Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

Rasmus Jn Frandsen,Jens A Andersson,Henriette Giese,Matilde B Kristensen

doi:10.1186/1471-2199-9-70

Rasmus Jn Frandsen, Jens A Andersson + Show 2 more

Open Access

https://doi.org/10.1186/1471-2199-9-70

Copy DOI

Abstract

BackgroundThe rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene.ResultsHere, we present a USER Friendly cloning based technique that allows single step cloning of the two required homologous recombination sequences into different sites of a recipient vector. The advantages are: A simple experimental design, free choice of target sequence, few procedures and user convenience. The vectors are intented for Agrobacterium tumefaciens and protoplast based transformation technologies. The system has been tested by the construction of vectors for targeted replacement of 17 genes and overexpression of 12 genes in Fusarium graminearum. The results show that four fragment vectors can be constructed in a single cloning step with an average efficiency of 84% for gene replacement and 80% for targeted overexpression.ConclusionThe new vectors designed for USER Friendly cloning provided a fast reliable method to construct vectors for targeted gene manipulations in fungi.

Highlights

The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes
We have shown that Agrobacterium tumefaciens mediated transformation (ATMT) is an excellent method for site-directed genome modifications in F. graminearum, with 200 transformants per 106 spores, with 60% targeted integrations, using 2 kb homologous recombination sequences (HRS)'s [15]
The constructed pRF-HU2 and pRF-HU2E vectors allow for the simultaneous directional cloning of two inserts in a four fragment assembly with an efficiency of 84.1% and

Summary

Introduction

The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion of coding sequences with fluorescent markers such as GFP are essential for this process. The wide interest in filamentous fungi has led to the sequencing and annotation of more than 30 fungal genomes, with another 130 on the way http://www.ncbi.nlm.nih.gov/ Genomes/. This resource constitutes a huge potential for future advances in the basic understanding and industrial exploitation of fungal biology. A key limiting factor in future work will be the speed by which site-directed genome modifications, such as gene deletion, promoter replacement, fusion of coding sequences with reporter genes

Methods

Results

Conclusion