Objective:To clone the full-length cDNA sequences of gibberellic oxidase genes in Paris polyphyllavar.yunnanensiswhich encode the key enzymes involved in GA biosynthetic pathway and analyze their bioinformatic properties.Methods:The sequences of candidate genes in our previous transcriptomic database of Paris polyphyllavar.yunnanensis were used to design the gene-specific PCR primers for RT-PCR cloning of their full-length coding regions.The amplified cDNA fragments were transformed into pMD18-T vector and E.coli DH5α competent cells for sequencing.A series of bioinformatic analysis were carried out,including sequence comparison of homologies,reconstruction of GAox phylogenetic tree,domain search,and 3D structure prediction.Results:Sequencing results showed that the full-length CDS of six PpGAoxgenes were about 1000 bp.Six genes,named as PpGA2ox1,PpGA2ox2,PpGA2ox3,PpGA2ox4,PpGA20ox1 and PpGA3ox1,belonged to three GAox subfamilies,encoding 300-400 amino acids.Their theoretical pI values were about 4-7 and in a positive charge.The proteins of PpGA2ox1,PpGA2ox2 and PpGA2ox4 were unstable and PpGA2ox3,PpGA20ox1 and PpGA3ox1 were stable.However,all of them were hydrophilic,signal peptide and had no transmembrane domain.PpGA2oxl was predicted to be located in the chloroplast,and the rest d in the cytoplasm.PpaGAox proteins had higher homologous sequences with those of other species w and strong domain conservatism.Conclusion:The full-length cDNA sequences of six GAoxwere cloned for the first time from P.polyphylla var.yunnanensis and the bioinformatic analysis were carried out.%目的:克隆编码滇重楼赤霉素合成代谢途径中关键酶GA氧化酶(Gibberellic oxidase,GAox)的全长cDNA序列,并进行生物信息学分析.方法:根据滇重楼转录组测序序列,设计特异性PCR引物,利用RT-PCR技术扩增目标基因编码区的全长序列连接至pMD18-T载体上,进行序列测定及序列同源性比较分子系统进化树构建、结构域搜索及蛋白质三维结构预测等生物信息学分析.结果:成功克隆了滇重楼的的6条GAox基因,大小在1000 bp左右,分别命名为PpGA2ox1、PpGA2ox2、PpGA2ox3、PpGA2ox4、PpGA20ox1和PpGA3ox1.6条基因分别属于GA氧化酶三个不同的亚家族.滇重楼GAox蛋白氨基酸数量均在300～ 400,理论等电点在4～7左右,带正电荷.PpGA2ox1、PpGA2ox2和PpGA2ox4为不稳定蛋白,PpGA2ox3、PpGA20ox1和PpGA3ox1为稳定蛋白,均为没有跨膜结构域和信号肽的亲水蛋白.除了PpGA2ox1亚细胞定位在叶绿体,其他蛋白均定位在细胞质.滇重楼GAox蛋白与不同物种同源序列的序列相似性较高,保守性比较强.结论:首次从滇重楼中克隆了6条GAox基因并对其进行了初步的生物信息学分析.
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