西洋参PqGA2ox基因的克隆与序列分析

Cloning and sequence analysis of PqGA2ox gene in Panax quinquefolium

目的克隆、分析西洋参Panax quinquefolium种子萌发过程的赤霉素2-氧化酶(gibberellin 2-oxidase,GA2ox)基因。方法从前期由高通量测序得到78207条unigenes基因的注释信息中挖掘出与赤霉素合成和分解代谢相关的基因序列,得到11条GA2ox基因相关序列,通过序列比对分析确定GA2ox的转录本。根据选定的unigene序列设计引物,采用PCR方法扩增西洋参GA2ox的cDNA全长;利用生物信息学方法分析所得基因,并用实时荧光定量PCR技术进行表达分析。结果得到一条长度为987bp,编码328个氨基酸残基的西洋参GA2ox基因,命名为PqGA2ox。生物信息学预测PqGA2ox蛋白不含跨膜区,不含信号肽,具有依赖于2-酮戊二酸和Fe2+的双加氧酶超家族的保守结构域。实时荧光定量PCR结果显示在形态休眠中期和生理休眠中期的表达量低于其休眠起始期和解除休眠期。结论首次获得西洋参种子PqGA2ox基因的编码区序列,为进一步研究西洋参种子解除休眠的分子机制奠定基础。

Objective To clone and analyze the gibberellin 2-oxidase（GA2ox） gene of Panax quinquefolium in seed germination. Methods Gene sequences about gibberellins synthesis and catabolism were found out from annotation information of 78 207 unigenes obtained by high-throughput sequencing in the early study. Then one transcript coding GA2 ox was obtained from 11 unigenes related to GA2 ox. Primers were designed according to selected sequence to get the full-length c DNA of P. quinquefolium using PCR method. Predictive analysis and expression analysis of Pq GA2 ox were obtained by bioinformatics and real-time PCR. Results A GA2 ox gene containing 987 bp encoding 328 amino acids was cloned and named as Pq GA2 ox. Bioinformatics analysis showed that Pq GA2 ox had no transmembrane domain or signal peptide, but had the 20G-Fell_Oxy conserved domains. The expression level of Pq GA2 ox was lower in the metaphase of morphological dormancy and physiological dormancy than that in the intitial period and release period based on real-time PCR analysis. Conclusion The Pq GA2 ox gene from the seeds of P. quinquefolium is cloned for the first time, which will provide a foundation for the molecular mechanism of dormancy release of P. quinquefolium.