Construction Of T-DNA Insertion Mutagenesis System For Phanerochaete Chrysosporium

As is known to all,the key technology during high efficient utilization of straw is pretreatment,while the most prospective pretreating method is microbial pretreatment.Nowadays,there are several weak points which seriously hindered the development of microbial pretreatment,such as low efficiency,long cycle time and small scale treatment.Our aim is to to achieve molecular modification towards model of white-rot fungi Phanerochaete chrysosporium through T-DNA random insertion mutagenesis,and obtain improved srains which could be used in high efficiency pretreatment of straw.On the basis of genetic engineering principle,we succeeded in constructing a binary vector.This vector contains the hph gene of Escherichia coli as the selective trait, governed by a heterologous fungal promoter from Phanerochaete chrysosporium genome and the Phanerochaete chrysosporium lip6 terminator. For a complete heterologous Hygromycin B resistance gene was integrated, It can be used in a system to infect Phanerochaete chrysosporium.The efficiency of transformation in agrobacterium varies from different protocols. The method through liquid nitrogen refrigeration resulted in few transformant. Attempts through various conditions such as different liquid nitrogen treating time, different amount of plasmid and different choice of receptor bacterium also generated no trransformant,which showed that it was rather hard to obtain agrobacterium through liquid nitrogen refrigeration protocols. The binary vector pC61300 was easily transformed into agrobacterium LBA4404 through Triparental-mating protocol and large amount trnasformants were obtained.Till then,a complete binary vector system was finally constructed.We tried to transform model of white-rot fungi Phanerochaete chrysosporium with the infection system which we previously constructed.Unfortunatly, as the fungal start material neither the conidate nor protoplast result in any transformant.For lots of false positive transformants put off process of construction of binary vector,we didn't have enough time to try many other factors which impacting the efficiency of transformation.