Study On The Genomic Rearrangement Breeding Of High-yield Tanshinone ?A Strain

Genome shuffling is a new breeding technology establishing on the DNA Shuffling theory,which combines traditional random mutagenesis with cell fusion.It is a recursion fusion technique,and its recombination object is the whole genome.With the superiority of effective,quickly,and strong practicality,it has been broadly used in the improvement of industrial microbial strains in recent years.Tanshinone IIA is an orange-red needle-like crystal and can easily be dissolved in organic solvents.It has some inhibitory capabilities including antioxidant,anti-thrombosis,anti-hypertension,and antitumor.Recently,various biological benefits of the medicine have been found,such as its ability to promote the circulation of the blood and treat hemorrhages,menstrual disorders and miscarriages.At present,the way to get the material is mainly from medicinal plants.However,the natural source of this medicinal plant is now in short supply because of the length of growth cycle and unstable quality.To provide an alternative source of natural tanshinone IIA,we isolated the endophytic fungus TR21 with low tanshinone IIA production rate.The purpose of this paper was to acquire a strain with high production of tanshinone IIA by the genome shuffling techniques.The main results are as follows:(1)This research had explored the highly effective protoplast preparation and regeneration means:the optimum age of the strain was 14 hours cultivated in the temperature 28,enzymolysised 2 hours by 1.5%cellulase+2.0%snailase+1.5%lysonzyme in the temperature 30,the optimum osmotic pressure stabilizer is 0.8mol/L MgSO4,and separated on the high infiltrate PDA culture medium plate.The results show that the protoplast yield reaches 80%,and the regeneration rate of protoplast reaehes 45%.(2)The condition of inactivating the parent protoplasts was that the purified protoplast under UV lamp from 30 cm distance for 10 min or incubation at 50? for 50 minutes.(3)The mutant strains' protoplasts fused 30 minutes by 30%PEG4000 in 30?.After three rounds of recursive-protoplast fusion,we have obtained a high production strain.The activity of tanshinone IIA production was improved up to 18.827±0.22 mg/g,which was 11.06-fold higher than that of the starting strain TR21.After serial passage,the high yield of the strain can stable inheritance.(4)The agreement of the classical examination and molecular analysis determined that F3-4 belonged to Emericella foeniculicola.Analysis by RAPD indieated that the existence of genetic differences among the mutant strains,shuffled strains and the original strain,which achieved the breeding purpose on the whole.(5)In order to produce more tanshinone IIA by the shuffling strain F3-4,the optimization tests were done to gain the best culture medium.Through the test of different basic culture mediums,we found that bean sprouts liquid medium was propitious to F3-4 both in mycelium growth and tanshinone IIA accumulation.By the analysis of RSM,the best carbon resource was approved to be sucrose.Among three element seclected,the results showed that carbon resource was most influencing element to the germinate of the mycelia and the yield of tanshinone IIA.After optimizing the culture medium,the the yield of tanshinone IIA was 1.6-fold higher than that of the PDA culture medium.