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皱纹盘鲍(Haliotis discus hannai Ino)胚胎RNA分离和cDNA文库构建
  • ISSN号:0029-814X
  • 期刊名称:《海洋与湖沼》
  • 时间:0
  • 分类:Q132[生物学—普通生物学]
  • 作者机构:[1]中国科学院海洋研究所,青岛266071, [2]中国科学院研究生院,北京100039, [3]浙江大学分析测试中心,杭州310029
  • 相关基金:国家自然科学基金项目,30271018号;国家863计划项目,2004AA626070号;青岛市自然科学基金项目,03-2JZP-9号资助.
中文摘要:

从皱纹盘鲍胚胎样本中分离RNA并构建了cDNA文库。分别收集孵化8、10和12h的皱纹盘鲍胚胎,用抽滤除菌的海水反复悬浮胚胎,将这3个发育阶段的胚胎样本等量混合后用TRIZOL试剂提取总RNA,将提取物用酚-氯仿-异戊醇再抽提2次,分离获得高质量的总RNA。从总RNA中分离mRNA,构建了皱纹盘鲍混合胚胎的cDNA文库,未扩增胚胎文库的滴度为5.0×10^6pfu/ml,重组率为94.12%,插入片段均大干400bp,70.6%克隆的插入片段分布在1000—1500bp之间,扩增后的文库滴度为3.06×10^9pfu/ml。

英文摘要:

In order to clone genes from embryos and compare gene expression differences in different developing stages of Pacific abalone, Haliotis discus hannai Ino, technology of sampling and RNA isolation from embryos were developed, and full length cDNA expression library of whole embryos of the abalone was repor- ted in this paper. Wild adult Pacific abalones from Japan have been used as bloodstock in this experiment. The animals were maintained in China for almost 10 months under natural condition and fed with fresh Laminaria japonica every day. Spawning was carried out in October 2002 for matured abalones. The animals were exposed to air at 75%-80% moisture for desiccation in darkness at 20℃ for 30min. Then each single individual was put into a 20L tank separately and treated with 10L of ultra-violet-irradiated [ 300mW/(h·L) ] seawater at the temperature of 23℃. The UV-treated seawater in each tank was renewed every 30 min until spawning. Artifi- cial fertilization was conducted at 20℃ with the oocytes and sperms from 3 donor parents. After inspecting under a light microscope, three females with high quality oocytes were selected and the eggs isolated from each abalone were combined. Eggs were filtrated by a plankton net to remove impurity such as secretion from the donor abalone. Keep the tank still and let the eggs settle down on the tank bottom, then removed the supernatant carefully and suspended the eggs with fresh seawater at 20℃ for fertilization. Meanwhile, three males were selected and their sperms were combined in the same way as the ooyctes, then diluted the combination by fresh seawater at 20℃. The diluted sperm was added to the egg suspension to a density of about 10 sperm per egg and mixed them gently. 10 minutes after fertilization, zygotes were washed with fresh seawater. Embryos were incubated at 20℃ at the density of 20 eggs in each milliliter by periodical washing at every 40 minutes till trochophore stage. Embryos were respectively sampled at 8, 10, and 12 h after fertilization.E

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期刊信息
  • 《海洋与湖沼》
  • 北大核心期刊(2011版)
  • 主管单位:中国科学院
  • 主办单位:中国海洋湖沼学会
  • 主编:孙松
  • 地址:青岛市南海路7号中科院海洋所内
  • 邮编:266071
  • 邮箱:
  • 电话:0532-82898753
  • 国际标准刊号:ISSN:0029-814X
  • 国内统一刊号:ISSN:37-1149/P
  • 邮发代号:2-421
  • 获奖情况:
  • 2000年中国科学院优秀期刊一等奖,2000年山东省科委、新闻出版黑署评估优秀级,中国期刊方阵“双百”期刊
  • 国内外数据库收录:
  • 美国化学文摘(网络版),英国农业与生物科学研究中心文摘,英国动物学记录,日本日本科学技术振兴机构数据库,中国中国科技核心期刊,中国北大核心期刊(2004版),中国北大核心期刊(2008版),中国北大核心期刊(2011版),中国北大核心期刊(2014版),中国北大核心期刊(2000版)
  • 被引量:21021