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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 order to explore the cold-induced haploid development in male mice without any oocyte inactivation, Takifugu rubripes were selected as the research object. The orthogonal design of three factors and three levels was designed using L_9 (3-4) The treatment temperature was set to 0,2,4 ℃, the treatment duration was set to 30,45,60 min, the treatment start time was set to 2,5,8 min, a total of nine test groups, and its offspring using a single embryo chromosome Counting method for chromosome number and microsatellite analysis. The results showed that the haploid rate induced by cold shock was the highest in the seventh and ninth experimental groups, which were respectively treated for 30 min at 4 ℃ and 60 min after the fertilization at 8 ℃ for 8 min after fertilization, The haplotype rates were 86.7% and 80.0%, respectively. According to the results of orthogonal test, the optimal combination of 3 factors for the cold-induced haploid development of C. orientalis was the treatment temperature of 4 ℃, the duration of treatment 60 min, and the initial treatment time was 8 min. The order of the three factors influencing the cold shock-induced haploid was treatment temperature> treatment start time> treatment duration. Microsatellite analysis showed that all the haploid-carrying genes The genetic parents. The results showed that the cold-shock method could successfully induce haploids in the male and female male of red-fledged fin, and the haploid rate of male and female nucleus was up to 86.7%, which laid the foundation for further study on the diploid diploid of the male and female .