原生质体融合选育耐高温耐盐红螺菌的研究

Study on the breeding of Rhodospirillaceaebacteria with salt-resistance and high temperature-resistance by protoplast fusion

为选育耐高温耐盐红螺菌用于其粉状制剂研制,以降解水产养殖水体亚硝基氮功能达90%以上的耐盐红螺菌与能在60～70℃下生长的嗜热脂肪芽孢杆菌为出发菌株,通过正交试验探讨2种菌株的原生质体融合条件。分别对融合子进行温度、生长盐度和净化水质等功能测定,检出性能较优的融合子,同时采用电镜、细胞色素吸收光谱等方法,将融合子优良株与2种亲株进行比较鉴别。实验结果表明,2种菌株原生质体融合的最优条件为温度37℃,作用时间3 h,聚乙二醇(PEG)浓度为40%。在该优化条件下原生质体融合率为1.536%,筛选获得3株能在30～50℃良好生长的融合子(a、b、c)。融合子生长耐受温度较耐盐红螺菌亲株提高66%,但耐受盐度、降解养殖水体氨氮与亚硝基氮功能均与耐盐红螺菌亲株相近。其中融合子a菌株具有生长更快、性能更稳定的特点,其细胞形态特征、革兰氏染色属性、色素吸收峰等均与耐盐红螺菌相似。

In order to breed Rhodospirillaceae bacteria with high temperature-resistance and salt-resistance for powdery agent-manufacture,the conditions of protoplast fusion between salt-resistant Rhodospirillaceae bacteria which could degrade N-nitroso over 90% and Bacillus stearothermophilus which grew at 60-70 ℃ were studied using orthogonal test.The physiological characteristics of fusants such as temperature,growth salt and purifying the water were determined and the identifications between parent strains and the excellent fusant by electron microscope and absorption spectroscopy were studied.The results showed that the optimal conditions of protoplast fusion were as follows： temperature 37 ℃,reaction time 3h and the adding amount of PEG 40%.Under these conditions,the rate of protoplast fusion was 1.536% and three fusants（a,b,c） which could grow well at 30-50 ℃ were obtained.The tolerance of growth temperature of three fusants was improved by 66% than salt-resistance Rhodospirillaceae bacteria,but some characteristics of salt-resistance,degrading N-ammonia and N-nitroso were similar to it.Fusant a grew faster and steadier than other fusants and its characteristics of cell shape,Gram stain and pigment absorbing peak were similar to salt-resistant Rhodospirillaceae bacteria.