铁皮石斛HDR基因克隆及真菌诱导子对其表达和生物碱含量的影响

Cloning of the HDR Gene and the Effect of the Fungus Fusarium oxysporum elicitor on the Expression Levels and Contents of Alkaloids in Dendrobium officinale Kimura et Migo

以铁皮石斛原球茎为材料,采用同源克隆的方法,成功得到了铁皮石斛Do-HDR基因的全长,并通过q PCR对其相对表达量进行分析。结果表明:Do-HDR基因全长1 784 bp(Gen Bank登录号KJ946381),5′UTR为40 bp,3′UTR为361 bp,开放阅读框为1 382 bp,编码460个氨基酸。生物信息学分析结果表明,该基因编码的蛋白为稳定的亲水蛋白,不具有跨膜结构,不含信号肽,定位于内质网。采用灭活的尖孢镰刀菌菌液作为诱导子,分别以0、100、200、500、1 000 mg/L的浓度培养3 d后测定总生物碱含量,结果表明,总生物碱含量呈现先上升后下降的趋势;当诱导子浓度为200 mg/L时,总生物碱含量达到最大值。在此基础上通过q PCR分析Do-HDR基因的相对表达量,结果发现Do-HDR基因相对表达量在诱导子浓度为200 mg/L时也到达最大值。因此,推测Do-HDR基因与生物碱的合成与积累有关。

In this experiment, the PLBs were used as experimental materials, to clone the full-length of Do-HDR gene by homology cloning techniques, and the relative expression levels were analyzed by q PCR in Dendrobium officinale Kimura et Migo. The full-length of Do-HDR was 1 784 bp（Gen Bank accessionr number is KJ946381）,5′UTR was 40 bp, 3′UTR was 361 bp, the ORF was 1 382 bp, encoding 460 amino acids. Bioinformatics analysis indicated that Do-HDR encoded a stable hydrophilic protein, without transmembrane structure and without the signal peptide, and it was located in endoplasmic reticulum. Then, the inactivated fungus Fusarium oxysporum was used as the elicitor to study the effect of the elicitor on the total alkaloids, and the treated concentrations were 0,100, 200, 500, 1 000 mg/L for 3 days＇ culture, and the determined the contents of the total alkaloids. The result showed that the contents of the total alkaloids rose first,then fell and finally reached the maximum. On this basis,q PCR was used to analyzed the relative expression levels of gene Do-HDR, and the results showed that when the concentration of elicitor was 200 mg/L, the relative gene expression levels reached the maximum, which was the same as the trend of the total alkaloids. So, it was speculated that the Do-HDR gene was associated with alkaloid synthesis and accumulation.