摘要
以海洋低温蛋白酶生产菌株YS-9412-130为研究对象,从传代、菌龄、溶菌酶浓度与酶解时间、甘氨酸与EDTA预处理以及稳定剂对该菌原生质体形成与再生的影响进行研究。结果表明,在相同条件下,第一代菌至第五代菌原生质体的形成率分别为86.9%、70.3%、66.8%、63.4%、60.2%,再生率分别为10.5%、18.6%、17.9%、18.2%、17.8%;取该菌对数生长前期、对数生长中后期、稳定期部分菌液制备原生质体,原生质体的形成率分别为72.1%、70.4%、60.5%,再生率为13.4%、18.9%、10.4%;溶菌酶浓度为2.5 mg/mL5、mg/mL1、0 mg/mL、20 mg/mL,酶解60 min,原生质体的形成率分别为40.6%、70.8%、81.8%、95.6%,原生质体的再生率为19.0%、19.2%、19.0%、10.6%;使用10 mg/mL溶菌酶酶解YS-9412-130菌30 min、60 min、90 min、120 min,原生质体的形成率分别为30.8%、81.3%、81.7%、82.9%,原生质体的再生率分别为19.2%、19.3%、16.9%、11.2%;在细菌培养液中添加终质量浓度为5 mg/mL、10 mg/mL、20 mg/mL和40 mg/mL的甘氨酸,培养该菌16 h后制备原生质体,原生质体的形成率分别为81.0%、81.1%、90.3%、90.8%、90.6%,再生率为19.1%、19.0%、19.3%、12.0%、9.0%;EDTA对原生质体的形成稍有促进作用,但作用超过30 min会影响原生质体的再生;分别以0.6 mol/L KCl、0.3 mol/L KCl+0.3 mol/L蔗糖、0.6 mol/L蔗糖作为稳定剂,原生质体的再生率分别为10.9%、19.6%、25.9%。结论认为,YS-9412-130原生质体制备的最佳条件为选用第2代YS-9412-130菌,在培养基中添加终质度浓度为10 mg/mL的甘氨酸培养16 h后,再将菌体置于含有0.05 mol/L EDTA的高渗溶液中30℃预处理30 min,用10 mg/mL溶菌酶,30℃酶解60 min,再用0.6 mol/L蔗糖作为原生质体再生培养稳定剂,比较适合于YS-9412-130原生质体的制备与再生。这一试验结果将为通过原生质体技术对YS-9412-130菌进行遗传改良提供重要参数。[中国水产科学,2006,13(1):106-111]
The effects of several factors on protopla^t formation and regeneration of strain YS-9412-130 were studied such as strain generations, age, lysozyrne concentration, enzyrnolysis time, the pretreatment by Gly or EDTA and osmotic stabilizer. The results showed that the protoplast formation rates of the first passage bacteria to the fifth passage ones were 86.9 %, 70.3 %, 66.8 %, 63.4 % and 60.2 % respectively, and the protoplast regeneration rates were 10.5 %, 18.6 %, 17.9 %, 18.2 % and 17.8 %. The bacteria obtained at their prophase, met anaphase and stabilization phase of logarithmic growth were prepared as protoplasts; the protoplast formation rates were 72.1%, 70.4% and 60.5% and regeneration rates were 13.4%, 18.9% and 10.4% respectively;the results also revealed that when enzyrnolysis on the bacteria by 2.5 mg/ mL, 5 mg/mL, 10 mg/mL, 20 mg/mL lysozyrne for 60 rain respectively; the protoplast formation rates were 40.6%, 70.8%,6, 81.8% and 95.6% , and the regeneration rates were 19.0%, 19.2%, 19.0% and 10.6% respectively. As the same way, when enzyrnolysis on the bacteria by 10 mg/mL lysozyrne for 30 min, 60 rain, 90 rain and 120 min, the protoplast formation rates of the bacteria were 30.8%, 81.3%, 81.7% and 82.9%, and the regeneration rates were 19.2%, 19.3%, 16.9% and 11.2% respectively. Another experiment indicated that the strain cultured in media containing 5 mg/mL, 10 mg/ mL, 20 mg/mL and 40 mg/mL Gly, its protoplast formation rates were 81.0 %, 81.1%, 90.3 %, 90.8 % and 90.6% ,and regeneration rates were 19.1%, 19.0%, 19.3%, 12.0% and 9.0% respectively. It demonstrated that EDTA had good effects on protoplast formation, but affected protoplast regeneration when its action time beyond 30 rain. Selecting 0.6 mol/L KC1, 0.3 mol/L KC1 ＋ 0.3 mol/L sucrose and 0.6 mol/L sucrose as osmotic stabilizer, the protoplast regeneration rates were 10.9 %, 19.6 % and 25.9 % respectively. It was concluded that the second passage strain YS-9412-130 were cultured in culture media with 10 mg/mL Gly for 16 h, then the bacteria were pretreatment in 0.05 mol/L EDTA at 30 ℃ for 30 min before enzyrnolysis at 30 ℃ for 60 rain by 10 mg/mL lysozyrne, subsequently 0.6 mol/L sucrose was chosen as osmotic stabilizer. All conditions above were suitable for the protoplast formation and regeneration of strain YS-9412-130. These results and data would provide important parameters for the strain improvement of strain YS-9412-130 through the protoplast technology.
出处
《中国水产科学》
CAS
CSCD
北大核心
2006年第1期106-111,共6页
Journal of Fishery Sciences of China
基金
国家"863"引导项目资助(2003AA001028)
