Effect of gene order in DNA constructs on gene expression upon integration into plant genome

M Aydın Akbudak,Vibha Srivastava

doi:10.1007/s13205-017-0729-2

M Aydın Akbudak, Vibha Srivastava

Open Access

https://doi.org/10.1007/s13205-017-0729-2

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Abstract

Several plant biotechnology applications are based on the expression of multiple genes located on a single transformation vector. The principles of stable expression of foreign genes in plant cells include integration of full-length gene fragments consisting of promoter and transcription terminator sequences, and avoiding converging orientation of the gene transcriptional direction. Therefore, investigators usually generate constructs in which genes are assembled in the same orientation. However, no specific information is available on the effect of the order in which genes should be assembled in the construct to support optimum expression of each gene upon integration in the genome. While many factors, including genomic position and the integration structure, could affect gene expression, the investigators judiciously design DNA constructs to avoid glitches. However, the gene order in a multigene assembly remains an open question. This study addressed the effect of gene order in the DNA construct on gene expression in rice using a simple design of two genes placed in two possible orders with respect to the genomic context. Transgenic rice lines containing green fluorescent protein (GFP) and β-glucuronidase (GUS) genes in two distinct orders were developed by Cre-lox-mediated site-specific integration. Gene expression analysis of transgenic lines showed that both genes were expressed at similar levels in either orientation, and different transgenic lines expressed each gene within 1–2× range. Thus, no significant effect of the gene order on gene expression was found in the transformed rice lines containing precise site-specific integrations and stable gene expression in plant cells could be obtained with altered gene orders. Therefore, gene orientation and integration structures are more important factors governing gene expression than gene orders in the genomic context.

Highlights

Plant cell transformation involves integration of single or multiple gene(s) to express the encoded proteins
When combining genes in a binary vector, the gene order is determined by the T-DNA borders, right border (RB) and left border (LB), since the transfer of T-DNA starts at RB, and most T-DNA fragments are intact at RB but contain variable lengths at the LB end
The gene order effect was analyzed through site-specific integration of green fluorescent protein (GFP) and GUS genes placed in two possible orders at the T5 locus through Cre-lox-mediated sitespecific integration (Fig. 1a)

Summary

Introduction

Plant cell transformation involves integration of single or multiple gene(s) to express the encoded proteins. Designing DNA constructs consisting of multiple genes is fundamentally tied to the success of plant transformation projects. When assembling two or more genes, investigators face the question of the order in which the genes should be assembled to obtain optimum expression upon integration into plant cells. This basic part of the construct design is guided by the principles of stable gene expression in transformed plant cells as the goal of the experiment is mostly to express the gene(s) at high levels. Tail-to-tail orientation, i.e., two genes having converging transcriptional direction are not desired as

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Results and discussion

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Materials and methods

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