PURIFICATION, CHARACTERISATION AND CLONING OF -1,3 GLUCANASE GENE FROM Trichoderma harzinum

usarium wilt, a vascular disease caused by several soil borne fungal pathogens are wide spread and serious in many crops cultivated in different soil types. Wilt pathogens such as Fusarium solani and Fusarium oxysporium are reported to attack plant roots causing serious losses in seed germination and plant stand as well (Srivastava et al., 2014). Traditionally, this disease is controlled by the application of synthetic fungicides. But the indiscriminate use of fungicides resulted in the accumulation of residual toxicity, environmental pollution and altered the biological balance in the soil by over killing the non-targeted microorganisms. Increase awareness of fungicide-related hazards has emphasized the need of adapting biological methods as an alternative disease control method. Fungal species belonging to the genus Trichoderma (Ascomycota, Hypocreales) are widely spread in the soil and plant root ecosystems. Many Trichoderma species are effective biological control agents against a range of crop diseases (Abo-Elyousr et al., 2014). The capability of Trichoderma spp. to suppress plant diseases usually is attributed to their direct antagonistic effects on the fungal pathogen, and especially their ability to produce lytic enzymes (Benítez et al., 2004). As chitinase and β1, 3-glucanase are two main hydrolytic enzymes associated with fungal cell wall lysis (Pandey et al., 2014). In the last few years, commercially available Trichoderma products (biopesticides, biofertilizers and soil amendments) have been used as part of environmental friendly protocols to defend crops against plant pathogenic organisms and to increase yields (Mukherjee et al., 2012). The aim of the present study was to amplify and clone the β 1,3 glucanase gene (gn13.1) from Trichoderma harzianum and investigate its physical characteristics.

usarium wilt, a vascular disease caused by several soil borne fungal pathogens are wide spread and serious in many crops cultivated in different soil types.Wilt pathogens such as Fusarium solani and Fusarium oxysporium are reported to attack plant roots causing serious losses in seed germination and plant stand as well (Srivastava et al., 2014).Traditionally, this disease is controlled by the application of synthetic fungicides.But the indiscriminate use of fungicides resulted in the accumulation of residual toxicity, environmental pollution and altered the biological balance in the soil by over killing the non-targeted microorganisms.Increase awareness of fungicide-related hazards has emphasized the need of adapting biological methods as an alternative disease control method.Fungal species belonging to the genus Trichoderma (Ascomycota, Hypocreales) are widely spread in the soil and plant root ecosystems.Many Trichoderma species are effective biological control agents against a range of crop diseases (Abo-Elyousr et al., 2014).The capability of Trichoderma spp. to suppress plant diseases usually is attributed to their direct antagonistic effects on the fungal pathogen, and especially their ability to produce lytic enzymes (Benítez et al., 2004).As chitinase and β-1, 3-glucanase are two main hydrolytic enzymes associated with fungal cell wall lysis (Pandey et al., 2014).In the last few years, commercially available Trichoderma products (biopesticides, biofertilizers and soil amendments) have been used as part of environmental friendly protocols to defend crops against plant pathogenic organisms and to increase yields (Mukherjee et al., 2012).The aim of the present study was to amplify and clone the β 1,3 glucanase gene (gn13.1)from Trichoderma harzianum and investigate its physical characteristics.

Fungal isolates
Three isolates of Trichoderma sp.(T.harzianum, T. viridi and T. longibrachiatum), which were kindly provided by Microorganisms Identification and Evaluating Pesticides Unit, Agriculture Research Centre, Giza, Egypt, were used in this study as bioagents.Pathogenic fungal isolates were obtained from disease of legumes crops Department, Agriculture Research Centre, Giza, Egypt.All of the fungal isolates were maintained on potato F dextrose agar (PDA) medium for further use.

