太子参2个赤霉素2-氧化酶基因的克隆与序列分析

Molecular cloning and sequence analysis of two GA2ox genes in Pseudostellariae Radix

目的克隆太子参Pseudostellariae Radix赤霉素2-氧化酶(gibberellin2-oxidase,GA2ox)基因,并进行生物信息学和表达分析。方法通过对太子参转录组数据库的分析,得到2个太子参GA2ox基因PhGA2ox1和PhGA2ox8。。采用反转录PCR(RT-PCR)和特异PCR技术获得2个基因的蛋白编码区(coding sequence,CDS),并对其编码的蛋白进行生物信息学分析。采用实时荧光定量PCR(qPCR)检测基因的表达模式。结果生物信息学分析表明,PhGA2ox1和PhGA2ox8的CDS区分别长981和1 056 bp,编码326和351个氨基酸残基,蛋白相对分子质量分别为36 871.3和40 169.9,理论等电点为8.66和6.91,具有GA2ox的保守结构域DIOX_N和20G-Fell_Oxy,但均无明显疏水区,无跨膜结构域,无信号肽,为稳定蛋白。序列系统进化分析显示植物GA2ox蛋白可被分为3个亚类,PhGA2ox1和PhGA2ox8分别归入Class I和Class III亚类。qPCR分析显示,PhGA2ox1在不同组织器官中的表达基本恒定,而PhGA2ox8在块根木质部的表达量却显著高于在其他组织器官中的表达量(P<0.05)。结论首次获得PhGA2ox1和PhGA2ox8基因的编码区序列,为进一步分析2个基因在太子参生长发育过程中的作用奠定了基础。

Objective To clone the gibberellin 2-oxidase（GA2ox） genes from Pseudostellaria heterophylla and perform the bioinformatic and expression mode analysis. Methods According to P. heterophylla transcriptome annotation, two transcript codings of GA2ox were identified. The full-length c DNA PhGA2ox1 and PhGA2ox8 were determined using RT-PCR. Then the bioinformatic analysis of these genes and encoded proteins were performed. The coding sequences of PhGA2ox1 and PhGA2ox8 were amplified by PCR. And then analyzed the bioinformation of these sequences. The expression levels of PhGA2ox1 and PhGA2ox8 were analyzed using qPCR. Results Bioinformatic analysis showed that PhGA2ox1 contained 981 bp and encoded a predicted protein of 326 amino acids with molecular weight of 36 871.3 and isoelectric point（PI） of 8.66; PhGA2ox8 contained 1 056 bp and encoded a predicted protein of 351 amino acids with molecular weight of 40 169.9 and PI of 6.91. Both PhGA2ox1 and PhGA2ox8 contained GA2ox conserved domains DIOX_N and 20G-Fell_Oxy, without obvious hydrophobic region, transmembrane domain and signal peptide. Phylogenetic analysis showed that the GA2ox of plant could be divided into three classes, PhGA2ox1 bolongs to Class I and PhGA2ox8 belongs to Class III. The qPCR. showed that the expression of PhGA2ox1 in different tissues and organs of P. heterophylla was constant, but the expression of PhGA2ox8 in root xylem was significantly higher than that in other tissues and organs（P〈0.05）. Conclusion The PhGA2ox1 and PhGA2ox8 genes are cloned for the first time, and provide a foundation for they may play an important role in the growth and development process of P. heterophylla.