Studies On The Establishment Of The Genetic Transformation System Of Eucalyptus Entophytic Fungi

Eucalyptus has been cultivated as quick growing species of trees. It is very important forresearch on Eucalyptus molecular biology and exploration of its commercial values to establish thegenetic transformation system. However, few works was reported in this area. In this project, wechoose the eucalyptus entophytic fungi as research topic and established the genetic transformationsystem. The chief means of this work are:1) Glyceraldehydes-3-phosphate dehydrogenase (GPD) is expressed strongly and constitutively inL..edodes.The GPD promoter was isolated by PCR amplification from the genomic DNA ofL..edodes. The GPD promoter was fused respectively to ipt gene and bialaphos resistance gene(bar). Using the resulting construct pL321b-ipt, the protoplasts of eucalyptus entophytic fungi wastransformed in PEG solution, and bialaphos-resistant transformants were obtained, providing asimple and economical method for research on eucalyptus at the molecular level.2) Isopentenyl transferase is the first enzyme of cytokinin biosynthesis, and it is very important tothe growth of cell. The application of ipt gene in eucalyptus entophytic fungi is rarely seen, so weconstructed an expression vector of ipt gene and hope can promote the growth of eucalyptus byusing it.3) In this project we first developed an efficient procedure for isolation and regeneration ofprotoplasts from entophytic fungi, so the regenerative rate is increased and at the same time theregenerative time is cut down. Using the expression construct, an efficient transformation systembased on electroporation was established.Paving the way for genetic modification of eucalyptus entophytic fungi, these researches are ofgreat importance for the research on eucalyptus genetic engineering and for the exploration of thenutritional values of eucalyptus.