| Coniothyrium minitans is an important sclerotia mycoparasitic of Sclerotinia sclerotiorum.and the ablity to parasite and destroy hypha and sclerotia of S.sclerotiorum.so it is an important biocontrol fungi for sclerotinia of crop. Previously research shows that conidium spray into the ground part of rape in the early flowering period which can well controlled sclerotinia of rape; and if conidium spray into the soil which can effectively reduce the number of sclerotia in soil, and in the meantime inhibite germination of sclerotia. The sclerotia is a kind of dormancy structure, high permeability potential,C. minitans need to overcome the high permeability of sclerotia to invade and get nutrients from sclerotia of S.sclerotiorum.so improving the ability of adapting the hyperosmotic environment of C. minitans,which is able to promote sclerotia parasited and decayed.In order to research the molecular biology of C. minitans, screen and clone some functional genes from C.minitans related to hypertonicity, our laboratory established technology of agrobacterium-mediated genetic transformation and T-DNA insertion mutation library used wild strain ZS-1 as material in the early time.In this study,we prepared hyperosmotic medium with sugar,screened six mutation strains related hypertonicity from T-DNA labed random insertion mutants library of ZS-1.and cloned these genes from six hypertonicity related mutants through IPCR and hi-Tall PCR.In these genes, we find a gene have high homology withβ-glucosidase enzyme precursor in mutant TN542, named BGL-Like, then we do some researches about mutant TN542.Comparing mutant TN542 and strain ZS-1, we find they have no significant differences in growth speed and spore production quantity cultured on PDA plates with 20℃; Through microscopic observation, we also find they have no significant differences in the morphology of colony, hyphal and conidium.But significant differenty is that mutant TN542 have better growth sate than ZS-1 cultured on hyperosmotic medium with 1.2M sugar of 20℃.so we supposed that mutant TN542 have ability of adapting the hyperosmotic environment. In order to determine the mutant TN542 have such feature, we separately prepared hyperosmotic medium with 1.0M sorbitol and 1.0M NaCI, the results both showed mutant TN542 have better growth sate than ZS-1 cultured above medium with 20℃; In parasitic sclerotia, mutant TN542 have stronger ability than ZS-1 cultured on the high permeability sand with 20℃. Southern hybridization confirmed that T-DNA insetion is a single site in mutant TN542, Combining the whole genome sequence of ZS-1, we find that BGL-Like gene have 1556bp,3 introns,T-DNA insertion site have 1300bp away from translation initial site of BGL-Like gene, with biology software we infer that BGL-Like gene have a signal sequence, T-DNA maybe insert promoter area; then we use RT-PCR to detect expression of BGL-Like gene,results indicate that BGL-Like gene have expressed both in ZS-1 and mutant TN542,but the expression of BGL-Like gene have more stronger in the mutant TN542. |
