scispace - formally typeset
Open AccessJournal ArticleDOI

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

TLDR
The new vectors designed for USER Friendly cloning provided a fast reliable method to construct vectors for targeted gene manipulations in fungi.
Abstract
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. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene. Here, 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. The new vectors designed for USER Friendly cloning provided a fast reliable method to construct vectors for targeted gene manipulations in fungi.

read more

Content maybe subject to copyright    Report

Citations
More filters
Patent

Devices and methods for oligonucleic acid library synthesis

TL;DR: In this paper, devices and methods for de novo synthesis of large and highly accurate libraries of oligonucleic acids are provided, which include structures having a main channel and micro-channel, where the micro-channels have a high surface area to volume ratio.
Journal ArticleDOI

Elaborated regulation of griseofulvin biosynthesis in Penicillium griseofulvum and its role on conidiation and virulence

TL;DR: The analysis of gsfR1 promoter revealed that the regulation of griseofulvin biosynthesis is also controlled by global regulators in response to many environmental stimuli, such as carbon and nitrogen.
Journal ArticleDOI

Activation of biosynthetic gene clusters by the global transcriptional regulator TRI6 in Fusarium graminearum.

TL;DR: The results showed that the trichothecene genes are under direct control, while the gramillin gene cluster is indirectly controlled by TRI6 through its interaction with the pathway‐specific transcription factor GRA2.
Journal ArticleDOI

An efficient procedure for marker-free mutagenesis of S. coelicolor by site-specific recombination for secondary metabolite overproduction.

TL;DR: Using this method, part of the calcium-dependent antibiotic and actinorhodin biosynthetic gene clusters were deleted, and the rrdA encoding RrdA, a negative regulator of Red production, was also deleted and the final prodiginine production of the engineered strain was over five times that of the wild-type strain.
Journal ArticleDOI

The loss of the inducible Aspergillus carbonarius MFS transporter MfsA leads to ochratoxin A overproduction.

TL;DR: Phenotypical analysis of ΔmfsA deletion mutants revealed that the loss of mfsA leads to a slight growth reduction and increased OTA production, and it is hypothesized that MfsA could be a stress response transporter whose disruption could cause an increase in oxidative stress together with a stimulation of mycotoxin production.
References
More filters
Journal ArticleDOI

A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions

TL;DR: These procedures, which can circumvent the need for large-scale phage or plasmid growths, preparative gel-electrophoresis and the screening of molecular clones, can facilitate the rapid study of sequence-specific interactions of proteins and DNA.
Journal ArticleDOI

Fungal secondary metabolism — from biochemistry to genomics

TL;DR: Questions are addressed, including which evolutionary pressures led to gene clustering, why closely related species produce different profiles of secondary metabolites, and whether fungal genomics will accelerate the discovery of new pharmacologically active natural products.
Journal ArticleDOI

Ligation-independent cloning of PCR products (LIC-PCR)

TL;DR: A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones, and the procedure is applied for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.
Journal ArticleDOI

A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors.

TL;DR: This study describes a method for rapidly creating knockout mutants in which it makes use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics.
Journal ArticleDOI

Agrobacterium tumefaciens-mediated transformation of filamentous fungi.

TL;DR: It is reported that A. tumefaciens can also transfer its T-DNA efficiently to the filamentous fungus Aspergillus awamori, demonstrating DNA transfer between a prokaryote and a filamentous fungi.
Related Papers (5)