摘要
电子克隆是基因克隆的新策略。以小麦胞质葡萄糖 6 磷酸脱氢酶cDNA (Tagpdl克隆 )序列为信息探针 ,在GenBank水稻nr数据库中找到高度同源的水稻基因组序列 ,通过人工序列拼接及RT PCR确认得到了水稻该基因的全长cDNA序列 ,命名为OsG6PDH。OsG6PDH与小麦Tagpdl克隆的DNA一致率为 88% ,推导的氨基酸序列与小麦、番茄、烟草的胞质葡萄糖 6 磷酸脱氢酶基因的一致率分别为 89%、79%、80 %。经RT PCR表达谱分析 ,OsG6PDH在水稻幼穗、胚、根、叶中都有表达 ,在幼穗与根中表达略高。另外 。
In silico cloning was a new strategy of gene cloning developed with the development of genome, EST projects and bioinformatics. Using wheat glucose 6 phosphate dehydrogenase cDNA (clone:Tagpdl) sequence as a querying probe, one highly homologous BAC clone sequence was obtained from rice sequence database of GenBank and the putative cDNA sequence of rice glucose 6 phosphate dehydrogenase was assembled according to the wheat clone. Futhermore, the full length cDNA of rice glucose 6 phosphate dehydrogenase was cloned by RT PCR with two primers designed based on this assembled cDNA sequence. Since this fragment contained a complete ORF of 1515bp with a stop codon in its upstream and poly(A) signal in its downsteam, it could be concluded that a full length gene (GenBank accession number AY078072), which was named as OsG6PDH . Homology analysis of OsG6PDH showed a 88% identity with wheat and the deduced amino acid showed 89%, 79% and 80% omology with G6PDH from wheat, tomato and tobacco respectively. OsG6PDH was expressed in inflorescence, embryo, root and leaf of rice, with a slightly higher in inflorescence and root. It was also discussed in this paper that the application of in silico cloning in the isolation of functional genes from rice.
基金
教育部高校博士点基金资助项目~~
