滇重楼GAox基因的克隆与生物信息学分析

Cloning and Bioinformatic Analysis of GAox Genes from Paris polyphylla var. yunnanensis

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。

Objective： To clone the full-length cDNA sequences of gibberellic oxidase genes in Paris polyphyUavar, yunnanens/swhich 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 polyphyl- lavar, yunnanensis were used to design the gene-specific PCR primers for RT-PCR cloning of their full-length coding re- gions. The amplified eDNA fragments were transformed into pMD18-T vector and E. coli DI-I5ct competent cells for sequen- cing. 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 PpGA2oxl, PpGA2ox2, PpGA2ox3, PpGA2ox4, PpGA2Ooxl and PpGA3oxl, 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 PpGA2oxl, PpGA2ox2 and PpGA2ox4 were unstable and PpGA2ox3, PpGA2Ooxl and Pp- GA3oxl 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 eDNA sequences of six GAoxwere cloned for the first time from P. polyphylla var. yunnanensis and the bioinformatic analysis were carried out.