| Tremella fuciformis Berk, was call as White Jelly Fungus or White Wood Ear. It was cultivated in large scales and was known as tonic food or health food. In order to increase nutrient and health value of T.fuciformis, three genes of high lysine protein and human insulin and honeybee (Apis mettiferd) antibiotic peptide were transformated respectively applying of genetic engineering technology.The result of retrieval, there have not been reported on transformation of this fungus since 1976.1 Establishing the Genetic Transformation System of T.fuciformis CanyOlmOlliaillBf BOneSOf BMMtemHlMIBfllin; Antibiotic sensitivity of T.fuciformis and E. coli and A. tumefaciens was studies. Hygromycin and kanamycin were the only two sensitivity antibiotic to T.fuciformis among 13 antibiotic tested. Hygromycin and neomycin phosphotransferase gene were established as select marker in genetic transformation of this fungus. Based on results of the activity of P -galactosidase(lac Z) and P -glucosidase(GUS) of T. fuciformis tested, GUS gene was established as report gene for genetic transformation. Two auxotroph were obtained by induction of mutation. One of them was inositol auxotrph, it could be developped and applied as auxotrophic marker after further research.Established a system of protoplast prepare and regeneration: The protoplast preparecondition was optimized applying the experiment schedule of orthogonal design, the yield of protoplast have got up to 2.75 X 107 /mL in the optimized condition. The regeneration condition of protoplast were studies, the highest regeneration rate was 32.3%. Protoplast yield and regeneration rate above were reach the requirement of protoplast genetic transformation.Research and development 2 methods fer T. fuciformis protoplast genetictransformation. These methods are PEG-mediated and REMI-mediated. The rate of transformation of PEG and REMI were 1593 and 2140 transformants/ug DNA respectively when used 20~30 ug plasmid DNA, the rate could be reach 266000 and 30000 transformants /ug DNA when used race plasmid DNA(50ng) respectively.Resoarch and development 3 nettads ftr T. fuciformis Intact cell oenetictransformation. These method were LiAc-mediated and ultrasonic-mediated and physics-chemical-mediated. These methods does not need to prepare protoplast, intact ceil can be transformated. The rate of the three methods above were 304 and 750 and 2688 transformants/ug DNA respectively when used 7.5 ~30 ug plasmid DNA, the rate could be reach 17000 transformants/ug DNA when used race plasmid DNA(60ng) by means of ultrasonic-mediated . This transformation rate was 2833 times to it of Lentinus edodes (6 transformants/ug DNA).2 Transformation three quality genes respectivelyHigh lyslnt CCntaln pratBin gene MysV. Lysine is essential amino acid for human body. It is the first limit amino acid of people who take rice as major food. Lysine have important nutrient value. Large amount of antibiotic resistant colony were obtained applying REMI-mediated to transformation lys gene. GUS activity of 26 antibiotic resistant strains were tested, 22 strains shown positive and 4 strains shown negative. The tolerance of antibiotic of 17 strains were tested, all of them could grow in the medium containing 200 ug/mL hygromycin; 8 strains were verified by PCR of lys gene, GUS gene, hpt gene, 35S promoter and Tnos sequence. The results show that all of them were positive. Amino acid contain of 7 trangene strains were determined, the increase of lysine contain ranged 3.6%~16.6% comparing to the host strain TauSl 1, there were 3 strains that lysine contain increase more than 15%, the strain of Tau811-1 was the highest increment rate, that was 16.6%.HdBian Insulin Ul|ne \BCA\\ Insulin is the specific medicine for diabetes. The major source for drug production was pancreas of animal now. It was success to production recombined drug by means of gene engineering, and this drug was bring to clinic, but the production cost was higher. Insulin gene (BCA) was synthesized with plant codon bias in this studies,... |
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