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    郭婷, 黄赛, 吴茹茜, 安新民. 栾树查尔酮合酶基因克隆与表达分析[J]. 北京林业大学学报, 2021, 43(3): 27-35. DOI: 10.12171/j.1000-1522.20200377
    引用本文: 郭婷, 黄赛, 吴茹茜, 安新民. 栾树查尔酮合酶基因克隆与表达分析[J]. 北京林业大学学报, 2021, 43(3): 27-35. DOI: 10.12171/j.1000-1522.20200377
    Guo Ting, Huang Sai, Wu Ruqian, An Xinmin. Cloning and expression analysis of chalcone synthase gene from Koelreuteria paniculata[J]. Journal of Beijing Forestry University, 2021, 43(3): 27-35. DOI: 10.12171/j.1000-1522.20200377
    Citation: Guo Ting, Huang Sai, Wu Ruqian, An Xinmin. Cloning and expression analysis of chalcone synthase gene from Koelreuteria paniculata[J]. Journal of Beijing Forestry University, 2021, 43(3): 27-35. DOI: 10.12171/j.1000-1522.20200377

    栾树查尔酮合酶基因克隆与表达分析

    Cloning and expression analysis of chalcone synthase gene from Koelreuteria paniculata

    • 摘要:
        目的  查尔酮合酶(CHS)是苯丙烷途径的限速酶之一,在植物次生代谢物的合成中起着重要的作用。本研究通过对栾树CHS 基因进行克隆与生物信息学分析,以及分析栾树 CHS 基因表达与类黄酮合成的关系,期望为后续深入研究栾树类黄酮代谢途径其他相关基因、 CHS 基因家族以及锦叶栾呈色机制提供参考。
        方法  以栾树叶片为材料,采用RT-PCR技术进行查尔酮合酶基因的克隆并进行生物信息学分析;通过实时定量PCR(qRT-PCR)技术分析 CHS 基因在栾树不同组织以及在5月、7月、9月的栾树和锦叶栾叶片中的表达模式;通过代谢组测定筛选出栾树与锦叶栾的类黄酮差异代谢物。
        结果  克隆获得两个 CHS 基因的全长DNA,命名为 KpCHS1 和 KpCHS2 。其中 KpCHS1 序列全长为2 492 bp,ORF为1 173 bp,编码含有390个氨基酸的蛋白质; KpCHS2 序列全长为1 321 bp,ORF为1 182 bp,编码含有393个氨基酸的蛋白质;进一步的序列比对和系统发育分析表明,KpCHS1和KpCHS2蛋白高度同源,具有四个CHS特异性保守基序和一个查尔酮合成酶活性位点; KpCHS1 和 KpCHS2 在栾树根、茎、叶、种子中都普遍表达,其中, KpCHS2 在种子中的表达量最高, KpCHS1 在叶片中表达量高,而在根和茎中,两个基因的表达量相似且较低;表达模式分析显示,在栾树和锦叶栾叶片中,随着月份增加, KpCHS1 的表达量呈现出下降的趋势,而 KpCHS2 表达量未表现出明显的规律。在7月份的叶片样本中, KpCHS1 基因在锦叶栾中表达量显著高于栾树;代谢组结果显示,山奈酚-7-O-葡萄糖苷、7-羟基香豆素、槲皮素-3β-D-葡萄糖苷、以及类黄酮生物合成途径重要的中间产物山萘酚、柚皮苷等黄酮类物质在锦叶栾叶中含量显著升高。
        结论   KpCHS1和KpCHS2属于栾树查尔酮合酶家族并且高度同源,但在系统进化树上分布在很远分支上,推测这两个蛋白在氨基酸活性催化功能上可能存在较大差异;KpCHS1和KpCHS2在根、茎、叶、种子中均有表达,且在叶和种子中较高。研究结果初步显示,KpCHS1基因与栾树类黄酮的生物合成高度相关。

       

      Abstract:
        Objective  Chalcone synthase (CHS) is one of the rate-limiting enzymes of phenylpropanoid pathway which plays superior roles in the production of secondary metabolites. In this study, by cloning and bioinformatics analysis of CHS gene and analyzing the relationship between CHS gene expression and flavonoid synthesis of Koelreuteria paniculata, we hope to provide reference for further study of flavonoid biosynthesis pathway related genes, evolution of CHS gene family and coloration mechanism of Koelreuteria paniculata ‘Jinye’.
        Method  CHS genes were isolated and characterized by RT-PCR from Koelreuteria paniculata. And the expression patterns of CHS gene in different tissues of Koelreuteria paniculata and in the leaves of Koelreuteria paniculata and Koelreuteria paniculata ‘Jinye’ in May, July and September were analyzed by qRT-PCR; the differential flavonoid metabolism between Koelreuteria paniculata and Koelreuteria paniculata ‘Jinye’ was screeidues ether.
        Result  Two full-length DNA of CHS genes were cloned named KpCHS1 and KpCHS2 . The KpCHS1 gene sequence was found to be 2 492 bp and comprised an open reading frame of 1 173 bp, encoding for 390 amino acid residues, the KpCHS2 gene sequence was found to be 1 321 bp and comprised an open reading frame of 1 182 bp, encoding for 393 amino acid residues ether. Alignment of the predicted amino acid sequence of KpCHS2 had been shown to have high sequence similarity with KpCHS1, with four CHS specific conserved motifs and one chalcone synthase active site. Furthermore, KpCHS1 and KpCHS2 were generally expressed in roots, stems, leaves and seeds of Koelreuteria paniculata. Among them, the expression of KpCHS2 was the highest in seeds, while that of KpCHS1 was higher in leaves. In roots and stems, the expression levels of the two genes were similar and lower. The expression pattern analysis showed that in Koelreuteria paniculata and Koelreuteria paniculata Jinye’, the expression of KpCHS1 decreased with the increase of months, while the expression of KpCHS2 did not show obvious regularity. In the July plant samples, the expression of KpCHS1 gene in Koelreuteria paniculata ‘Jinye’ was higher than that in Koelreuteria paniculata. Besides, we analyzed the metabonomics of Koelreuteria paniculata and Koelreuteria paniculata ‘Jinye’ leaves in July, and screened out the different flavonoids. It was found that kaempferol-7-o-glucoside, 7-hydroxycoumarin, quercetin-3β-D-glucoside, and kaempferol, naringin, which were important intermediate products in flavonoid biosynthesis, were significantly increased in Koelreuteria paniculata ‘Jinye’ leaves.
        Conclusion  KpCHS1 and KpCHS2 belong to the chalcone synthase family of Koelreuteria paniculata and are highly homologous, but they are distributed in far branches of the phylogenetic tree. It is speculated that the two proteins may have great differences in the catalytic function of amino acid activity. KpCHS1 and KpCHS2 are expressed in roots, stems, leaves and seeds, and higher in leaves and seeds of Koelreuteria paniculata. Our results indicate that the expression of KpCHS1 gene is highly related to the synthesis of flavonoids in Koelreuteria paniculata.

       

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