Dual culture tests (Antagonistic activity)
The antagonistic potentialities of the Trichoderma isolates were determined against Fusarium sp by dual culture technique described by Morton and Stroube (1955).About 7-days old culture, mycelial disc (5 mm) from each of the Trichoderma isolates and test pathogens were placed on the same plate opposite to each other equidistant from the periphery and then incubated at 25C.During 5 days of the incubation period, radial growth of pathogen was recorded and inhibition percentage was calculated in relation with control according to the following formula: For enzyme production, T. harzianum isolate was grown in 100 ml liquid mineral synthetic medium (MSM) containing: MgSO 4 •7H 2 O, 0.2; K 2 HPO 4 , 0.9; KCl, 0.2; NH 4 NO 3 , 1.0; FeSO 4 •7H 2 O, 0.002; MnSO 4 , 0.002 and ZnSO 4 , 0.002 (in g l -1 ), and supplemented with 0.1% cell walls of FOL to induce cell wall enzymes production, or 0.1% glucose as control (Mondejar et al., 2011).The cultures were grown at 150 rpm on a rotary shaker for 6 days at 25C.The mycelia were harvested by filtration through Whatman No. l filter paper and the filtrate centrifuged at 4C for 10 min at 5000xg.The supernatant was decanted and stored at −20C until used to assay enzyme activity (El-Katatny et al., 2000).
β-1,3 Glucanase activity was determined by a colorimetric method (Burner, 1964).The amount of reducing sugar released from laminarin (Sigma Chemical Co.St. Louis, MO) was measured.The standard assay contained 10 µl of the crude enzyme solution and 90 µl of 5mg/ml laminarin in 0.1M sodium acetate buffer pH 5.0.After incubation at 40C with gentle shaking for 10 min, the reaction was stopped by boiling for 5min and 0.2 ml of 1% dinitro salicylate (DNS) and 0.2 ml of sodium acetate buffer were added and boiled for another 5min, then placed in an ice bath and 0.9 ml distilled H 2 O was added.The optical absorption was measured at 540 nm.The amount of reducing sugar released was calculated from a standard curve prepared with glucose and the glucanase activity was expressed in units (µmol glucose equivalent/min).Proteins concentrations were determined by the method of Bradford (1976) and bovine serum albumin was used as standard.

Purification of β 1-3 glucanase
A supernatant of the T. harzianum

Characterization of β 1-3 glucanase
The effect of temperature and pH on enzyme activity was determined.The standard assay conditions were used for 30 min at temperatures between 20 and 90C.The pH of the reaction mixture was varied between 3 and 11 using 50 mM buffer (sodium acetate, pH 3.0-6.9,Tris-HCl, pH 7.0-8.9;and glycine buffer, pH 9.0-11.0).

Sodium dodecyle sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE was performed in 12.5% polyacrylamide gels according to the method of Laemmli (1970).The separated proteins were stained with Coomassie Brilliant Blue R-250 (Fluka, Switzerland) and their molecular weights were determined by comparison with low range molecular weight markers (Bio-Rad).

Isolation of total DNA from Trichoderma harzianum isolates
DNA was extracted from 50 mg of mycelium using Qiagen Kit for DNA extraction.The extracted DNA was dissolved in 100 µl of elution buffer.The concentration and purity of the obtained DNA was determined by using "Gen qunta" System-Pharmacia Bio-Tech.The purity of the DNA was between 90-97%.
Concentration was adjusted at 50 ng/µl using TE buffer pH 8.0.

Isolation of β 1-3 glucanase gene
PCR amplification was carried out using glu forward and reverse specific primers; A synthetic oligonucleotide primer based on the sequence of an internal peptide was designed to clone the cDNA corresponding to BGN13.1.The amplification reaction includes approximately 50 ng of fungal genomic DNA as template, one unit of Taq DNA polymerase, 0.2 mM each dNTPase, 1× PCR buffer, 3 mM Mgcl 2 and 10 pmol of each primer in total volume 25 µl, using T-gradient thermal cycler (Biometra).PCR was carried out for 35 cycles (94C, 1 min; 56C, 1 min; 72C, 3.5 min) followed by a 10 min extension at 72C.The amplified product were separated on 1.5% agarose gel at 100 constant volt to check for product size and purity, using 1× TBE buffer followed by staining in ethidium bromide solution (1 µg/ml) and determine with UV transilluminator.Gene clean purification system (QiAquick Gel Extraction Kit, Qiagen) was used to purify DNA fragment from the agarose gel according to manufacture procedure.

