method, generating a plastid transformation vector for expression of the model protein green fluorescence protein (GFP) from the tobacco plastid genome
Controlling the gfp reporter gene by the tobacco rRNA operon promoter combined with the 5′untranslated region (UTR) from gene10 of bacteriophage T7 (NtPrrn:T7g10) and the 3′UTR from the E. coli rrnB operon (TrrnB), we show that the transplastomic plants generated with the iVEC-assembled transformation vector efficiently express the transgene, reaching protein accumulation levels of 9% of the total soluble protein (TSP)

Summary

Introduction

Plastid transformation has become a promising approach for both basic research and plant biotechnology applications. In vivo Assembly of Vectors for Plastid Transformation avoiding gene silencing and position effects (Maliga, 2004; Bock, 2007, 2015; Maliga and Bock, 2011). Design and construction of suitable transformation vectors are inherent steps in plastid genome engineering. Identification of suitable combinations of the sequence elements involved in the control of plastid transgene expression (e.g., promoters, 5′ and 3′ untranslated regions) for optimized expression of the transgenes (i.e., adjustment of transgene expression to the desired level) usually requires construction of a set of different plastid transformation vectors and their test in vivo. The aforementioned cumbersome cloning procedures involved are becoming a limiting step in plastid genome engineering

Methods

Results

Conclusion