PJB-2023-577
Fatima Shahid
Abstract
For human survival, crop cultivation is vital to accomplish their dietary requirements. However, crops plants are continuously at the risk of attack by various pathogenic fungi. To protect themselves, plants produce different pathogenesis related (PR) proteins and phytoalexins. β-1,3-glucanases, the antifungal proteins belonging to PR proteins, are induced in response to a pathogenic attack therefore, functioning as a plant defense system. Wheat (Triticum aestivum) is the major staple food grain of Pakistan and is one of the largest producer of wheat globally. Our study reports the cloning of 1211bp β-1,3-glucanase (TaGlb2) gene into E. coli DH5α using pTZ57R/T vector. After conformation through colony PCR and restriction analysis, gene was subcloned into expression vector pET22b(+) and resulting pET22b-wheat TaGlb2 construct was introduced into E. coli BL21. The transformed E. coli BL21 cells harboring TaGlb2 gene showed maximum expression of recombinant gene with 1mM IPTG after 6 hours of incubation and auto induced with 10mM lactose. Purified recombinant β-1,3-glucanase possessed 34.6 U/mg specific activity with percentage recovery of 45 and displayed ~38 kDa band on SDS-PAGE and zymogram. Further studies on this enzyme would provide a valuable data for efficient utilization in the improvement of fungal resistance in different crop plants. Additionally, its role in hydrolyzing grains cell wall β-glucans, which cause a number of problems during brewing process, seems to have considerable potential in brew industry.
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