摘要DSN（duplex specific nuclease）是最近发现的一种热稳定核酸酶，该酶能够降解双链DNA和DNA-RNA的杂交体中的DNA而对单链核酸分子几乎没有作用。基于DSN的核酸均一化技术是一项操作简单，均一化效果好的新技术。为高效获得与绿僵菌产孢相关的全长cDNA，采用DSN均一化技术与SMARTTM (switching mechanism at 5′end of RNA transcript)建库技术相结合的方法，构建了杀虫真菌绿僵菌菌株CQMa102产孢时期的均一化全长cDNA文库。经检测，原始文库滴度为2.1×106 cfu/mL，库容量超过6×106。随机挑取100个克隆，PCR方法测得文库重组率大于95％, 插入片断平均长度1500bp。小规模测序分析表明，全长基因的比例超过60％。对组成性表达基因3-磷酸甘油醛脱氢酶G3PDH和微管蛋白β-tubulin 均一化前后丰度检测表明，其丰度均有大幅度的下降。
Abstract：DSN (duplex-specific nuclease ) displays a strong preference for cleaving ds DNA and DNA in DNA–RNA hybrid duplexes compared with ss DNA and RNA irrespective of sequence length , as described recently. The technique termed DSN is a simple and effective cDNA normalization method. In order to screem and isolate new and full-length sporulation-specific genes of entomopathogenic fungus Metarhizium in the sporulation stage with great efficiency, a combined DSN normalization method with the SMARTTM (switching mechanism at 5′end of RNA transcript) library construction method was used to construct a normalization and full-length cDNA library of entomopathogenic fungus Metarhizium in the sporulation stage. The titer of unamplified cDNA library was 2.1 ×106 cfu/mL and contained 6×106 independent clones.The average cDNA inserts size was 1500bp with a recombination rate of 95%.Sequencing results and bioinformatics analysis of random picked clones indicated that the ratio of full-length cDNA was about 60％.The presentation levels of the abundant transcripts G3PDH and β-tubulin decreased obviously compared with non-normalized samples.
张石柱 王中康 彭国雄 殷幼平 谢磊 刘静 夏玉先. 利用DSN和SMARTTM技术构建绿僵菌产孢时期均一化全长cDNA文库[J]. , 2007, 15(5): 0-.
. Construction of normalization and full-length cDNA library of Metarhizium in the sporulation stage based on DSN and SMARTTM. , 2007, 15(5): 0-.