Cloning of β 1-3 glucanase gene
The purified DNA was ligated into pGEM-T-Easy vector (system 1) (Promega, Madison, WI, USA).The ligation mixture transformed into competent JM109 E. coli cells (Promega USA) with subsequent ampicillin selection.QiA prep R Miniprep Kit (Qiagene) was used to isolate pure super-coiled plasmid DNA with high yields (15 µl) according to instruction manual.Validation of cloning took place by PCR to select the transformed colonies with recombinant pGEM -T-Easy vector by using specific glu forward and reverse primers as well as the universal primers (M13 forward and reverse).The amplified product was separated on 1.5% agarose gel at 75 constant volt to check for product size and purity, using 1×TBE buffer followed by staining in ethidium bromide solution (1 µg/ml) and was determined by UV transilluminator.

Nucleotide sequencing of β 1-3 glucanase
Partial nucleotide sequence of β 1-3 glucanase gene was sequenced by ABI PRISM TM 310 genetic analyzer by using dye-primer and dye terminator method at Gene Link DNA Sequencing service, New York, USA and Gene Analysing Unite.The resulting sequences gene was then compared to published sequences in the Gene Bank.

RESULTS AND DISCUSSION
The main objective of the present study was to identify the most promising Trichoderma isolate for the management of root rot.Therefore, dual culture technique was carried out for studying the interaction between Trichoderma species viz.T. harzianum, T. viride and T. longibrachiatum with Fusarium oxysporium.
Different species of Trichoderma with respect to suppression of mycelial growth of the test pathogen was recorded.It is evident from the data that T. harzianum suppressed the radial growth of Fusarium significantly (79.30%) followed by T. viride (68.5%) and T. longibrachiatum (44.3%).As shown in Fig. (1) The promising antagonistic activity of the isolates T. harzianum, T. viride and T. longibrachiatum on dual culture may be due to mycoparasitism.The ability of Trichoderma to inhibit the growth of plant pathogens like Fusarium sp. has been reported by Srivastava et al. (2014).The antagonistic action of Trichoderma species against phytopathogenic fungi might due to secretion of extracellular hydrolytic enzymes (Schirmbock, 1994).The present finding was also supported by several workers (Obaiue and Oti, 2007).Singh et al. (2013) also revealed that 30 isolates of Trichoderma collected from various districts of U.P. were found highly antagonist against three of the tested pathogens (Fusarioum oxysporium f. sp.udum, Fusarioum oxysporium f. sp.cicero, Fusarioum oxysporium f. sp.lenties).
β-1,3-glucanase has been purified from many plant species, usually by multistep procedures employing ion exchange columns followed by gel filtration.There is increasing evidence that they play important roles in mycoparasitism (Kauffmann et al., 1987;Kurosaki et al., 1991).In the present study, it was found that when the dialyzed protein solution, obtained by ammonium sulphate precipitation of the culture supernatant was loaded on DEAE-Sephacel column, the three major peaks with 11.5 purification fold and 23.7% recovery were detected (Table 1 and Fig. 2A).
As the eluted peaks of high Glucanase activity were subsequently submitted to further purification in Sephadex G-100, single activity peak was observed with final enzyme activity of 21 U mL -1 , specific activity of 4 U mg -1 pro-tein, purification fold 20 and recovery 11.9% of the crude preparation (Fig. 2B).The apparent molecular mass of the purified Glucanase was estimated to be 30 kDa by SDS-PAGE (Fig. 3).The molecular masses of β-1,3-glucanases produced by Trichoderma appear to vary considerably, not only between organisms, but also within the same species.Molecular masses of exo-β 1,3-glucanases from T. harzianum T-Y were in a high range of 75 kDa (Ramot et al., 2000), while in the range of 29, 31 and 40 kDa β 1,3glucanase also have been isolated and characterized (Noronha and Ulhoa, 2000).The optimal pH for the enzyme activity was found to be 7.5 (Fig. 4) meanwhile Pitson et al. (1993) found the most optimal activities of fungal β 1,3-glucanases are usually in the range of 4.0 and 7.0.The optimum temperature for the enzyme activity was found to be 55C (Figure 5), and it is in agreement with exo-β-1,3glucanases from T. harzianum TC (Noronha and Ulhoa, 2000).

