Establishment Of Trinal-fusants And RAPD Analysis Of Bacillus Thuringiensis, Sporocytophaga And Bacillus Subtilis

In this research, Bacillus thudngie that can kill same parasites, Bacillus subtiliswhich can enhance the immunity of aquatic animal, better the quality of water andpromote growth of fish, and Sporocytophaga myxococcoides with the function ofdegradation of cellulose were tried to integrate into a fusant by protoplast fusion,expecting obtains the probiotics with three parents fine characteristics. In the end, amultifunctional fusant which can extinguish the parasite, and degrade intensely thecellulose, meanwhile strengthen the aquatic animal immunity. Speciticmeasures andresearch results were showed as follows:1. Bacillus thuringiensis were separated and purified from soil, on which physicsbiochemistry characteristic was appraised. As a result, Bacillus thuringiensisbacterial strain BS-12 was obtained, which had been able to produce spore2. The growth curve of Bacillus thuringiensis, Sporocytophaga and Bacillus subtilishad been tested separately, which showed that logarithm growth medium term ofBacillus thuringiensis, Sporocytophaga and Bacillus subtilis were 4.0 hours, 5.5hours and 6 hours respectively3. Discussing factors which affect the formation rate and regeneration rate ofprotoplast of BS-12,BS-3 and SP-2 by means of one-way experiment. The resultsshowed that: The glucose, glycine and penicillin can affect the formation andregeneration of protoplast; The best lysozyme concentration of BS-12 and BP-1were 5.0mg/mL, 6.0mg/mL; the best enzyme treatment time were 60min,60min. on the condition of enzyme treating solution and time, the percentages ofprotoplast formation and protoplast regeneration of the BS-12 bacterial strain were82.7%, 32.4%;BP-1 bacterial strain were 84.6 %, 34. 6%.4. The protoplast of BS-12 was treated by the heat inactivation, and binary fusantbacterial strain BP-1 was treated by the ultraviolet inactivation. The resultsshowed that: the treating time of the protoplast of BS-12 was 25min and the deathrate was 100%; and the treating time of the protoplast of BP-1 was 5min and thedeath rate was 100%.5. The protoplast of BS-12 and BP-1 were be fusing 60min on the condition ofPEG6000 and Ca²⁺ at 37℃in the huge hot water bath tank, then the stablefusants were screened. 68 trinal-fusants were isolated on revival medium andmake them exist from the fist generation to the tenth generation, getting a strain ofstable trinal-fusants FM-20-1.6. The fusant was identified preliminarily by comparing to the appearance ofcolony, micrososmic configuration, biochemical characteristics and enzyme activity identification of the parent and fusant.7. FM-20-1,BP-1,BS-12,Sporocytophaga and Bacillus subtilis were analysed byRAPD, the results showed that: it's had common bands, and the geneticdistance of FM-20-1 and BP-1 was the most close.