鰤鱼诺卡氏菌与黄粉色诺卡氏菌原生质体融合的研究  

Protoplast Fusion of Nocardia seriolae and Nocardia flavorosea

作　　者：王英华[1] 陈建林[1] 徐亮[1] 张红莲[1] 夏立群[1,2,3] 

机构地区：[1]广东海洋大学水产学院,广东湛江524088 [2]广东省水产经济动物病原生物学及流行病学重点实验室,广东湛江524088 [3]广东省教育厅水产经济动物病害控制重点实验室,广东湛江524088

出　　处：《海南大学学报（自然科学版）》2016年第4期350-355,共6页Natural Science Journal of Hainan University

基　　金：广东省国际科技合作项目(2016A050502061);广东省教育厅特色创新项目(2014KTSCX075);深圳大鹏新区产业发展专项资金(KY20160207);广东省攀登计划项目(pdjh2016b0234);"海之帆-起航计划"大学生科技创新培育专项(hzfqhjhzrkx2015b03)

摘　　要：鰤鱼诺卡氏菌是鱼类诺卡氏菌病的主要病原,黄粉色诺卡氏菌为一种近缘无毒环境诺卡氏菌.本实验研究主要以鰤鱼诺卡氏菌和黄粉色诺卡氏菌为两亲本,开展了原生质体融合研究.针对诺卡氏菌细胞壁厚的特性,本实验对原生质体制备时的溶菌酶脱壁时间进行了筛选,确定鰤鱼诺卡氏菌和黄粉色诺卡氏菌的最佳脱壁时间分别是65 min和100 min.用聚乙二醇促使两亲本菌的原生质体融合后,通过选择性培养基培养、菌落形态筛选、革兰氏染色观察、16S rRNA测序等方法,对融合菌株进行了初步鉴定.鉴定结果表明:融合菌株的菌落形态较两亲本发生了改变,为革兰氏阳性菌,通过16S rRNA基因序列比对,其与鰤鱼诺卡氏菌的同源性为99%.融合菌株的毒力较鰤鱼诺卡氏菌野生株有所降低.本研究为鰤鱼诺卡氏菌活疫苗的研制提供了新思路,对鱼类诺卡氏菌病的防治具有一定的潜在价值.Nocardia seriolae is the main pathogen of fish nocardiosis, while N. flavorosea is an avirulent related environmental Nocardia spp. In the study, N. seriolae and N. flavorosea were selected as parent strains and protoplast fusion was carried out. According to the cell wall characteristics of the two Nocardia spp , which were thick, the cell wall treating time by lysozyme during protoplast preparation were optimized, and the optimal treating time for N. seriolae and N. flavorosea was 65min and 100min, respectively. After the two kinds of protoplast were fused by using polyethylene glycol （PEG） as fusogen strains were identified by selective medium culture, colony morph under high concentration of Ca2＋ , the fusion ology filtering, Gram staining, and 16s rRNA sequencing. The resuhs indicated that the fusant was Gram-positive bacteria, and whose colony morphology was different with that of the parent strains, the 16S rRNA sequence of fusant was 99% identical to that of N. seriolae. Compared with wild strains N. seriolae ZJ0503, the virulence of fusant was decreased. Our study provides a new idea for N. seriolae live vaccine development, and is valuable for the prevention and treatment of fish nocardiosis.

关 键 词：原生质体融合 鰤鱼诺卡氏菌 黄粉色诺卡氏菌 

分 类 号：S943.2[农业科学—水产养殖]