Study On Breeding Up High-Yield Strain Of Taxol By Protoplast Mutagensis And Regulation Of Taxol Biosynthesis

Nowadays, tumors and cancers seriously threaten human's life, and they are one of the persistent ailments. For the human being, it is the second main cause of death that is just after heart and brain blood vessel diseases. For a long time, scholars made many attempts and explorations to prevent and cure all kinds of cancers in the world, but their results were dissatisfactory. Now, it is believed that the taxol is a new kind of anti-cancer drug, and it is very effective for many kinds of cancers. Therefore, the production of taxol becomes a hotspot in the field of research and exploitation of taxol around the world. Compared with other methods of producing taxol, the advantage of using microbiological fermentation to produce taxol is much more superior. In this experiment, the fermentation of taxol-producing fungus UV40-19 was investigated through single factor test , and the optimized fermentation conditions were obtained; The effects of precursors, elicitors, inhibitors and their cooperation on the regulation of taxol biosynthesis were studied by using both single factor and orthogonal design experiment, respectively; At the same time, the breeding of high-yield strain of taxol was performed by protoplast mutagensis of strain UV40-19 using combined treatment of UV and NTG; In addtion, genetic differences between the mutants UV_40-19 ,UL_50-6 and their parent strain NCEU-1 were primarily compared through random amplified polymorphic DNA (RAPD), AFLP, ITS sequence and isozyme technique; Moreover, it was primarily discussed that if taxadiene synthase also existed in the biosynthesis pathway of microbiological fermentation method to produce anti-drug taxol by Southern blotting. In the field of using taxol-producing fungi to produce taxol , the report correlative to the experiment has not been found yet. Therefore, the progress we made provides a strong academic guidance for regulation of taxol biosynthesis and building high-yield strain of taxol by microbiological fermentation method to produce anti-drug taxol; at the same time, it shows a great prospect for producing taxol industrially and creating enormous economic and social benefit in the near future.The main results of the experiment were listed below.1 The optimized fermentation conditions are: fermentation liquid initial pH value 5.5, culture temperature 25℃, rotational speed 150r/min;2 The syntheses of taxol was significantly promoted by adding some precursors, elicitors and inhibitors into the S-7 medium with an appropriate concentration;3 The optimized medium allowed the yield of taxol to be increased from 396.43μg/L to 493.74μg/L;4 The optimal conditions for the preparation and regeneration of the Nodulisporium Sylviforme taxol producing fungus UV40-19 were 3% lywallzyme+4% snailase+1% lysozyme + 3% cellulose at 30℃bath, pH5.5-6.0 with enzymolysis time at 5 hours, then the protoplasts were collected and cultured on the PDA regeneration medium composed of NaCl : KCl :glucose ( 5:3:2, v/v/v, final concentration 0.7mol/L) by bilayer-plate culture;5 The suitable conditions for the mutagenesis of the Nodulisporium Sylviforme taxol producing fungus UV40-19 was as follows: the protoplast suspension was treated with 0.8mg/mL NTG for 15min; then irradiated by UV ( 30w, 30cm away) for 40s under magnetic stirring; 6 The mutagenesis strain, UN05-6, which was stable hereditarily with taxol output at (478.12±11.36)μg/L, was screened from resistance plate containing 90mg/mL NTG;7 A new, quick and effective method was explored for extracting genome DNA of Nodulisporium sylviforme taxol-producing fungus in the study;8 the genetic differences were very obviously between the parent strain and its mutants and between the two mutants, which laid foundation of molecular mechanism for the study of genes related to the taxol biosynthesis and mutants for raising the taxol yield;9 The gene fragment of taxadiene synthase was cloned from Taxus cuspidate. The sequence was analyzed by Blast program on NCBI GeneBank database , and it was compared with the homologous sequences collected from GeneBank. The result showed that the sequence that was cloned in the experiment had highly homologous with sequences (98%) collected from GeneBank;10 It was verified that taxadiene synthase also existed in the biosynthesis pathway of using microbiological fermentation method to produce anti-drug taxol for the first time.