皱纹盘鲍(Haliotis discushannai Ino)胚胎RNA分离和cDNA文库构建  

ISOLATION OF RNA AND CONSTRUCTION OF FULL-LENGTH CDNA LIBRARY OF EMBRYO HALIOTIS DISCUS HANNAI INO

作　　者：刘晓[1] 赵敏[1,2] 高其康[3] 张国范[1] 

机构地区：[1]中国科学院海洋研究所 [2]中国科学院研究生院北京100039 [3]浙江大学分析测试中心

出　　处：《海洋与湖沼》2006年第4期355-360,共6页Oceanologia Et Limnologia Sinica

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;032JZP9号资助

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5.0×106pfu/ml,重组率为94.12%,插入片段均大于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3.06×109pfu/ml。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

关 键 词：皱纹盘鲍 胚胎 CDNA文库 基因表达 

分 类 号：Q132[生物学—普通生物学]