Direct cloning of differential expression genes with fulllength by a modified solid subtractive hybridization
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摘要: 该文集多种分离差异表达基因方法的优点于一体,自行优化了一种基于固相扣除杂交和聚合酶链式反应(PCR)技术直接克隆全长差异表达基因的策略.该策略中的所有操作步骤,包括poly (A)+mRNA的分离,cDNA的合成和扣除杂交都是在磁珠上进行的,操作简单易行.扣除杂交是用结合在磁珠上的以oligo(dT)为引物合成的对照材料(Driver)的cDNA第一链,直接扣除加有接头的处理材料(Tester)cDNA第二链中的共有序列.多次反复使用Driver一链与Tester二链进行杂交,能有效去除Tester二链中的共有序列.磁珠的分离能最小限度地减少每次杂交造成的损失.最后一轮扣除后的Tester二链中,含有大量差异表达序列,用Tester二链特有的接头引物进行扩增,能进一步提高扣除效率.该文选用此扣除方法,成功分离到了低温驯化沙冬青幼苗和对照苗2个群体的18个差异表达克隆,其中有4个克隆是全长cDNA序列.经序列分析发现,这些基因都与低温相关,进一步说明,该方法可用于克隆未知差异表达基因.Abstract: A simple and efficient cDNA subtraction technique was developed based on solid_support magnetized beads and polymerase chain reactions to identify the differential expression genes. This strategy is efficient, easy to use and the whole process including mRNA isolation, cDNA synthesis and subtractive hybridization is performed on magnetized beads. It can be applied to any comparative studies on gene expression in different populations. Driver mRNA isolation and first strand cDNA synthesis were performed by biotinylated oligo(dT) primer on magnetized beads. Tester mRNA isolation, doublestrand cDNA synthesis and adapter ligation were also performed by biotinylated oligo(dT) primer on magnetized beads.Then the second strand was eluted and subtracted by first strand of driver cDNA hung on the beads for the efficient removal of common cDNAs. Differentially expressed Tester cDNA was specifically amplified with adapter primer and oligo(dT) primer and then cloned. Dot blotting confirmed that randomly selected clones were differential transcripts of Tester. In the present study, some differentially expressed genes, including several fulllength cDNAs, were isolated and screened from cold-acclimated Ammopiptanthus mongolicus seedlings by this modified method. Sequence analysis showed that the screened cDNAs shared homologies with previous reported genes involved in plant cold adaptation. 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