迷人杜鹃花青素还原酶基因RaANR1克隆及功能分析

Cloning and functional analysis of Rhododendron agastum anthocyanidin reductase gene RaANR1

  • 摘要: 【目的】 克隆迷人杜鹃花青素还原酶(ANR)基因RaANR1,并分析RaANR1基因对花青素和原花青素合成的调控功能,为迷人杜鹃花色遗传改良及ANR1基因在植物代谢工程中的应用提供理论依据。【方法】 以迷人杜鹃花朵为材料,克隆获得RaANR1基因,利用生物信息学软件对其进行分析,利用蘸花法和注射法将RaANR1分别转入野生型拟南芥和烟草中,经抗性筛选和反转录PCR(RT-PCR)验证获得转基因植株后,使用分光光度计检测拟南芥幼苗和烟草花朵花青苷和原花青素含量。利用实时荧光定量PCR检测烟草花青苷和原花青素合成过程中内源基因的相对表达量。【结果】 成功克隆获得RaANR1基因,编码区(CDS)全长为1002 bp,编码333个氨基酸残基,属于稳定的非分泌型亲水性蛋白,其二级结构中α-螺旋和无规则卷曲占比较高,含有ANR保守的NADPH/NADP结合域、活性位点及底物结合位点。RaANR1与茶树CsANR、柿子DkANR和兔眼蓝莓VaANR的氨基酸序列相似性分别为82.49%、80.88%和89.79%。RaANR1与VaANR的亲缘关系最近且保守基序最相似。与野生型拟南芥相比,2个拟南芥转基因株系幼苗子叶和下胚轴颜色由紫色变为绿色,且花青苷含量较野生型极显著降低(P<0.001),但2个转基因株系的原花青素含量较野生型显著(P<0.05,下同)或极显著(P<0.001)升高。与野生型烟草相比,2个烟草转基因株系花色明显变浅,且其花朵中的花青苷含量较野生型均极显著降低(P<0.001、P<0.01),但原花青素含量极显著(P<0.001)或显著升高。与野生型相比,2个转基因株系中NtF3'5'HNtDFRNtANSNtANR1基因显著上调表达,而NtF3'HNtANR2基因与野生型无显著差异(P>0.05)。【结论】 RaANR1基因的过表达使拟南芥幼苗子叶及烟草花朵中的花青苷含量大幅降低,而原花青素含量大幅升高,表明RaANR1基因可通过调控原花青素和花青素的合成改变花朵颜色。

     

    Abstract: 【Objective】 The aim of this study was to clone Rhododendron agastum anthocyanidin reductase (ANR) gene RaANR1, analyze its regulatory function in the biosynthesis of anthocyanidins and proanthocyanidins, and provide theoretical basis for the genetic improvement of flower color in Rhododendron agastum and the application of RaANR1 gene in plant metabolic engineering.【Method】 RaANR1 gene was cloned using Rhododendron agastum flowers as material. Bioinformatics analysis on RaANR1 gene was performed using relevant softwares. RaANR1 was transfected into wild-type Arabidopsis thaliana and tobacco by dipping method and injection method respectively. After resistance screening and reverse transcription PCR (RT-PCR) verification,anthocyanin and proanthocyanidin contents in transgenic Arabidopsis thaliana seedlings and tobacco flowers were detected using spectrophotometer. The relative expression of endogenous genes in the synthesis of anthocyanins and proanthocyanidins in tobacco was also detected by real-time fluorescence quantitative PCR.【Result】 RaANR1 gene was successfully cloned. Its coding region (CDS) was 1002 bp,encoding 333 amino acids. The encoded protein was a stable, non-secretory and hydrophilic protein. In its secondary structure,α-helices and random coils were the most abundant, and it contained the conserved NADPH/NADP binding domain,active site and substrate-binding site of ANR. The amino acid sequence similarities of RaANR1 with Camellia sinensis CsANR,Diospyros kaki DkANR and Vaccinium ashei VaANR were 82.49%,80.88%,and 89.79% respectively. RaANR1 was most closely related to VaANR and they shared the most similar conserved motifs. Compared with wild-type Arabidopsis thaliana, seedlings of the two transgenic Arabidopsis thaliana lines exhibited a color shift from purple to green in both cotyledons and hypocotyls, and the anthocyanin content was significantly lower than that of wild-type plants (P<0.001), whereas the proanthocyanidin content in the two transgenic lines was significantly (P<0.05, the same below) or extremely significantly (P<0.001) higher than wild-type plants. In two transgenic tobacco lines, flower color was distinctly paler than that of the wild-type tobacco, and anthocyanin content in flowers were extremely significantly reduced (P<0.001, P<0.01) compared to wild-type tobacco, while proanthocyanidin content was extremely significantly (P<0.001) or significantly increased. Compared with the wild-type tobacco, the expression levels of NtF3'5'HNtDFRNtANS, and NtANR1 genes were significantly up-regulated in the two transgenic lines, whereas no significant difference was observed for NtF3'H and NtANR2 genes (P>0.05).【Conclusion】 Overexpression of RaANR1 gene drastically decreases anthocyanin content in Arabidopsis thaliana seedling cotyledons and tobacco flowers, while significantly promotes proanthocyanidin content. These results indicate that RaANR1 gene modulates flower color by regulating the synthesis of proanthocyanidins and anthocyanidins.

     

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