芍药PlFLS基因生物信息学分析及对拟南芥的遗传转化
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江苏省农业三新工程项目(SXGC2017-297);江苏省优势学科基金项目(PAPD)


Bioinformatics analysis of Paeonia lactiflora PlFLS gene and its genetic transformation in Arabidopsis thaliana
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    摘要:

    【目的】探讨类黄酮合成途径关键基因黄酮醇合酶基因(FLS)在芍药Paeonia lactiflora花色调控中的作用。【方法】克隆获得芍药PlFLS基因,对其进行生物信息学分析,构建PlFLS基因的过表达载体,通过农杆菌介导的Floral-dip法进行拟南芥遗传转化研究。【结果】生物信息学分析表明,芍药PlFLS基因氨基酸序列与茶树相似性较高,存在2个功能结构域,但不存在信号肽位点。芍药PlFLS蛋白预测模型揭示了其蛋白三级结构中存在1个2-氧代戊二酸配体,并与多条肽链相连接。试验成功获得了转PlFLS基因纯合拟南芥植株,GUS染色和PCR鉴定证实了目的基因已经整合转基因植株基因组,qRT-PCR分析显示,相对于野生型,PlFLS基因在遗传转化植株中显著高表达(P<0.05)。色谱分析结果显示,转PlFLS基因拟南芥植株叶片中花黄素含量显著增加(P<0.05)。【结论】成功构建转PlFLS基因拟南芥,并证明PlFLS基因可以显著影响拟南芥类黄酮合成途径。

    Abstract:

    【Objective】 To investigate the molecular mechanism of flavonol synthase (FLS) gene in peony flower (Paeonia lactiflora) color formation.【Method】 We cloned PlFLS gene and carried out bioinformatics analysis. The over-expression vector of PlFLS gene was constructed and transformed as heterologous gene into Arabidopsis by floral-dip method with recombinant agrobacterium transformants.【Result】 The bioinformatics analysis results suggested that the amino acid sequence of PlFLS gene was highly similar with Camellia sinensis, and there were two structure domains but no potential signal peptide. The tertiary structure prediction of PlFLS protein showed that there was a 2-oxoglutaric acid ligand connecting with several peptide chains. Transgenic Arabidopsis plants expressing homologous PlFLS gene were obtained successfully. GUS staining and PCR detection confirmed the integration of PlFLS gene into Arabidopsis genome, and qRT-PCR analysis showed PlFLS gene was expressed at significantly higher level in transgenic lines compared with wide type (P<0.05). Through HPLC detection, the anthoxanthin content in transgenic plants significantly increased compared with wide type (P<0.05).【Conclusion】 PlFLS gene has been transformed into Arabidopsis successfully and it plays an important role in regulating the synthesis of flavonoids.

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史旻,孙静,陶俊.芍药PlFLS基因生物信息学分析及对拟南芥的遗传转化[J].华南农业大学学报,2018,39(5):93-100

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  • 收稿日期:2018-02-10
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  • 在线发布日期: 2018-07-18