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三角梅二氢黄酮醇-4-还原酶基因的克隆及表达特异性分析

孙蓉 刘桃 潘凯越 刘姗 刁毅 曾道萍

孙蓉,刘桃,潘凯越,等. 三角梅二氢黄酮醇-4-还原酶基因的克隆及表达特异性分析 [J]. 福建农业学报,2023,39(X):1−7
引用本文: 孙蓉,刘桃,潘凯越,等. 三角梅二氢黄酮醇-4-还原酶基因的克隆及表达特异性分析 [J]. 福建农业学报,2023,39(X):1−7
SUN R, LIU T, PAN K Y, et al. Cloning and expression characteristics of BsDFR gene of Bougainvillea spectabilis [J]. Fujian Journal of Agricultural Sciences,2023,39(X):1−7
Citation: SUN R, LIU T, PAN K Y, et al. Cloning and expression characteristics of BsDFR gene of Bougainvillea spectabilis [J]. Fujian Journal of Agricultural Sciences,2023,39(X):1−7

三角梅二氢黄酮醇-4-还原酶基因的克隆及表达特异性分析

基金项目: 四川省自然科学基金面上项目(2023NSFSC0145);大学生创新创业训练计划项目(S202311360061)
详细信息
    作者简介:

    孙蓉(1987 —),女,博士,讲师,主要从事干热河谷特色植物资源的开发利用研究,E-mail: sunrong@pzhu.edu.cn

    通讯作者:

    刘姗(1980 —),女,博士,教授,主要从事植物次生代谢相关研究,E-mail: liushan@pzhu.edu.cn

  • 中图分类号: Q 781;Q 786

Cloning and expression characteristics of BsDFR gene of Bougainvillea spectabilis

  • 摘要:   目的  克隆分析三角梅二氢黄酮醇-4-还原酶(DFR)基因,探讨其在三角梅苞片呈色中的作用。  方法  基于三角梅转录组数据,利用PCR技术克隆BsDFR基因,并通过生物信息学工具分析其分子特性;通过分子对接技术预测BsDFR底物特异性;采用实时荧光定量PCR分析该基因在不同颜色三角梅中的表达量差异。  结果  三角梅BsDFR基因(GenBank ID:ON417750)编码区全长987 bp,编码328个氨基酸。BsDFR理论相对分子质量为36.48 kDa,pI为6.33。具有DFR特有的NADPH及底物特异结合位点,属于Asn型DFR,不具有跨膜结构及信号肽,定位于细胞质中,α螺旋为占比最多的二级结构,三级结构预测显示为二聚体蛋白,底物对接模拟预测BsDFR对二氢山奈酚(DHK)、二氢槲皮素(DHQ)和二氢杨梅素(DHM)三种底物均具有催化活性,与结构分析相吻合。进化树分析其与石竹目植物聚为一类。qRT-PCR分析发现其在橙色系三角梅中含量较高,进一步推测其主要底物为DHK,催化生成橙色系花青素——天竺葵素前体物质,无色天竺葵素苷元。  结论  BsDFR基因是一个典型的植物二氢黄酮醇-4-还原酶基因,主要与橙色系三角梅苞片色素合成有关。
  • 图  1  盛花期不同品种三角梅

    Figure  1.  Blooming stage of different Bougainvillea cultivars

    图  2  RNA提取及PCR扩增电泳图

    A中,M:D2000 DNA Marker;1:RNA样品;B中,M:D2000 DNA Marker;1:目的条带。

    Figure  2.  Electrophoresis detection result of RNA and PCR amplification products

    In A, M: D2000 DNA Marker; 1: RNA sample; in B, M: D2000 DNA Marker; 1: target band.

    图  3  BsDFR与其他植物DFR多序列比对

    下划线:NADPH结合区域;箭头线:底物特异性结合区域;红色方框:134位天冬酰胺。

    Figure  3.  DFR amino acid sequences comparison of B. spectabilis and other plants

    Under line: NADPH binding site; Arrow line: substrate specificity site; Red box: Asn134.

    图  4  BsDFR二级结构预测

    Figure  4.  Prediction of the secondary structure of BsDFR proteins

    图  5  BsDFR蛋白的三级结构

    Figure  5.  Tertiary structure of BsDFR protein

    图  6  BsDFR与3种底物的对接情况

    绿色虚线:氢键;粉色虚线:π-alkyl;红色虚线:unfavorable donor-donor作用。

    Figure  6.  Molecular docking simulation between BsDFR and three substrates

    Green dotted lines: hydrogen bonds; Pink dotted lines: π-alkyl; Red dotted lines:unfavorable donor-donor.

    图  7  BsDFR与其余植物DFR同源蛋白系统进化树

    Figure  7.  Phylogenetic relationships of BsDFR and DFRs from other plants

    图  8  BsDFR在不同颜色三角梅盛花期苞片中的表达量差异

    不同小写字母代表样品间差异显著(P<0.05)

    Figure  8.  Analysis of BsDFR expression levels in different color B. spectabilis at blooming stage

    Small letter(s) above the bars indicate significant differences (P<0.05) among the samples.

    表  1  引物信息

    Table  1.   Primers used in this study

    引物 Primers序列 Sequence (5′-3′)
    基因克隆引物 Gene cloning primers
    DFR F:ATGAGTGGAGGAGAAGAGCAAG
    DFR R:TAAACTCTCCACTGTATCCTTGA
    荧光定量引物 Gene expression primers
    18S F:CAGAACATCTAAGGGCATCACA
    18S R:TAGTTGGTGGAGCGATTTGTCT
    DFRq F:AAGGCTCTGATGTGATGTGGTATG
    DFRq R:ACTATCGTTGAGGGTTGGTTGC
    下载: 导出CSV
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  • 收稿日期:  2023-09-26
  • 修回日期:  2023-11-10
  • 网络出版日期:  2023-12-21

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