| Summary: | 碩士 === 東吳大學 === 微生物學系 === 106 === In order to reduce the cost of the production for antibacterial peptides in aquaculture, to establish recombinant strains that exhibit the effect of both antibacterial peptides and probiotics is our aim. In this study, the cDNA of an antibacterial peptide ALF B9, which is a mutant form of MrALF from freshwater Macrobrachium rosenbergii, was transformed into the yeast Kluyveromyces maxianus (KM), a human probiotic, to express ALF B9. By feeding method, prawn M. rosenbergii was the model animal to evaluate the possibility of using KM and the recombinant strain expressing ALF B9 as prawn probiotics.
The non-secreting strain rKM-B9, secreting strain rKM+B9 and KM were continuously fed to shrimp for five days. The mortality of shrimp in KM, rKM-B9, rKM+B9, KM+YPG and rKM+B9+YPG were 50%, 38%, 47% 42% and 36%, respectively. There result were significantly lower than the control group (73%) of the non feeding strains. Bacterial DNA was extracted from the shrimp feces before and after uptaking KM and rKM-B9 and the Next Generation Sequencing was used to analyze the bacterial composition. The results showed that the intestinal bacreial community in shrimp feces after uptaking KM and rKM-B9 were highly different from those in feces from before uptaking KM and rKM-B9. In addition, the relative abundance of pathogen L. garvieae derived from feces was reduced after the shrimp uptake KM and rKM-B9.
The possible physiological factors of shrimp resistance to pathogens include proPO and Pon , gene associated with antioxidant SOD-Mn and GPx, the gene associated with stress HSP70, the activity of specific phenol oxidizing enzymes (phenoloxidase, PO), and superoxide dismutase (Superoxide dismutase, SOD). The result that the mRNA expression of five genes of shrimp uptaking KM, rKM-B9, and rKM+B9 decreased significantly (P < 0.05). The activity of activated PO (POS) and POT in the plasma and the activity of POT were higher than that of shrimp before feeding, and the SOD activity of the shrimp in each feeding group was significantly higher than that of the shrimp before feeding or after stopping. According to the above results, two recombinant strains of recombinant ALF B9 resistant to microbial peptide, rKM-B9 and rKM+B9, were superior to the original strain KM. These strains could enhance the PO activity in the shrimp's immune related enzymes and the activity of antioxidant enzyme SPD, and reduce the number of pathogenic bacteria in the shrimp's intestines. Therefore, it is believed that these strains have potential to be a probiotics for shrimp production.
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