Cloning And Expression Analyses Of A β-1, 3-glucanase Gene From Wheat

Hydrolytic enzymes such asβ-1,3-glucanases are considered to constitute part of the general array of defense genes induced by pathogen infection in higher plants.Wheat is one of the important crops in China.Fungal diseases have been one of the principal causes of damaging wheat growth and wheat yield losses,and thus effective control of fungal diseases has been one of the concerned questions by the scientists.Since human started to cultivate wheat plants,a lot of measures have been applied to control the fungal diseases,such as breeding the fungus-resistant cultivars by traditional breeding ways,the application of pesticides,and colligated ways to reduce dosage of agrochemicals and rudimental agrochemicals.But due to the limited genetic germplasm resources,the high variability of fungi and the pollution of the pesticides etc,the technology of gene engineering to breed resistant cultivars becomes one of the recent research focuses of agricultural biological technology.In this study,aβ-1,3-glucanase gene(Glu) was amplified by RT-PCR and subcloned into an expression vector pGEX,to construct pGEX-Glu for bacterial expression.A SDS-PAGE analysis indicated that the clonedβ-1,3-glucanase gene was highly expressed in bacteria after induction by IPTG.Enzymetic assays confirmed that the glucanse displayed a clearβ-1,3-glucanase activity.In vitro antifungal assay revealed that the glucanase showed s strong inhibitory activity towards a pathogenic fungus Rhizoctonia solani.The expression pattern of Glu was studied by Northern blot analysis.The results revealed that the glucanase gene expression was developmentally regulated and the Glu transcripts were detectable in both vegetative and reproductive tissues such as spikes and stems,with peaks in spikes one week before floweing.The lowest expression level was observed in spikes one week after floweing.Infection of wheat seedlings by Fusarium graminearum and Verticillium dahliae induced the Glu gene expression.There would be 4 to 6 copies of the Glu gene in wheat genome revealed in Southern blot analysis.The Glu gene and GFP gene were fused together with Glu in the N-terminus and subcloned into plant expression vector pTR.The pTR-Glu-GFP were transformed into in Arabidopsis thaliana.Some transgenic plants were obtained.The present results provide valuable data for subsequent investigation of inhibitory mechanisms of this glucanse and further use of the gene in genetic engineering of fungal resistance in plants.