A new vector for efficient gene targeting to the pyrG locus in Aspergillus niger

Abstract

Background: The possibility for efficient gene targeting for the controlled integration of DNA constructs is an important tool in fungal genetics.

Findings: In this study, we report a new targeting vector based on the pyrG marker in Aspergillus niger. The DNA sequence to be targeted is surrounded by two fragments of the pyrG gene to allow homologous recombination of the recombinant DNA at the pyrG locus. The 5' end of the targeting cassette contains a non-functional truncated pyrG open reading frame (first 112 bases deleted) and the 3' untranslated region (3' UTR). At the 3' end, the targeting cassette consists of the 3' flanking region of the pyrG gene. A unique NotI site between the flanks allows the insertion of a gene of interest. The linearized targeting cassette is transformed to the A. niger pyrG mutant strain AB4.1 or a derivative thereof. By using a constitutively expressed luciferase reporter gene (mluc) as an example, it is shown that the targeting system is efficient as 4 out of 6 (67%) AB4.1 transformants and 51 out of 66 (77%) MA169.4 (ku70^- ) transformants contained the reporter gene at the pyrG locus. A luciferase (lux) activity assay, performed with independently obtained transformants in which the mluc reporter was integrated at the pyrG locus, showed comparable and reproducible lux activities.

Conclusion: The new pyrG targeting vector is an important improvement to the existing method for gene targeting in A. niger. Although the vector is specific for A. niger, the presented design and approach is easily applicable for constructing integration vectors for other fungi.

Keywords: Luciferase activity; Promoter analysis; Reporter gene.

Figure 1

Schematic representation of integration of a reporter construct after a single crossover event using the pyrG* targeting system. This system was described previously by van Gorcom and van den Hondel [9]. Strain AB4.1 contains a base deletion at position 378 in the pyrG open reading frame indicated with a *. A circular plasmid containing the reporter gene and the mutated pyrG gene (pyrG ^BamHI) is transformed to AB4.1 and a single cross over at the pyrG locus leads to integration into the genome. Note that the entire vector is integrated in this system. The pyrG* fragment is a located on a 3.8 kb XbaI subclone and can be inserted in a vector for targeted integration.

Figure 2

Schematic representation of integration of a reporter construct after a double crossover event using the pyrG** targeting system. A) The truncated pyrG gene (-112) + 3’ UTR fragment (1255 bp) was amplified by PCR using primers ABpyrGP12f and ABpyrGP10r. The 3’ pyrG fragment (684 bp) was amplified by PCR using primers ABpyrGP11f and ABpyrGP13r. Both PCR products were digested (EcoRI-NotI for pyrG-3’ UTR and NotI-SstII for 3’ pyrG) and ligated in EcoRI-SstII opened pBluescriptSK, yielding plasmid pMA334. The mluc reporter cassette was obtained by PCR using SL1 and TtrpCP2rev-NotI as primers and pMA313 (containing PgpdA-mluc-TtrpC-AOpyrG, unpublished vector) as template. pMA334 was opened with NotI and the NotI digested Pgpd-mluc-TtrpC fragment was inserted, giving plasmid pMA349. Both plasmids have been deposited at Fungal Genetic Stock Centre. pMA349 was digested with AscI to release the complete pyrG** targeting transformation DNA. B) Integration of the pyrG** targeting construct via a double cross over at the pyrG locus.

Figure 3

Analysis of A. niger MA317 transformants containing the Pgpd-mluc reporter construct using the pyrG** targeting method. A) Southern blot analysis. Genomic DNA was restricted with EcoRI or KpnI and size fractioned by electrophoresis on a 1.0 % agarose gel. For hybridisation, ³²P-labelled pyrG probe (1255 bp, Figure 2A) or 3’ pyrG probe (684 bp, Figure 2A) were used. When digested with EcoRI and using the pyrG probe (upper panel), a signal of 9.0 kb corresponds with the wild-type pyrG locus, while a signal of 4.9 kb corresponds with integration of the Pgpd-mluc cassette at the pyrG locus. When digested with KpnI and using the 3’ pyrG probe (lower panel), a signal of 3.3 kb corresponds with the wild-type pyrG locus, while a signal of 4.8 kb corresponds with integration of the Pgpd-mluc cassette at the pyrG locus. Strains MA317.1 and MA317.3 have the wild-type pyrG locus, while strains MA317.2 and MA317.4-6 contain the Pgpd-mluc cassette at the pyrG locus. B) Lux activity assay. The lux activity assay described by Meyer et al. [12] has been slightly modified. Briefly, 100 μL of 2 x Minimal Medium [5] with 0.006 % yeast extract (w/v), 76 μL deionized water, 4 μL 25 mM luciferin (Promega, E1605) and 20 μL spore suspension (7.5*10⁵ spores/mL) was pipetted together (in triplicate) in a well of a white, clear bottom, 96 wells plate (Greiner Bio-one, ref 655095) and incubated for 24 hours at 30 °C in the EnSpire Multiplate Reader (Perkin) with continuous measuring of lux and OD. Lux activities after 16 h of incubation are shown here. Strains MA317.1 and MA317.3 have no lux activity, while strains MA317.2 and MA317.4-6 show comparable lux activities.