Cloning And Expression Of A Wheat β-1,3-glucanase Gene Induced By Puccinia Striiformis F.sp. Tritici In E.coli

Plantβ-1,3-glucanases are pathogenesis-related proteins which are implicated in plant defense responses against pathogen infection. The enzyme catalyzes the hydrolysis of a major structural component of the cell walls of many pathogenic fungi,β-1,3-glucan (a polymer ofβ-1,3-linked glucose residues). Although the activity ofβ-1,3-glucanase was proved to be associated with the wheat resistance to rust fungi by biochemical analysis, the role of this enzyme in resistance of wheat to stripe rust is not yet fully understood. As an initial step in understanding the roles ofβ-1,3-glucanase in the wheat-stripe rust interaction system, the cloning and expression of a wheatβ-1,3-glucanase gene induced by Puccinia striiformis f.sp. tritici were studied in this research.1. The encoding region was engineered via reverse transcription polymerase chain reaction (RT-PCR), using specific primers designed based on a wheatβ-1,3-glucanase gene full length cDNA acquired previously.2. The amplified fragments were first cloned into pGEM T-easy vector. After restriction and sequence analysis, the gene was inserted into pET-32a right behind T7 promoter and then the GLU-pET-32a recombinant was transferred to an E.coli strain BL21(DE3). After IPTG induction, the product was expressed to be a 49 kDa fusion protein.3. Under optimum inducement condition (37℃, 0.3 mmol/L IPTG, 4 h), the content of recombinant protein could reach to 19% of total cell protein tested by Gene Genius Bio Imaging System.4. The recombinant protein was expressed mainly as inclusion body in E.coli when expressed at 37℃, 50% of them could be soluble after induced 20 h by 0.01 mmol/L IPTG at 20℃.5. Triton X-100 and Urea were both used to extract the inclusion bodies. Urea appeared to be more efficient than Triton X-100, and was efficient at 5 mol/L.6. Soluble recombinant protein could be purified by HisTrap affinity columns using ?KTA HPLC systerm to 15.23% of total cell protein. The specific activity of purification products could be 0.146 U/mg, the purification factor is 73.The expression and purification system of wheatβ-1,3-glucanase in E.coli could be very...