Detection the Effect of Cu on Transcription of glox Gene in Myceliophthora verrucosa Using RT-PCR

Reverse transcriptase PCR was used to detect the transcription of the glox gene in a local isolate of M. verrucosa, in two different growth media. The results indicated the presence of gene transcription in this strain with no effect to Cu on gene expression. This means that this fungus is suitable for use in various fields such as a bioremediation. This method depends on RNA to detect gene expression under various conditions. ــ ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ نيج خاسنتسا ىلع ساحنلا تانويا ريثأت نع فشكلا glox رطفلا يف Myceliophthora verrucosa لسلستملا يفعاضتلا لعافتلل يسكعلا خاسنتسلاا ةقيرط مادختساب


‫ﺠﻴﻥ‬ ‫ﺍﺴﺘﻨﺴﺎﺥ‬ ‫ﻋﻠﻰ‬ ‫ﺍﻟﻨﺤﺎﺱ‬ ‫ﺍﻴﻭﻨﺎﺕ‬ ‫ﺘﺄﺜﻴﺭ‬ ‫ﻋﻥ‬ ‫ﺍﻟﻜﺸﻑ‬
. glox homologous were found in human, plant pathogenic fungi and plants but not in other mammals or yeast. It was reported (Leuthner et al. 2005) that Ustilago maydis is gene produces H 2 O 2 and the membrane bound glo 1 protein is involved in filamentous growth and pathogenicity of U. maydis. However the biological role of glox in plants remains unclear, (Zhao et al., 2012).
In previous study (Khalil et al., 2013) the Iraqi local isolate Myceliophthora verrucosa was the only strain able to produce Laccase enzyme and show a high activity of this enzyme within 24 hours compared to the standard strain P. chrysosporium which need 7 days to produce the same enzyme. There is a strong relation between laccase and glox enzymes; both of them belong to ligninolytic enzymes system (LES) (Maciel et al., 2010). The LES has an important roles in biodegradation and biological function (Singh, 2006). M. verrucosa may have other enzymes and can be considered an optimum local strain that has a complete LES (Khalil et al., 2013).
glox gene expression can be detected by using reverse transcriptase polymerase chain reaction (RT-PCR). RNA can also serve as a template for PCR amplification after conversion to cDNA, RNA-PCR or RT-PCR is a modified PCR procedure designed for analyzing RNA transcript. The RT-PCR is more sensitive than other methods used for DNA analysis especially in response to medium components effect (Bridge et al., 1998). Copper has been reported to be a strong enzyme inducer in several species, it is known that Cu induces both lactase transcription and activity and the increase in activity is proportional to the amount of copper added (Levin et al., 2002).
Due to the good ability of the local strain of the fungus M. verrucosa to produce laccase enzyme (Khalil et al., 2013), this study was designed to detect another enzyme, the glox gene transcript by using the RT-PCR technique and study the effect of the copper in glox gene transcription.

MATERIALS AND METHODS Fungal strain:
M. verrucosa was previously isolated from plastic garbage and identified by using the rDNA method (Khalil et al., 2014).

Preparation of the fungal spore inocula:
The spores were harvested in distilled water from a 7 days old fungal plate and then Tween 20 was added to a concentration of 0.01% then mixed by vortex and used to inoculate flasks of liquid media (Probha et al., 2013).

Medium of growth:
Two media differing in the presence and absence of the Cu ++ ion were used in this study, Submerged cultivation of the fungus was carried out on a rotary shaker at 140 rpm and 30 ° C in 250 ml flasks which contained 200 ml of standard medium g/l : glucose 15 ; pepton 3 ; KH 2 PO 4 0.8 ; K 2 HPO 4 0.4 ; MgSO 4 .7H 2 O 0.5 ; CaSO 4 .5H 2 O 0.125 ; CuSO 4 .5H 2 O 0.125 ; yeast extract 4. The pH was 5.5 and incubation was for 10 days, (Kenkebashvili et al., 2012).

Isolation of RNA
At the end of the incubation period, the fungal mycelium was filtered through sterilized muslin and ground in liquid nitrogen to break open all hyphal constituents. The RNA was extracted by using the Micro RNA isolation kit (miRNA) from Omega, and all other reagents were provided by BioLab, England, and the work was done according to the company protocols. The purity and concentration were measured by using Biodrop. The RNA concentration was adjusted at 300 ng/µl to be equal for all samples and then diluted for RT-PCR reaction.

M-MULV Reverse Transcriptase:
Moloney Murine Leukemia Virus (M-MULV) a Reverse Transcriptase (RT) is an RNA-and DNAdependent DNA polymerase, the enzyme possesses a ribonuclease H activity specific to RNA in RNA-DNA hybrids, M-MULV RT incorporates modified nucleotides, E.coli cells with a cloned fragment of the pol gene encoding Moloney Murine Leukemia Virus reverse transcriptase (Sambrook and Russell, 2001).

