| Morchella importuna is a rare edible and medicinal fungus with high economic value,but its life history and mechanism of nutrition transformation and utilization have not been fully clarified,and its cultivation technology is still immature.It is of great significance for the sustainable and healthy development of morel industry to improve the ability of degrading cellulose and reduce the amount of wheat grains in nutritional bags.The expandin-like protein Cerato-platanin(CP)is involved in the process of fungal cell wall remodeling during fungal growth and development,and has the ability to loose cellulose substrates.In this study,two CP family genes,MiCP1 and MiCP2,were found in the genome information of M.importuna,and the functions of the two genes were studied.The results are as follows:(1)The structure and physicochemical properties of MiCP1 and MiCP2 proteins were analyzed by bioinformatics methods,and a phylogenetic tree was constructed.The expression levels of MiCP1 and MiCP2 genes at different growth and development stages were detected by q RT-PCR.The results showed that MiCP1 was only significantly expressed in the mycelial growth stage,while the expression level of MiCP2 in the primordium stage was 4.7 times higher than that in the young mushroom stage,indicating that the CP protein is not only related to the vegetative growth of M.importuna,but may also be involved in the growth and development of the fruit body.(2)By removing the signal peptide,the expression vectors of PET30a-MiCP1 X,PET30a-MiCP2 X,p MAL-c2X-MiCP1 X and p MAL-c2X-MiCP2 X were constructed,and their expression was induced in Escherichia coli BL21(DE3)strain.As a result,only p MAL-c2X-MiCP1 X protein was successfully induced to express,but MiCP2 X protein did not,which may be related to the expression vector used.In addition,a subcellular localization vector of MiCP1 protein was constructed,and by transient expression in rice protoplasts,it was found that MiCP1 protein was localized in the plasma membrane,which was consistent with the predicted results of subcellular localization.This lays the foundation for the next in vitro study of CP protein.(3)In order to verify the gene functions of MiCP1 and MiCP2,the two genes were cloned from M.importuna.Using the original vector in the laboratory,four overexpression vectors of these two genes,p1391-e GFP-CP1,p1391-e GFP-CP2,p Hyg-m Cherry-CP1,p Hyg-m Cherry-CP2,were constructed.The monospora strains A2and A50 of M.importuna were transformed by Agrobacterium-mediated method.After screening for hygromycin resistance,stably growing transformants were tested.Three transformants with the most obvious up-regulation of MiCP1 and MiCP2 genes of A2and A50 strains were selected,and the phenotypes of the overexpressed transformants were analyzed.The results showed that compared with the wild type,the A2 strain transformants had a slightly thicker colony edge and a faster growth rate on the straw and wood chips medium,and the cellulase activity decreased in the liquid medium.The A50strain on the straw and sawdust medium obviously thickened the colony edge,but the growth rate did not change significantly,the cellulase activity increased.It is speculated that the MiCP1 and MiCP2 genes are related to the growth and develpment of the fruit body and cellulose degradation of Morchella tertiaryensis(4)MiCP1 and MiCP2 gene silencing vectors p1391-i-CP1 and p1391-i-CP2 were constructed,and transformed into M.importuna strains A2 and A50 to obtain five MiCP1 and MiCP2 silencing transformants,respectively.The phenotype analysis of the transformants found that the phenotype of the gene silencing transformants was not significantly different from the wild type.This result is similar to the pathogenic fungi,which may be due to the fact that after the gene silencing of CP,its function is compensated by other expantin-like proteins in fungi,so that it has no obvious effect on the growth and development of fungi.In this study,the function of CP gene in macrofungi was studied for the first time,and it was preliminarily revealed that MiCP1 and MiCP2 were involved in the growth and development process and cellulose degradation of morel,and it was found that CP gene showed different phenotypes after up-regulated expression in different monospora strains.This study laid a preliminary foundation for further analysis of CP protein expression in vitro and breeding of new varieties of M.importuna. |
