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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%。
The shell of Haliotis discus hannaiIno is an outstanding representative of biomineralized tissue where biomineralized proteins play a key role in its structure and properties. In view of the lack of information on the biomineralization of functional genes of Haliotis discus hannai, a full-length cDNA expression library was constructed based on the SMART (Switching Mechanism At5 ’end of RNA Transcript) technique. The Trizol kit was used to extract the total RNA from the mantle tissue of abalone Haliotis discus hannai. The first strand cDNA was reverse transcribed by PowerScript reverse transcriptase. The first strand cDNA was used as a template, Synthesis of double-stranded cDNA. The double-stranded cDNA product was digested with SfiI and fractionated on a ChromaSpin 400 column, ligated with the λTriplEx2 vector, and packaged in vitro to obtain a cDNA library of abalone mantle cDNAs. The titer was 1.2 × 106pfu / mL, and the recombination rate was 95%. The length of inserted cDNA fragment was between 0.5-2kb and the average was 1.4kb. The library was amplified. The amplified cDNA library had a titer of 4 × 109pfu / mL and a recombination rate of 93.6%.