Localization And Identification Of Ultrastructural Components Of The Secretory Pathway For Endoglucanase In The Edible Mushroom, Volvariella Volvacea

The edible straw mushroom, Volvariella volvacea, produces a family of cellulases that hydrolyse the cellulose component for fungal growth .However, since the cellulose polymer is too large to enter the fungal hypha, the cellulolytic enzymes have to be secreted to the external environment. The previous studies showed that V.volvacea can produces three kind of cellulase which are endoglucanse(EC3.2.1.4), cellobiohydrolase(EC3.2.1.9.1),β-glucosidase(EC3.2.1.21). The special research on EndoglucanseI(EGI) showed it has a molecular mass of 42KDa and an isoelectric point of 7.65, its maximal PH and temperature are 7.5 and 55℃respectively.In the experiment, we first have the research that EgI expression in V.volvacea is regulated at the transcriptional level by both induction and catabolite repression, and hereα-lactose serves as both inducer and repressor. EgI expression was observed with RT-PCR assays after 3h following addition ofα-lactose to V.volvacea mycelium pre-grown for 72 hr in basal medium containing 1% sorbitol. After 6hr the expression is strongest, then after 9hr and 12hr, the expression became weaker. During the process of RT-PCR, we use gpd gene as house-keeping gene.In the experiment, we used gel chromatography and preparative gel electrophoresis protocol yielded EG1 in pure form and the recombinant enzyme was active. Then we use the purified recombinant enzyme to raise the antiserum and purified the primary anti-endoglucanase antibody with affinity chromatography. We also did western blotting to confirm the purity and specificity of the primary antibody.Confocal laser scanning microscopy in combination with immonolabelling using the anti-endoglucanse antibody and fluorescent dye conjugated (Rhodamine) secondary antibody studies investigating the hyphal distribution of endoglucanase during an enzyme induction and repression cycle revealed a strong immunofluorescent signal variation at the hyphal cell wall. After 3h induction, a weak but uniform immunofluorescence signal was evident throughout the fungal hypha. After 6 to 8 hours induction, a stronger, continuous immunofluoresccnce was clearly observed in the region of the hyphal wall. Labelling of hyphac harvested after 9 hours and 12 hours was less intense and more disposed. No labeling was observed in control hypha.Electron microscopy in combination with immunolabelling using anti-endoglucanase primary antibody and anti-rabbit secondary antibody gold conjugates revealed that enzyme production was associated with the endoplasmic rcticulum during an enzyme induction and repression cycle. And also the transport of cndoglucanse within the hyphal cells is carried out via vesicles which fuse with the plasma membrane and released the endoglucanse enzymes through cell wall.