皱纹盘鲍外套膜cDNA表达文库的构建  

Construction of Mantle cDNA Expressive Library from Abalone Haliotis discus hannai Ino

作　　者：李静[1,2] 张文兵[1] 麦康森[1] 张璐[1,3] 艾庆辉[1] 徐伟[1] 谭北平[1] 马洪明[1] 刘付治国[1] 

机构地区：[1]中国海洋大学教育部海水养殖重点实验室,山东青岛266003 [2]中国石油大学华东生物工程与技术中心,山东青岛266555 [3]广东粤海饲料有限公司生物工程中心,广东湛江524017

出　　处：《中国海洋大学学报（自然科学版）》2008年第5期744-748,共5页Periodical of Ocean University of China

基　　金：国家自然科学基金项目(30200215)资助

摘　　要：皱纹盘鲍(Haliotis discus hannaiIno)的壳是生物矿化组织的杰出代表,其中的生物矿化蛋白质对其结构和性能发挥了关键性的作用。鉴于对皱纹盘鲍生物矿化功能基因信息研究的缺乏,文中采用SMART(Switching Mechanism At5’end of RNA Transcript)技术,构建皱纹盘鲍外套膜组织的全长cDNA表达文库。按Trizol试剂盒的操作程序提取皱纹盘鲍外套膜组织总RNA,以此为模板,通过PowerScript逆转录酶逆转录合成第一链cDNA;再以第一链cDNA产物为模板,用LD-PCR法合成双链cDNA。双链cDNA产物经SfiⅠ酶切和ChromaSpin 400柱分级分离后,与λTriplEx2载体进行连接,体外蛋白包装,即获得皱纹盘鲍外套膜cDNA原始文库。测定其滴度为1.2×106pfu/mL,重组率为95%。PCR扩增方法测得插入cDNA片段长度在0.5～2 kb之间,平均为1.4 kb。进行文库的扩增,扩增后的cDNA文库的滴度为4×109pfu/mL,重组率93.6%。In order to understand the molecular mechanism of the shell biomineralization in abalone, the SMART（Switching Mechanism At 5＇end of RNA Transcript） technology was used to construct eDNA expressive library of the mantle from abalone Haliotis discus hannai Ino. Total RNA from mantle was isolated using Trizol reagent. Moreover, the PowerScript reverse transcriptase was used to synthesize and anchor the firststrand cDNA. Following reverse transcription, LD-PCR was performed using a modified oligo（dT） and an anchor primer to enrich the cDNA population for full-length sequences. The ds cDNA products were digested by sfi Ⅰ enzyme and fractionated with CHROMA SPIN 400 column. The cDNAs with size of larger than 0.5 kb were recovered and ligated to λTriplEx2 vector. The λ phage packing reaction for the ligation was performed to produce an unamplified library. The unamplified library contained 1.2 × 10^6 independent clones, in which the recombined clones with an average insert size of 1.4 kb were range form 0.5 to 2 kb. The percentage of recombination is as high as 95 %. The titer of amplified library is 4 × 10^9 pfu/mL and the percentage of reeombinants is 93.6%.

关 键 词：皱纹盘鲍 外套膜 CDNA表达文库 生物矿化 

分 类 号：S917[农业科学—水产科学]