Research On Agrobacterium Tumefaciens-mediated Transformation Of Human Insulin Gene BCA To Tremella Fuciformis

In ordor to obtain Hygromycin B sensitive strains, logarithmic phase yeast-like conidia of Tremella fuciformis Tr21 was treated with UV. 12180 colonies were selected and 5 mutants were obtained after two rounds selection. After 10 generations of subculture, one strain of the six turned to be resistant to Hygromycin B while the others remained sensitive, which suggested that those five mutans were of genetic stability.Transform plasmid pCB-BCA and pBHg-BCA, both carrying human insulin gene BCA and hygromycin resistance gene hph, into Agrobactrium tumefaciens EHA105 and GV3101 respectively by freeze-thaw method. The promoter used to initiate transcription of hph gene and BCA gene in pCB-BCA was CaMV 35S promoter while gpd promoter of Agaricus bisporus was used in pBHg-BCA. Different Agrobactirium tumefaciens carrying different plasmid with different promoter cocultured with logarithmic phase yeast-like conidia of Tremella fuciformis Tr21-01, transformants were selected on selective medium containing 50μg/mL hygromycin B and 400μg/mL Cefotaxime. resitant Resistent colonies could be observed on SM plate that Tr21-01 and EHA105 carrying pBHg-BCA were cocultured, which may demonstrate that Tremella fuciformis could recognize gpd promoter of Agaricus bisporus, and different Agrobacterium tumefaciens strains showed host-specificity in transformation of heterogeneous gene.Agrobacterium tumefaciens-mediated transformation can be affected by many factors resulting in different transformation efficiency. Influencing factors such as cocultured period, AS concentration, concentration of Tremella fuciformis and Agrobacterium tumefaciens were optimized. High transformation efficiency can be observed when AS concentration was 500μmol/L, yeast-like conidia 108/mL, OD value of Agrobacterium tumefaciens 0.5, and cocultured for 2 days, and the transformation rate was about 310 transformants/108 yeast-like conidia.The integration of the human insulin gene and hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing of 5 randomLy selected putative transformants. The mRNAs of human insulin gene were detected by RT-PCR. Southern blot analysis of 3 randomLy selected transformants showed that BCA gene was integrated as a single copy into the fungal genome of T. fuciformis. All the transformants still show resistance to hygromycin B after 5 generations of subculture, which means that heterogeneous gene can be inherited stably.