A.niger has a transcriptional activator which regulates the expression of xylanolytic and some cellulolytic enzyme. The xInR gene encodes an 875 amino acid polypeptide which contains a zinc binuclear cluster domain and belongs to the superfamily of GAL4 transcription factors. The binding site for XInR is 5?-GGCTAAA-3? (confirmed in vivo and in vitro) and is a consensus sequence found in various Aspergilli and Penicillium chrysogenum.In filamentous fungi, the induction of xylanolytic genes is through a cis-acting element. The transcription factor affects the gene expression of enzymes involved in the degradation of xylan, arabinan and cellulose, including genes encoding endoxylanases (xInB and xInC) and beta-xylosidases (xInD) .
It was also found to regulate the expression of two endoglucanases; eglA and elgB. In terms of cellulolytic degradation, XInR has been shown to regulated the expression of ?- and ?-galactosidase genes (aglB and lacA) as well as cbhA and cbhB, two cellobiohydrolase genes[28, 43]. However, genes containing the XInR binding site within its promoter sequence are not automatically regulated by XInR, an example being endo-beta-1,4-glucanase A gene in A.nidulans, where gene expression was not detected in the presence of xylose.
Carbon Catabolite Repression Catabolite repression in Aspergillus is mediated by a repressor protein, CreA at the transcriptional and post-transcriptional level [45, 46]. The protein, CreA, has a zinc finger motif which allows binding to target gene promoter sites containing SYGGRG. A similar carbon catabolite repressor protein is seen in T.reesei called CRE1 and is 46% similar to CreA in A. nidulans.CreA repression occurs on glucose, xylose and other carbon sources. If A.
niger is carbon-starved, the addition of glucose to media causes transcription of creA genes within minutes but if carbon sources are low, transcription levels of creA are down-regulated by itself. At low concentration levels of xylose, XInR activates genes encoding CHs however if xylose concentration increases to above 1mM, CreA-mediated repression takes place. Nevertheless, the effect of high xylose concentration is weaker than glucose in terms of catabolite repression. CreA does not just affect the expression hemicellulolytic enzymes but also arabinose catabolic enzymes.Aim This study aims to determine the gene expression of An01g11670 (eglA), which contains a CBM domain and An03g06550 (glaA) when Aspergillus niger is grown wheat straw, model carbon source, through a carbon (straw) timeline and a no carbon timeline. By comparing the expression of these proteins with control proteins (cbhA, cbhB and yefC) through a process of RNA extraction, RT-PCR and gel electrophoresis, it will be possible to deduce the responses of these proteins under the specified conditions. Growth Conditions for Aspergillus niger Potato dextrose agar (PDA) (Oxoid), premade in house, was heated until melted. 100ml of the liquid PDA poured into 8 labelled tubes with the date and allowed to cool in an airing cupboard.
The A.niger strains used were N402 (ref) and maintained on potato dextrose agar (Oxoid). Slopes were incubated at 28oC until the cultures had conidiated and there was a thin layer of spores across the slope. Spores were resuspended in 0.
1% (v/v) Tween 20 (Sigma). The cultures were inoculated with 106 spores/ml and grown in 100ml of minimal medial (see below for recipe). The appropriate carbon source was added to a final concentration of 1 % (w/v) in 250 ml conical flasks at 28 °C and shaken at 150 r.
p.m.For Carbon Timeline The time course for the A.niger spores consisted of 48 hours of growth in 1 % (w/v) glucose media (as a carbon source) which allowed a mycelial mass to be formed.
Mycelia were removed by filtration (Nalgene and MERCK), washed with water and transferred to straw media which contained 1 % (w/v) ground wheat straw (as a carbon source) and no glucose. Incubation was continued for another 24 hours. At selected hour intervals [Glucose/Initial, 0.5, 1, 2, 3, 6, 9, 12, 24] A.niger was extracted for RNA extraction.
For the carbon timeline, Glucose was then added exogenously to a final concentration of 1 % (w/v) (S+G) and incubation continued for 6 hours. This was also extracted for RNA extraction.For No Carbon Timeline The time course consisted of growth for 48 h in 1 % (w/v) glucose media which allowed the A.niger to grow to its mycelia stage. Mycelia were harvest by filtration (Nalgene and MERCK), briefly washed with water and transferred to fresh AMM solution. Incubation was continued for another 24 hours. At selected hour intervals [Glucose/Initial, 0.5, 1, 2, 3, 6, 9, 12, 24] A.
niger was extracted for RNA extraction.Making Aspergillus Minimal Media solution This solution does not contain carbon source (wheat straw). For a litre solution: NaNO3 (Sigma), 6 g; KCl, 0.52 g; MgSO4. 7H2O, 0.52 g; KH2PO4, 1.52 g; Na2B4O7.
10H2O, 0.08 mg; CuSO4. 5H2O, 1.6 mg; FePO4.H2O, 1.6 mg; MnSO4.
4H2O, 1.6 mg; NaMoO4.2H2O, 1.6 mg. The pH of the solution was maintained at pH 6.5 (Hanna pH meter instruments). If a carbon source was added, the solution was autoclaved at 1770C for 30 minute and if glucose was added, the solution was filter sterilised using Making Straw Media for Carbon Timeline Wheat straw (Sutton Bonnington) was added to the AMM solution to a final concentration of 1% (w/v) which was then autoclaved at 1770C for 30 minutes.The wheat straw was of the cordiale variety.
It was composed of 37% cellulose and 32% hemicellulose and was ball milled using a Laboratory bill. Reverse Transcription Polymerase Chain Reaction RNA extraction A.niger spores were allowed to grow until the mycelia stage. At each time period, mycelia was extracted, frozen and ground under liquid nitrogen using a pestle and mortar. The RNA material extracted using the TRIzol reagent protocol (Invitrogen).
An additional clean-up was done using the RNEasy Mini Kit (Qiagen), following the manufacturer’s RNA Clean-up protocol, including the additional on-column DNAse digest.Finding the Gene sequence CAZy The sequence of each gene was found through Cadre-Genome website. The cDNA (without intron) sequence was exported from the website and using the primer sequence (forward and reverse), the replicated product produced (in PCR) was found. To the gene size, Primer Blast was used. cDNA synthesis For the cDNA synthesis mixture: 500ng of extracted RNA (Nanodrop Spectrometer, SLS) from each time point was added with 1µl of 50µM oligo (dt)20 (Invitrogen), 1µl of 10mM dNTP mix (10mM each of dATP, dGTP, dCTP, dTTP at neutral pH, in house) and water was added to make up to a final volume of 15µl.
The tube (Invitrogen) was heated on heat block (Grant) for 5 minutes at 65oC, placed on ice for 5 minutes then spun down (Spingene, SLS). Finally, 4µl of 5x First-strand buffer, 1µl of 0.1M DTT and 1µl of Superscript III RT (200 units/µl) was added to the tube. Using the PCR machine (Techne TC-512), the contents of the tube was heated at 50oC for 60 minutes then 70oC for 15 minutes to deactivate the enzyme.PCR For 19µl master mix: 0.5µl of dNTP mix (10mM each of dATP, dGTP, dCTP, dTTP at neutral pH, in house), 4µl of 5X Phusion HF Buffer (NEB), 0.2µl of Phusion® High-Fidelity DNA Polymerase (NEB), 0.5µl of 500nM of the appropriate forward primers (Sigma), 0.5µl of 500nM of appropriate reverse primer (Sigma) and finally 1µl of the appropriate cDNA [Glucose/Initial, 0.5, 1, 2, 3, 6, 9, 12, 24, S+G, gDNA] was added.