Detection of β 1-3 glucanase gene from T. harzianum
In this study, we isolated βglucanase gene (bgn13.1)from T. harzinum which had previously exhibited the highest antagonistic effect.To isolate the bgn13.1 gene, the oligonucleotide primers were designed based on the related DNA sequences available in the GenBank database.PCR amplification that was performed on the genomic DNA generating a specific band of approximately 600 bp.(Fig. 6).The primer pair specif-ic to β 1,3 glucanase gene was used in order to amplify a PCR product of about 600 bp from the sample which is agreement with Shinya et al. (2006).It has been suggested that β-1,3-glucanases contribute to the mycoparasitic activities of several fungal species by facilitating penetration through host cell wall structures (Lorito et al. 1994).
The strategy used for cloning of β 1-3 glucanase into E. coli was based on a direct gene cloning of the generated DNA fragments after gene clean using QiAquick Gel Extraction Kit (Qiagene) eluted into 50 µl of autoclaved deionized water and quantified by spectrophotometer and gel electrophoresis.The DNA concentration was checked by measuring the OD at 260/280 nm.The eluted DNA was ligated into prokaryotic expression pGEM-T-Easy-vector.The ligation reaction was transformed into competent E. coli JM109.The obtained ampicillin resistant white colonies containing recombinant plasmid were further selected for isolation of DNA plasmids containing the β 1,3 glucanae gene by plasmid minipreparation using Qiaprep ® miniprep kit (Qiagene).
One ampicillin resistant colony, named T1glu, containing recombinant plasmids was validated by PCR to confirm the presence of β 1,3 glucanase gene in the right orientation.Plasmid was amplified using glu forward and reverse primers as well as forward and reverse of universal primers M13.The results showed amplified product of 1500 bp by M13 primers, where amplified product was 600 bp by the specific primers as shown in Fig. (7).
T1Glu clone was sequenced using forward primer used in PCR technique.Partial nucleotide sequence of β,1-3 glucanase from T. harzianum was performed on an applied Biosystems 310 genetic analyser (Applied Biosystems) using Big Dye terminator cycle sequencing ready reaction mix according to manufacturer's instruction.The obtained sequence was aligned to published β 1,3 glucanase in Gene bank using DNAMAN V 5.2.9 package.Madison.Wisconsin, USA.Comparison of partial nucleotide sequence of T1Glu showed 97% sequence homology with other published sequence of T. harzianum mRNA for endo1,4 glucanase under the accession number X84085.1 and 96% sequence homology with the other sequences Trichoderma viride clone T1#36endo1,3(4) -glucanase mRNA, complete cds (Accession no.Kj603460) and Trichoderma viride -,3glucanase (glu) gene, complete cds (Accession no.EF176582), as shown in Fig. (8).
The most pathogenesis related proteins have damaging action on the structure of parasite PR1 and PR5 interact with the plasma membrane.Whereas β-1,3 glucanase (PR2) and attack β 1,3 glucans which is component of the cell walls in most higher fungi such as reported by Odjakova and Hadjiivanova (2001).Most of these proteins have been shown to exhibit antifungal activity in vitro (Ponstein et al., 1994).
With regard to the importance of yield losses due to the contribution of fungal diseases, some research has been conducted to develop transgenic crop plants that have increased expression levels of pathogen related (PR) protein genes in hopes of producing fungal disease resistant varieties (Lawrence et al., 2000;Vleeshouwers et al., 2000;Gau et al., 2004).Our study showed that T. harzianum isolate is a high glucanase enzyme activity source, from which Trichoderma glucanase gene, bgn13.1, was isolated and amplified.There are several reports indicating transgenic plants expressing bgn13.1 gene have shown enhanced fungal disease resistance in different species including apple (Bolar et al., 2000(Bolar et al., & 2001)), creeping bent grass (Wang et al., 2003), and rice (Liu et al., 2004).This fragment was sub cloned in order to transfer it into plant vector.Various strategies have been suggested to utilize genes encoding cell wall-degrading enzymes of mycoparasitic fungi.First, superior fungal strains that exhibit greater biocontrol activities have been produced by genetic transformation, (Migheli et al., 1998;Limon et al., 1999).Second, transgenic plants that exhibit increased resistance to fungal pathogens have been obtained by introducing genes of fungal origin (Lorito et al., 1998;Bolar et al., 2000).Finally, expression of these genes in heterologous hosts may yield enzyme production at a commercially reasonable scale (Margolles et al., 1996).This work is still in progress to reach the ultimate goal of this study.In order to produce transgenic plants with these defense genes.

