水稻幼苗SAGE文库的构建和测序

Constructing and Sequencing of SAGE Libraries of Rice Seedlings

基因表达系列分析(Serial analysis of gene expression,SAGE)是功能基因组学研究中的一项功能强大的工具.对水稻幼苗SAGE文库的构建进行了探索试验,其主要操作步骤为:以磁珠法提取PolyA mRNA用于合成双链cDNA,再经过一系列的酶切、加接头序列、连接、PCR扩增,获得到26-bp的水稻双标签体,再将26-bp Ditag连接成标签链接体,并将其克隆进pZero-1载体中用于测序.DNA测序结果证明获得了水稻的SAGE标签.试验的各个中间步骤的结果均经过PCR验证.

Serial analysis of gene expression （SAGE） is a powerful tool in functional genomics. In this experiment,a rice SAGE library is succeeded constructed following the protocols as described in the SAGE kit manual. Briefly, 2-strands cDNA is synthesized from mRNA prepared by Magnetic Beads method. By a serial enzymatic cutting, ligating, adding adaptor sequence and PCR amplification,the 26-bp rice Ditags were obtained. Subsequently, the 26 -bp Ditags were connected into concatemers, which were then cloned into pZero-1 vector for sequencing. The DNA sequencing results demonstrate that the tag sequences were derived from rice mRNA species. During the process of constructing SAGE library, the result of every experiment was confirmed by PCR before starting the subsequent step.