三丁基锡暴露条件下杂色鲍肝胰腺均一化cDNA文库的构建  

Construction of Normalized cDNA Library from Hepatopancreas of Abalone Haliotis diversicolor supertexta Exposure to Tributyltin

作　　者：贾锡伟[1,2] 张子平[3] 邹志华[2] 王艺磊[2] 

机构地区：[1]厦门大学近海海洋环境科学国家重点实验室厦门大学环境科学研究中心,厦门361005 [2]集美大学水产学院福建省高校水产科学技术与食品安全重点实验室,厦门361021 [3]德克萨斯州立大学化学与生物化学系,美国圣马科斯tx78666

出　　处：《生物技术通报》2009年第11期136-139,144,共5页Biotechnology Bulletin

基　　金：厦门市科技计划项目(3502Z20055024);国家自然科学基金(20877034);集美大学创新团队基金(2008A001)

摘　　要：用RDP试剂提取三丁基锡暴露诱导的杂色鲍肝胰腺总RNA,经Oligotex纯化得到mRNA;应用SMART技术合成双链cDNA,双链特异核酸酶(DSN)进行双链cDNA的均一化,构建了杂色鲍三丁基锡暴露诱导下的均一化cDNA文库。原始文库的库容为4.3×106CFU/ml,重组率为97.9%。从文库中随机挑选了3288个克隆进行测序,得到3048个高质量EST序列,其中有370条Contigs,2103条Singlets,Unigenes共2473条,冗余率为18.86%。以上结果说明该文库质量较好,为进一步筛选相关功能基因打下基础;较低的冗余率说明该文库值得继续使用大规模ESTs测序的方法寻找相关功能基因,并为进一步使用基因芯片技术研究相关功能基因的表达谱提供便利。A normalized cDNA library from hepatopancreas of abalone Haliotis diversicolor supertexta exposure to Tributyhin was constructed. Total RNA were prepared using RDP reagent. Oligotex （QIAGEN） was used to separate the mRNA from total RNA. The first strand cDNA was synthesized by transcription of mRNA with the SMART technique. The LD-PCR was performed using a modified SMART primer as the primer set,and the first -strand cDNA as the template to synthesize double strand cDNA. Double strand cDNA was normalized using Duplex-Specific Nuelease （DSN）. The containing capacity of the unamplified cDNA library was 4.3 × 10^6 CFU/ ml with a recombinant rate of 97.9%. A total of 3 288 clones were random selected to be sequenced, and 3 048 high quality ESTs were generated. After proeessing,a total of 2 473 unigenes comprising 370 eontigs and 2 103 singlets were obtained. The redundancy was 18.86%. These results indicate that the normalized eDNA library is suitable to be used for further cloning and analysis of genes related to tributyltin exposure.

关 键 词：三丁基锡 杂色鲍 均一化cDNA文库 

分 类 号：Q78[生物学—分子生物学] X132[环境科学与工程—环境科学]