SUMMARY
Dual culture technique was used to evaluate the effect of three species of Trichoderma that showed a potential control of Fusarium oxysporium.Trichoderma harzianum showed maximum growth inhibition (79.3%) followed by Trichoderma viridi (68.5%) and Trichoderma longibrachiatum (44.3%).β-1,3-glucanases was purified from Trichoderma harzianum to homogeneity by ion exchange chromatography on DEAE-Sephacel and gel filtration on Sephadex G100.A typical procedure provided 20-fold purification with 11.9% yield.The apparent molecular mass was 30 kD and it was active on a broad pH range, however the maximal activity was detected at pH 7.5.The optimum temperature of the β-1,3-glucanase was 55C.
Polymerase chain reaction (PCR) was used to amplify a fragment about 600 bp from β-1,3 gluanase gene using specific glu forward and reverse primers.The eluted DNA was ligated into pGEM-T-Easy vector and transformed into competent E. coli JM109.White transformed colony, named T1glu, containing recombinant plasmid was validated by PCR using both glu forward and reverse and M13 forward and reverse primers to confirm the presence of β 1,3 glucanase gene insert in right orientation whereas, the fragment amplified with glu forward and glu reverse primers was 600 bp.Partial sequence of the amplified DNA fragment showed 97% sequence homology with the other published sequences.

L=
of the radial growth of the pathogen towards opposite side in control plate T = radius of the radial growth of the pathogen towards the opponent antagonist in test plate β 1-3 glucanase assay isolate was obtained after low speed cen-trifugation.All purification steps were carried out at 4C.Proteins were precipitated from the supernatant with 80%saturation of ammonium sulphate and centrifuged at 9000xg for 15 min.The pellet was dissolved in 50 mM Tris-HCl buffer, pH 7.5 and dialyzed in the same buffer.The enzyme solution was loaded onto a (2.5 cm × 25 cm) column with DEAE-Sephacel (Pharmacia) equilibrated with 50 mM Tris-HCl buffer, pH 7.5 at a flow rate of 18ml/h.The column was washed with 390ml of the same buffer and the proteins were eluted with 420ml of a linear 0-0.5MNaCl gradient collecting 3 ml fractions.Fractions containing glucanase were pooled, dialyzed against the same buffer and concentrated.The concentrated enzyme was applied to a Sephadex G-100 (Pharmacia, 1.3 cm × 60 cm) column equilibrated with 50mM Tris-HCl buffer, pH 7.5 and eluted with the same buffer at a flow rate of 6 ml/h collecting 1ml fractions.The active fractions were pooled, concentrated and then stored at 4C.