Study On Transformation Of Tremella Fuciformis With HLF Gene

Lactoferrin is a kind of iron combine glycoprotein which distributes widely many organs and tissues of people and mammal. Lactoferrin has many physiological function including stimulating iron absorption, adjusting iron balance, antibiotics, antivirus and so on. Transformation of human lactoferrin gene to Tremella fuciformis produced human lactoferrin which had bioactivity. So Tremella fuciformis was added new hygienical factor. Lactoferrin with high iron combination and high antibiotics can improve iron content and enhance antibiotics of transgenic Tremella fuciformis.In the study, on the basis of screening of genetic transformation marker and optimizing genetic transformation system of Tremella fuciformis, a human lactoferrin gene expression vector was constructed, and then transferred into Tremella fuciformis by electroporation. Molecular evidences demonstrated that human lactoferrin gene had been integrated into the genome of transgenic Tremella fuciformis strains. These results were following.1．Screening of genetic transformation marker of Tremella fuciformisInhibition of thirteen antibiotics was determined to yeast-like-conidia of Tremella fuciformis Tr21．The results showed Tr21 can resistant most of antibiotics and only had sensitivity to kanamycin, lincomycin, gentamycin, hygromycin. So kanamycin and hygromycin can be as selective antibiotics of Tremella fuciformis transformants. Tremella fuciformis has no GUS activity itself. GUS gene can be used reporter gene of genetic transformation of Tremella fuciformis.2．Construction of expression vector of Tremella fuciformisThe DNA fragment including 35S promoter, nos terminator and GUS gene was excised from plasmid pBI121 with HindⅢ和EcoRⅠenzymes and then was inserted into plasmid pCAMBIA1301．The middle recombinant plasmid was named as pCB. The human lactoferrin gene was excised from plasmid pUC-hlf and subcloned into expression vector pCB as an BamHⅠ/ SacⅠfragment instead of GUS gene. The resulting recombinant plasmid was named as pCB-hlf.3．Construction of high efficiency Tremella fuciformis genetic transformation systemThe optimum parameters for electroporation were studied. The results showed yeast-like-conidia of Tremella fuciformis needed pretreatment with 2% lywallzyme 1h. The optimal conditions were voltage 300V, electric resistance200Ω, capacitance 25μF. In addition electroporation buffer was added PEG4000, transformation efficiency can be improved and had 9．25×10~6 transformants perμg plasmid DNA. The concentration of PEG4000 was 7%.4．Transformation of human lactoferrin gene to Tremella fuciformis and its molecular evidenceTransformation of expression vector pCB-hlf with human lactoferrin gene to Tremella fuciformis by electroporation gained many antibiotic resistant transformants. These transformants bring with human lactoferrin gene, GUS gene by PCR determination. Southern hybridization demonstrated that human lactoferrin gene had been integrated into the genome of transgenic Tremella fuciformis strains, and the determination of RT-PCR of transformants showed human lactoferrin gene can transcribe in Tremella fuciformis.