摘要
采用Trizol试剂提取副溶血弧菌感染的九孔鲍血细胞的总RNA,经Oligotex纯化得到mRNA.根据SMART技术原理合成双链cDNA.双链cDNA采用双链特异核酸酶进行cDNA的均一化,构建成九孔鲍血细胞的均一化全长cDNA文库.原始文库的库容为3.4×106cfu/cm3,重组率为92.3%,扩增后文库的滴度为2.6×1011cfu/cm3以上.从文库中随机挑取897个克隆,测序获得高质量ES-Ts814条,其中有23条Contigs;762条Singlets,Unigenes共785条,冗余率只有3.56%.以上结果说明所构建的文库质量好,完全可以满足后续基因克隆和表达序列标签测序工作的需要.
A normalized full-length haemocytes cDNA library was constructed in order to clone genes involving in Vibrio parahaemolyticus defenses from Haliotis diversicolor supertexta. Total RNAs were prepared from haemocytes using TRZOL reagent. Oligotex （QIAGEN） was used to separate the mRNA from total RNA. The ＂anchor firststrand cDNA＂ containing a symmetrical sfiI restriction enzyme sites （A＆B） was synthesized by transcription of mR- NA with the SMART technique. Double strand cDNA was normalized using Duplex-Specific Nuclease （DSN）. The containing capacities of the unamplified cDNA library was 3.4 × 10^6 cfu/cm3 with a recombinant rate of 92. 3 % and that of amplified one was more than 10^11 cfu/cm3. A total of 897 clones was random selected to be sequenced and 814 high quality expressed sequence tags were obtained. Among them,there were 23 Contigs and 762 singlets,the redundancy was only 3.56%. The results indicated that a high quality library was meet the needs for the consequence gene clone and expressed sequence tag analvsis.
出处
《台湾海峡》
CAS
CSCD
北大核心
2008年第3期278-285,共8页
Journal of Oceanography In Taiwan Strait
基金
福建省自然科学基金资助项目(B0440003)
福建省教育厅基金资助项目(JA004241)
福建省科技重点资助项目(2007N0048)
集美大学优秀青年骨干教师基金资助项目(2006B001)
关键词
CDNA文库
均一化
九孔鲍
副溶血弧菌
血细胞
cDNA library
normalized
Haliotis diverslcolor supertexta
Vibrio parahaemolyticus.
haemocytes
