新疆雪莲全长cDNA文库构建及表达序列标签(ESTs)特性分析

以野生新疆雪莲(Saussurea involucrata Kar.et Kit)为研究材料,利用Creator SMARTTMcDNA Library Construction技术构建了雪莲全长cDNA文库,从野生雪莲中提取总RNA和mRNA,用分离到的微量mRNA合成cDNA第1链。通过长距离PCR扩增得到足够量的双链cDNA。将SfiⅠ酶切后并纯化的cDNA片段连接到经SfiⅠ酶切的噬菌体λTripIEX2上,并对噬菌体进行包装,建成cDNA的全长库。经大肠杆菌XL1-Blue平板检测,原始文库滴度达4.03×107(pfu/mL)。随机挑选500个克隆进行序列测定,PCR鉴定结果显示多数插入片段均在500 bp以上。将所测序列经GenBank检索和生物信息学比较,共有56个序列为非冗余数据。其中有14个cDNA片段序列在GenBank上无明显的同源性,42个片段与已报道的基因有较高的同源性。这些EST数据为进一步筛选和克隆雪莲特异性表达基因提供了材料平台,为雪莲基因组数据增补了大量信息。

cDNA library Construction and EST Analysis of Saussurea involucrata Kar.et Kit

A cDNA library from the Saussurea involucrata Kar.was constructed by using CreatorTM SMARTTM cDNA library Construction Protoco1.Total RNA and mRNA were extracted from Sasussured involucrata Kar.et Kir.After synthesizing the first-strand cDNA using limited amount of mRNA isolated from total RNA,the double-strand cDNA was amplified with long-distance PCR method.The ligation of the SfiⅠ digested cDNA to the SfiⅠdigested vector λTripLEx2 was performed and the phage was packed to construct the full length cDNA library.The titer of unamplified library titer of was estimated as 4.03×107(pfu/mL),which was about 500 times coverage of gene expressional profile,the PCR results showed that the size of most inserts was larger than by 500 bp.By searching the NCBI non-redundant nucleotide databases,56 ESTs had no significant similarity to any protein or DNA sequence in the databases.The results showed that 14 cDNA clones are expected to be novel genes,because no sequence homology with any known sequences was found in GenBank batabases.And 42 ESTs with known function were identified by Blastx searched against the NCBI non-redundant nucleotide databases.The results are good enough for further cloning of the specific expression genes and adding much geneties information of Saussurea involucrata Kar.