家蚕LongSAGE文库鉴定及延长标签的改进GLGI方法

Improved GLGI Method to Identify Tags from LongSAGE of Silkworm,Bombyx mori

结合标签的基因序列延伸(GLGI)是随着基因表达序列标签(SAGE)应运而生的一种鉴定新标签的技术。为了克服该技术操作繁琐、成本高的弊端,根据已经构建的家蚕限性茧色品种的2个LongSAGE文库,选择30个差异表达的标签(tag)进行GLGI延伸。尝试了几个简化,(1)用totalRNA代替mRNA进行dsDNA的合成;(2)调整磁珠吸附DNA的顺序;(3)在PCR反应中用普通的DNA聚合酶代替高保真酶(Pfu)。结果显示,30个标签有27个获得有效扩增,扩增序列真实包括了SAGE标签的17bp序列,3个标签的结果符合NCBI的家蚕数据库已有注释,13个未注释标签经过延长后得到了注释,11个标签序列为待确定的新基因序列。这种简化能够作为家蚕SAGE文库其他tag和新基因研究的有效简化操作体系。与传统的方法相比,可减少成本30%以上,缩短操作时间20%。

Generation of Longer cDNA Fragments from SAGE tags for Gene Identification（GLGI）being as a new tag identification technique was brought about with the serial analysis of gene expression （SAGE）. Its result is an EST in the cDNA isolated by the 3 ＇end and with a ployA tail. Their steps are complicated and the cost is relatively high. Based on the improvement details reported, some new improvement was conducted to aim directly at two LongSAGE libraries of silkworm constructed by our laboratory. 30 differentially expressed tags were selected to improve the GLGI method：（1） Total RNA was used to synthesis dsDNA instead of mRNA; （2） The order of beads adsorption of DNA was adjusted, so as to diminish loss of template and simplify process; （3） DNA ordinary polymerase in PCR reactions was used instead of the high-fidelity enzyme Pfu. Then sequence results were analyzed BLAST against Bombyx mori database on NCBI. Annotation result of 3 tags was conformity with known annotation, it provided a verification proof that improved test is reliable. There are also some tags without annotation, which can be taking as a foundation for further study of novel genes. A valuable reference system for other tags and novel genes research of SAGE library were tried to provide.