Study On The Cloning And Prokaryotic Expression Of Cellulose Hydrolase Enzyme Gene From Rapeseed Pathogen Sclerotinia Sclerotiorum And Development Of Sclerotia

Sclerotinia caused by Sclerotinia sclerotiorum is a serious fungal disease affecting the yield and quality of Brassica napus. Sclerotia is an important sources of primary infection for S.sclerotiorum. In order to deeply dissect the pathogenesis of S.sclerotiorum, the cellulose hydrolase genes from S.sclerotiorum and the relevant characteristics of the sclerotia have been systematically investigated:5cellulose hydrolase CDS were cloned, and expressed in E.coli. The conditions of the sclerotia formation, the structure and storage substances of the sclerotia, and the composition of liquid droplets on the surface of the sclerotia were analyzed. The results are summarized as followed:1. Based on S.sclerotiorum Database, five cDNA sequences of cellulose hydrolases, including HyMan, HyEnGlu, HyYoaJ, Hy-1,4-Man and Ex1Pre-2, were cloned from Brassica napus pathogen S.sclerotiorum culture by RT-PCR.2. HyMan, HyEnGlu, HyYoaJ, Hy-1,4-Man and Ex1Pre-2cDNAs were successfully introduced into prokaryotic expression vector pGEX-4T-1.3. The conditions of five above-mentioned expression systems were optimized. The highest amount of fusion proteins was induced by0.5mmol/L IPTG for2h at37℃. The solubility tests showed fusion proteins mainly existed in the form of inclusion bodies, no souble form detected.4. Studies on the space or nutritional conditions for sclerotia formation suggested:In the present of sufficient nutrition, the mycelium tended to form sclerotia with limited growth space, but failed to form sclerotia with enough growth space. In the absence of nutrition, the mycelium tended to form smaller sclerotia even with enough growth space. There was no sclerotia formation when lack of the carbon source.5. Microstructure and ultrastructure analysis showed that the sclerotia consisted of rind, cortex and medulla. The rind was complicated and composed of mycelial network and convex body. which includes convex film and the hollow spherules under it. Inside the rind, cortex was constituted by mycelia grown in parallel with the epiderma. Medulla was consisted of loose honeycomb structure formed by expanded abnormal mycelia, similar to the storage tissue of plants.6. Frozen section analysis showed that the storage material in sclerotia was mainly composed by soluble protein, no starch or fat detected. At least six kinds of protein compoents in secretion droplet of sclerotia were detected.