冷休克诱导红鳍东方鲀雄核发育单倍体的条件优化

Optimization of inducing conditions for cold shocked androgenetic haploid in redfin puffer Takifugu rubripes

为探索无需卵子失活仅用冷休克方法诱导雄核发育单倍体,以红鳍东方鲀Takifugu rubripes为研究对象,试验选用L_9(3~4)设计,进行3因素3水平的正交试验,处理温度设为0、2、4℃,处理持续时间设为30、45、60 min,处理起始时间设为2、5、8 min,共9个试验组,并对其后代采用单个胚胎染色体计数法进行染色体数目统计及微卫星分析。结果表明:冷休克诱导得到的单倍体率最高为第7试验组和第9试验组,分别为受精后8 min在4℃下处理30 min和受精后5 min在4℃下处理60 min,单倍体率分别为86.7%和80.0%;根据正交试验直观分析结果,得出冷休克诱导红鳍东方鲀雄核发育单倍体的3因素最优组合为处理温度4℃、处理持续时间60 min、处理起始时间8 min;影响冷休克诱导单倍体的3因素主次顺序为处理温度>处理起始时间>处理持续时间;微卫星分析结果表明,单倍体携带的遗传基因全部为父本的遗传基因。研究表明,仅用冷休克方法可以成功诱导红鳍东方鲀雄核发育单倍体,雄核发育单倍体率高达86.7%,为进一步研究诱导红鳍东方鲀雄核发育二倍体奠定了基础。

In a three factor and three level orthogonal experiment L9（34） , fertilized eggs of redfin puffer Takifugu rubripes were exposed to water temperature of 0, 2, and 4 ℃（A ） for 30, 45, and 60 min （B）, and initial treat-ment time of 2, 5, and 8 min （C ） in order to explore a method of inducing androgenetic haploid of redfin puffer by cold shocked without inactivation. Then the chromosome number was counted in one embryo and microsatellite a-nalysis was performed in the offspring. The results showed that the maximal haploid rate was observed in the embry-os 8 min after fertilization treated at 4 ℃ for 30 min （86.7% ） and 5 min after fertilization treated at 4 ℃ for 60 min （80. 0% ）. The intuitive analysis revealed that the optimal inducing androgenetic haploid was conducted under con-ditions of 8 min after fertilization at 4 ℃ for 60 min with initial treatment time of 8 min. The order of three factors influencing androgenetic haploid was expressed as treatment temperature〉treatment initial time〉treatment duration. The microsatellite analysis showed that the genes of androgenetic offsprings were all derived from male redfin puffer. The findings indicate that the redfin puffer had androgenetic haploid rate of 86.7% by cold shock treatment, and that lay the foundation for the further study of redfin puffer androgenetic diploid.