| This experiment used Aeromonas punctta f. intestinalis 58-20-9 strains,Myxococcs piscicola sp. nov G4 strains and Pesudomonas fluorescens 56-12-10 strains to operate as arch-strains to conduct protoplast fusion,established fusants and studied their morphological,configuration,physiologic,biochemical,enzyme character and pathogenicity, antigenicity and the sonsanguinity of DNA.it is in order to coform the technology of building trinal fusant and grope for the basic theory and feasibility of preparing fusants bacterins of fish pathogeny,which also supply for creating bacteria with many values. The main results were presented as follows:Using the qualitative and quantitative antimicrobial susceptibility experiment to screen the genetic markers of protoplast fusion between 56-12-10 strains and AM-1 strains,they are Smr(Streptomycin resistance) and Cmr(Ciprofloxacin resistance) respectively. In this protoplast fusion experiment between two bacteria,the applying density in selective culture medium of Streptomycin and Streptomycin are 300 Iu / ml and 100 Iu / ml.When the temperature of enzymolysis was 37C,we singled out the best enzymolysis time to be 60 minute and the best lysozyme density to be 5 mg/ml . In the condition of PEG operating as agent of accelerating fusion,AM-1 strains and 56-12-10 strains conducted protoplast fusion,the protoplast formation rates of 56-12-10 strains are 81.6% and the protoplast regenerative rates of 56-12-10 strains were 19.8%,the protoplast formation rates of AM-1 strains were 79.2% and the protoplast regenerative rates of AM-1 strains were 8.2%.Arch-screening protoplast fusion rates were 6%o.Breeding respectively 100 fusants colony of arch-screening on duplexing repel select culture medium and generating continuously 10 times,derived a steady fusant conoly.The fusants steady rates were 1%; preparated the threes' protoplast at the same time,at first let 58-20-9 strains and G4 strains conduct protoplast fusion,then mixed it with 56-12-10 strains and finished the second fusion. Arch-screening protoplast fusion rates were 3.5%o.After breeded on single repel select culture medium one by one,derived two strains.one is nomal,the other is aberrance.it can gain steady trinal fusion through the two methods,but the latter is more shortcut.The screened fusants are short bacilliform,they are similar to AM-1 strains.their average macro-axis are 4.52um,average brachy-axis are 0.75um,average volume are 1.33um3.Gram female,flagellum,motion.The colour of giga-colony which was cultured 30 days is roundness,wetness and lranslucency,the colony's center is thicker than rim.The colony is bulging and it's rim is orderliness.The fusants' characters of enzyne and physiologic and biochemical exist among three parental strains.Using fusant strains to infect manually to health grass carps which were born in just that year.After observing two weeks,the results showed that the fusant strains also had pathogenicity.Examined the disease and dead grass carps further,we found that the grass carps which were infected fusant strains had part symptom as bacterial enteritis disease, Bac terial gill rot disease and Red-skin disea.Studied on antigenicity of fusant strains,the results showed that after the grass carps which were immunized by fusanl strains infecting AM-lstrains,56-12-10 strains and three parental strains,their survival rates run to 64.9%, 68 % and 62.5%,valency of antibody upmost to 1:256 and 1:512 on the fourth week.Under the same experiment condition,we abstract the DNAof these five fusants and conduct withanalysis of agrose,the result revealed that the DNA content of binary fusants and trinal fusants are more than any parental strains.As to the gene's concrete integration and its express circumstance,awaiting the further trial. |
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