利用SMART技术构建烟草茎全长cDNA文库

Construction of tobacco stem full-length cDNA library with SMART technique

以烟草不同生长时期茎混合为材料,采用SMART(switching mechanism at5'end of RNA transcript)技术构建了茎全长cDNA文库.原始文库的库容为5.64×106个独立克隆,重组率达99%以上,插入片段集中在0.75-2.0 kb之间,平均长度约为1.2 kb.随机挑取部分克隆进行测序,并进行生物信息学分析,37.5%的序列烟草已有报道,62.5%为烟草未报道的基因,45.8%是未知功能的基因,表明所构建文库达到了用于目的基因分离筛选和新基因克隆的建库要求.

Using mixture of tobacco stems of different periods for the material,and modified SMART（switching mechanism at 5′end of RNA transcript） technology,we constructed a stem full-length cDNA library.The primary library capacity was 5.64×106,the recombination rate was more than 99%.The insert fragments were mostly 0.75-2.0 kb in size and the average size was approximately 1.2 kb.Part of clones were randomly selected,sequenced and preliminary analysis was performed using bioinformatics software,which indicated that 37.5% of sequences have been reported and 62.5% have not in tobacco,while 45.8% of them were unknown function genes.The results show that the constructed library should be qualified for the requirement for cloning and screening novel genes.