中间球海胆卵膜与受精膜去除方法及染色体核型分析  

Methods for egg membrane and fertilization membrane removal and karyotype analysis of the sea urchin(Strongylocentrotus intermedius)

作　　者：陈小慧[1] 张伟杰[1] 李雅娟[1] 刘雷 张宝警 丁君[1] 常亚青[1] CHEN Xiaohui;ZHANG Weijie;LI Yajuan;LIU Lei;ZHANG Baojing;DING Jun;CHANG Yaqing(Key Laboratory of Mariculture&Stock Enhancement in North China's Sea,Ministry of Agriculture and Rural Affairs,Dalian Ocean University,Dalian 116023,China)

机构地区：[1]大连海洋大学,农业农村部北方海水增养殖重点实验室,辽宁大连116023

出　　处：《中国水产科学》2020年第9期1033-1041,共9页Journal of Fishery Sciences of China

基　　金：国家自然科学基金面上项目(31672652);农业农村部农业科研杰出人才及创新团队项目。

摘　　要：去除卵膜及受精膜是成功制备棘皮动物染色体的关键步骤。为找到高效去除海胆卵膜与受精膜的方法,使用三氮唑(2 g/L)、盐酸海水溶液(pH 4.75)、二硫苏糖醇(3×10^-3 mol/L)和对氨基苯甲酸(3×10^-3 mol/L)等4种化学试剂分别对中间球海胆(Strongylocentrotus intermedius)卵子及各时期胚胎进行处理,比较了不同试剂、处理时间或处理时期的去膜率、去膜后受精率和胚胎畸形率等指标的差异;利用常规空气干燥法对经去膜处理的囊胚期胚胎制备染色体,并进行核型分析。结果表明,三氮唑、盐酸海水溶液、二硫苏糖醇和对氨基苯甲酸等4种试剂对中间球海胆的卵膜和受精膜均有去除效果,其中2g/L三氮唑处理未受精卵30min的去膜率为85.50%,去膜后受精率为97.25%,畸形率为1.75%,去膜效果优于其余处理组。利用经该方法去膜后的早期囊胚进行染色体制备效果较好,获得了61个分散良好、形态完整的染色体中期分裂相。核型分析表明,中间球海胆的二倍体染色体数为2n=42,核型公式为:2n=20m+20sm+2st,NF=84,即有10对中部着丝粒染色体,10对亚中部着丝粒染色体,1对亚端部着丝粒染色体,染色体臂数为NF=84。本研究可为海胆染色体制备及染色体操作育种提供技术参考。Strongylocentrotus intermedius is a commercially important sea urchin species in China and Japan.The chromosomes are difficult to prepare,so the karyotype of S.intermedius is unknown.Egg membrane or fertilization membrane removal is a key step in the preparation of the echinoderm chromosome.To identify the optimal method of egg membrane and fertilization membrane removal,four reagents[triazole solution(2 g/L),dithiothreitol solution(3×10^-3 mol/L),p-aminobenzoic acid solution(3×10^-3 mol/L),and hydrochloric acid solution(pH 4.75)]were used to treat the eggs and embryos for 10 min,20 min,30 min,40 min,50 min,and 60 min post-fertilization at the 2-cell,4-cell,8-cell,and 16-cell stages,respectively.The chromosomes of the blastocyst embryos whose membranes were removed were prepared by conventional air-drying,and the karyotypes were analyzed.The results showed that all four reagents could remove the egg membrane or fertilization membrane.The eggs treated with 2 g/L of triazole for 30 min had an 85.50%membrane removal,97.25%fertilization rate,and 1.75%deformity rate,indicating that this was the best method.Sixty-one metaphase cells with well-dispersed and well-preserved chromosomes were successfully prepared from the early blastocysts by the optimal membrane removal method.Karyotype analysis showed that the diploid chromosome number of S.intermedius was 42,and the karyotype was composed of 10 pairs of metacentric chromosomes,10 pairs of submetacentric chromosomes,one pair of subtelocentric chromosomes,and 84 chromosome arms.Thus,the karyotypic formula is 2 n=42=20 m+20 sm+2 st and NF=84.The chromosome number is the same as that in previous reports for this species and in other sea urchins,such as S.nudus and Hemicentrotus pulcherrimus.The karyotype is the same as H.pulcherrimus but is different from Paracentrotus lividus.In conclusion,this study compared the membrane removal efficiencies of four reagents,and the results showed that treating with 2 g/L triazole solution for 30 min was the optimal method.We also obtai

关 键 词：中间球海胆 去膜 染色体 核型分析 

分 类 号：S917[农业科学—水产科学]