Synthesis of AI-2 in vitro and effect of AI-2 on bacteriocin synthesis of Lactobacillus plantarum KLDS1.0391
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摘要: 本文采用单因素实验,优化AI-2合成的孵育温度、pH、酶浓度以及孵育时间。利用优化的合成条件,成功合成具有生物活性的AI-2,活性约为阳性对照的2倍。添加合成的AI-2到MRS培养基中,研究AI-2对植物乳杆菌KLDS1.0391生长和细菌素合成的影响,并采用实时荧光定量PCR分析培养15 h的植物乳杆菌KLDS1.0391的细菌素结构基因pln EF的转录水平。结果表明:AI-2合成的最优条件为:0.5 mg/m L Pfs蛋白、1.0 mg/m L Lux S蛋白、2 mmol/L SAH,p H7.5,37℃孵育5 h。添加合成的AI-2对植物乳杆菌的生长无明显影响;培养3 h后,相同培养时间时添加AI-2的实验组对枯草芽孢杆菌ATCC6633的抑菌圈直径显著高于空白对照和阴性对照组（p<0.01）;添加AI-2的实验组的pln EF基因的表达量上调至空白对照的2.89倍。本研究成功优化AI-2的体外合成条件;添加合成的AI-2对植物乳杆菌KLDS1.0391细菌素合成有促进作用,且这种促进作用可能与pln EF基因转录水平上调有关。Abstract: The factors including incubation temperature,p H,enzyme concentration and incubation time were optimized by single- factor experiment design. Then AI- 2 was synthesized under the optimum conditions. The biological activity of the synthesized AI- 2 in vitro,was about 1- fold higher than the positive control. In order to research effect of AI-2 on the growth and bacteriocin synthesis,Lactobacillus plantarum KLDS1.0391 was cultured in MRS broth supplemented with AI- 2 synthesized. The expression level of pln EF gene encoding bacteriocin of Lactobacillus plantarum KLDS1.0391 cultivated for 15 h was studied by RT-PCR.The results showed that the optimum conditions for the synthesis of AI-2 in vitro were determined as follows: the SAH concentration of 2 mmol / L,the Lux S protein concentration of 1.0 mg / m L,the Pfs protein concentration of 0.5 mg / m L,the incubation temperature of37 ℃,p H of 7.5,and the incubation time of 5 h.Growth rate of Lactobacillus plantarum had no significant change,and the inhibition zone against Bacillus subtilis ATCC6633 increased significantly after cultivated in the MRS broth supplemented with AI-2 synthesized for 3 h.And the expression level of pln EF gene of the experimental group was1.89- fold higher than the blank control.In this study,the conditions for the synthesis of AI-2 in vitro were optimized.AI- 2 played an obvious positive regulatory role on bacteriocin production,and the regulatory effect may be associated with the increases of transcriptional level of pln EF gene.
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Keywords:
- AI-2 /
- synthesis /
- bacteriocin /
- RT-PCR
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