酶解制备日本沼虾游离精子方法的研究  

Trypsinase Digestion in Acquiring Free Sperms from Macrobrachium nipponensis

作　　者：颜跃弟[1] 傅洪拓[1,2] 乔慧[2] 白鸿坤 孙盛明[2] 蒋速飞[2] 熊贻伟[2] 龚永生[2] 金舒博[2] 张文宜[2] 

机构地区：[1]上海海洋大学水产与生命学院,上海200000 [2]中国水产科学研究院淡水渔业研究中心/农业部淡水渔业和种质资源利用重点实验室,江苏无锡214081 [3]南京农业大学无锡渔业学院,江苏无锡214081

出　　处：《中国农学通报》2015年第2期107-111,共5页Chinese Agricultural Science Bulletin

基　　金：中央级公益性科研院所基本科研业务费专项资金项目"青虾性别决定机制及性别控制技术的研究"(2013JBFM15);江苏省基础研究计划(自然科学基金)青年基金"青虾精巢发育调控基因的鉴别;克隆及表达研究"(BK2012091);国家"十二五"支撑"青虾新品种选育技术研究"(2012BAD2604-05);江苏省科技支撑计划-农业部分"优质高产抗逆青虾新品种的选育"(BE2012334);江苏省三新工程工程项目"青虾新品种选育"(D2013-6)

摘　　要：为了获得数量多且存活的游离青虾精子,首次采用胰蛋白酶消化日本沼虾精荚的方法,获得了游离日本沼虾的精子。通过观察游离精子形态,并经台盼蓝染色,以每克精荚所获得形态正常、结构完整且不被染色的存活精子数为依据,探究胰蛋白酶处理的最佳条件。在p H 7.1的条件下,设计三因素四水平正交试验,设置不同酶浓度(0.2%、0.4%、0.6%、0.8%)、处理时间(5 min、10 min、15 min、20 min)和处理温度(30℃、35℃、40℃、45℃),根据正交试验结果确定酶处理最佳反应条件。结果表明,酶浓度对获得游离精子影响最大,其次是处理温度和处理时间。酶处理的最适条件为:酶浓度0.8%,处理时间10 min,处理温度40℃。经优化验证试验,表明上述理论组合确为最佳组合。本研究通过酶解法获得较多数量的存活游离精子,为进一步研究日本沼虾精子提供了获得游离精子的方法。The purpose of this paper was to acquire free sperms with high quantity from spermatophores of Mac- robrachium nipponensis by tryptic digestion. Viable sperms of spermatophore per gram were identified by nor- mal morphology, intact structure and not being colored by trypan blue, the optimum enzyme processing condi- tions were determined according to the results of sperm morphology observing and trypan blue dyeing. The re- search was conducted under the condition of pH 7.4 and different reaction temperatures （30℃, 35℃, 40℃ and 45℃, respectively）, trypsinase concentrations （0.2%, 0.4%, 0.6% and 0.8%, respectively） and reaction times （5 min, 10 min, 15 min and 20 min, respectively）. The best reaction condition of trypsinase digestion method was studied by orthogonal tests and the sperm amounts which got from each gram of spermatophores were used as a criterion. According to the results of orthogonal tests, we found that trypsinase concentration was the prima- ry factor, followed by reaction temperature and enzymatic reaction time. The best effect of trypsinase digestion was at trypsinase concentration 0.8%, time 10 rain and temperature 40℃. This method of acquiring free sperms is useful for further study on sperms of Macrobracbiu nipponensis.

关 键 词：日本沼虾 精荚 游离精子 胰蛋白酶 正交试验 

分 类 号：S931[农业科学—渔业资源]