PCR Primers:
Three set of primers differ in design methods, the first set designed according to complementary DNA, while the second primers set was designed according to genomic DNA and the third set designed by using National Centre of Biotechnology Information (NCBI) web site. All these Primers were used to amplify cDNA and these were : 1. F: 5'-GCGAATTCATGTTGTCGCTGCTAGCCGTAGT-3 'R: 3'-CGTACGTACTCCAGGGTCGGCGGAGGGT-5' (Son et al., 2012).

First strand cDNA synthesis:
The cDNA was synthesized by using the reaction mixture shown in (Table 1) in an eppendorf tube. The RNA denatured for 5 minutes at 70 °C and span briefly then 10 µl of the M-MULV reaction mix and 2 µl of the M-MULV enzyme mix were added, the reaction mixture incubated at 42 °C for one hour and the enzyme inactivated at 80 °C for 5 minutes. The reaction mixture was diluted to 50 µl with 30 µl H 2 O for PCR reaction, (Jalali et al., 2011).

RT-PCR reaction cDNA template preparation:
For each cDNA reaction, series (1:5, 1:10, 1:100) dilution of cDNA into RNase-free dH 2 O was made,working cDNA dilution depends on abundance of transcript so it may be necessary to make a dilution to determine optimum cDNA input dilution comparing with undiluted cDNA.
Primers were used to set the RT-PCR cDNA template in the reaction made as shown in (Table 2): The thermocycler programs consisted of an initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation (95°C for 1 min), and to avoid annealing temperature failure, the gradient primer annealing temperature was used in range 55 ±10 °C (50-60 °C) for 2 min and polymerization (72°C for 2 min). Final amplification was at 72°C for 10 min. (Khalil et al., 2014;Alrawi and Alsanjary, 2009).
The PCR products (glox gene bands) were separated on 2% agarose gel, stained with ethidium bromide and bands visualized were by gel documentation.

RESULTS AND DISCUSSION
The RNA extraction by miRNA isolation kit showed a high purity and concentration as shown in (Table 3). Pure RNA has an A260/A280 ratio of 1.9-2.1 (Nielson, 2011). The results showed that the first set of primers only gave pure bands for the most diluted cDNA samples and for both mycelium grown in standard medium or medium lacking Cu ++ (Fig. 1). The results also showed a pure and strong bands at 75 bp in 2, 3, 4 wells (standard medium) and 5, 7, 8 wells (medium without Cu + ), and the bands in 1, 6 were a week due to high concentration of cDNA ( because the difference in RNA concentration) which used undiluted cDNA, and the strong bands are from the high dilution ratio. The high concentration of cDNA leads to the reaction component may interact with primers annealing, and react week bands and making a series dilution can avoid the interaction. The pure bands indicates that the local strain M. verrucosa contains the glox gene. Its expression will not be affected by absence of Cu + . This enzyme is a member in ligninolytic enzyme system in white rot fungi, (Janusze et al., 2013).

Medium lacking Standard
The results also showed that there were no amplified cDNA by using the second and third primer sets. This may due to the primers design where these primers may contain intron / exon for genomic DNA while the complementary DNA doesn`t contain an Introns just exons and because of this the primers should be designed for exon only as in the first primer set (Fig. 2), (Zhong, 2003)

Fig. 2: primers design for reverse transcriptase PCR (Zhong, 2003)
Also the results indicated that the glox gene expression didn`t need the Cu + . This ion may be needed for the activation glox enzyme. However, how this happens is not clear. The effect of Cu + is it Cu + could be a component of the active site of the enzyme or simply an effector of the enzyme (Kersten, 1990).
The presence of the glox gene and enzyme in the local strain M. verrucosa point to the possibility of using this strain in bioremediation. The glox and Laccase (detected in a previous study) are very important enzymes in bioremediation; they have the ability to break the most strong bond between lignin atoms (Khalil et al., 2013).
The RT-PCR has become one of the most widely applied techniques in biomedical research (O`Connell, 2002). The ease with which the technique permits specific mRNA to be detected and quantified has been a major asset in the molecular investigation of disease pathogenesis (Nielson, 2011) . Disease-related imbalances in the expression of specific mRNAs can be sensitive parameter and a very useful tool for detecting variations in the expression of many genes under various condition, (O`Connell, 2002). In many cases the RT-PCR is a very important tool to detect gene expression in various condition especially with these induced during secondary metabolism under nutrient-limiting culture conditions and they cannot be detected by genomic DNA PCR (Singh, 2006).