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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 seed germination. Methods The gene sequences related to gibberellin synthesis and catabolism were excised from the annotation information of 78,207 unigenes genes obtained by high-throughput sequencing in the early stage. Eleven GA2ox gene-related sequences were obtained, and the transcripts of GA2ox gene were determined by sequence alignment . The primers were designed according to the selected unigene sequence, and the full-length cDNA of GA2ox in American ginseng was amplified by PCR. The gene was analyzed by bioinformatics method and analyzed by real-time fluorescence quantitative PCR. The results obtained a length of 987bp, encoding 328 amino acid residues GA2ox American ginseng gene, named PqGA2ox. Bioinformatics predicts that the PqGA2ox protein contains no transmembrane domain, no signal peptide, and a conserved domain that is dependent on the dioxygenase superfamily of 2-oxoglutarate and Fe2 +. The results of real-time PCR showed that the expression level in mid-term and mid-term dormancy was lower than that of dormancy and dormancy. Conclusion The coding region sequence of PqGA2ox gene in American ginseng seeds is obtained for the first time, which lays the foundation for further study on the molecular mechanism of American ginseng seed dormancy release.