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

doi:10.19303/j.issn.1008-0384.2024.01.005

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

doi: 10.19303/j.issn.1008-0384.2024.01.005

1.
攀枝花学院生物与化学工程学院，四川　攀枝花　617000

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: Q781；Q786
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出版历程
- 收稿日期: 2023-09-26
- 修回日期: 2023-11-10
- 网络出版日期: 2023-12-21
- 刊出日期: 2024-01-28

Cloning and Expression of BsDFR in Bougainvillea spectabilis

1.
College of Biological and Chemical Engineering，Panzhihua University，Panzhihua, Sichuan　617000, China

摘要

摘要: 目的克隆分析三角梅（Bougainvillea spectabilis）二氢黄酮醇-４-还原酶（Dihydroflavonol-4-reductase, DFR）基因（BsDFR），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，等电点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；二级结构中α螺旋占比最多，三级结构预测显示为二聚体蛋白。底物对接模拟预测BsDFR对二氢山柰酚（Dihydrokaempferol, DHK）、二氢槲皮素（Dihydroquercetin, DHQ）和二氢杨梅素（Dihydromyricetin, DHM）3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（Centrospermae）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）的前体物质——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。
- 三角梅 /
- BsDFR基因 /
- 生物信息学 /
- 表达量分析
Abstract: Objective The dihydroflavonol-4-reductase (DFR) gene in bracts of Bougainvillea spectabilis was cloned and characterized to study the role it plays in color formation. Method BsDFRwas cloned based on the transcriptome data on the ornamental plant to study the related bioinformatics. Molecular docking technology was employed to predict the substrate specificity, and qRT-PCR applied to examine the relative transcription levels of the genes in B. spectabilis of different colors. Result The full-length coding sequence of BsDFR (GenBank ID: ON417750) was 987 bp encoding 328 amino acids. The protein had a calculated molecular weight of 36.49 kDa and an isoelectric point of 6.33. It had the NADPH and substrate specific binding sites unique to DFR of Asn type without a transmembrane structure or signal peptide. The subcellular localization of the protein indicated it to be cytoplasmic. Alpha helices were the most abundant secondary structure of the protein, while the tertiary structure was a dimer. A substrate docking simulation, consistent with the structural analysis, predicted BsDFR to possess a catalytic activity on dihydrokaempferol, dihydroquercetin, and dihydromyricetin. The phylogenetic tree analysis grouped it along with caryophyllales plants. High expression of the gene was found in the orange B. spectabilis by qRT-PCR. It was speculated that the main substrate to be DHK, which was catalyzed by BsDFR into leucopelargonidin, a precursor of orange-colored anthocyanidin——pelargonidin. Conclusion BsDFR in B. spectabilis had typical molecular characteristics of the plant dihydroflavonol-4-reductase, which is associated with the pigment synthesis in the bracts of orange B. spectabilis.
- Bougainvillea spectabilis Willd /
- dihydroflavonol-4-reductase gene /
- bioinformatics /
- expression analysis

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图 1 盛花期不同品种三角梅

Figure 1. Various bougainvillea cultivars at blooming stage

下载: 全尺寸图片幻灯片

图 2 RNA提取及PCR扩增

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

Figure 2. Electrophoresis of RNA and PCR amplification products

In Fig. A, M: D2000 DNA marker; 1: RNA sample. In Fig. B, M: D2000 DNA marker; 1: target band.

下载: 全尺寸图片幻灯片

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 of B. spectabilis and other plants

Under lined is NADPH binding site; arrow lined, substrate specificity site; red boxed, Asn134.

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图 4 BsDFR二级结构预测

Figure 4. Predicted secondary structure of BsDFR

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图 5 BsDFR蛋白的三级结构

Figure 5. Tertiary structure of BsDFR

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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 3 substrates

Green dotted lines indicate hydrogen bonds; pink dotted lines, π-alkyl; red dotted lines, unfavorable donor-donor.

下载: 全尺寸图片幻灯片

图 7 BsDFR与其余植物DFR同源蛋白系统进化树

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

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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. BsDFR expressions in B. spectabilis of different colors at blooming stage

Different lowercase letters indicate significant differences between samples (P＜0.05).

下载: 全尺寸图片幻灯片

表 1 引物信息

Table 1. Primers applied

引物 Primers	序列 Sequence（5′-3′）	用途 Usage
DFR	F：ATGAGTGGAGGAGAAGAGCAAG	基因克隆 Gene cloning
DFR	R：TAAACTCTCCACTGTATCCTTGA	基因克隆 Gene cloning
18S	F：CAGAACATCTAAGGGCATCACA	荧光定量 Gene expression
18S	R：TAGTTGGTGGAGCGATTTGTCT
DFRq	F：AAGGCTCTGATGTGATGTGGTATG
DFRq	R：ACTATCGTTGAGGGTTGGTTGC

下载: 导出CSV

参考文献(18)

[1]	徐夙侠, 王亮生, 舒庆艳, 等. 三角梅属植物的生物学研究进展 [J]. 植物学通报, 2008, 43(4):483−490. XU S X, WANG L S, SHU Q Y, et al. Progress of study of the biology of the resource plant Bougainvillea [J]. Chinese Bulletin of Botany, 2008, 43(4): 483−490.(in Chinese)
[2]	常圣鑫, 杨光穗, 陈金花, 等. 世界三角梅产业发展历史及趋势 [J]. 热带农业科学, 2018, 38(1):71−77. CHANG S X, YANG G S, CHEN J H, et al. Development history and tendency of Bougainvillea industry all over the world [J]. Chinese Journal of Tropical Agriculture, 2018, 38(1): 71−77.(in Chinese)
[3]	GROTEWOLD E. The genetics and biochemistry of floral pigments [J]. Annual Review of Plant Biology, 2006, 57: 761−780. doi: 10.1146/annurev.arplant.57.032905.105248
[4]	HELLER W, FORKMANN G, BRITSCH L, et al. Enzymatic reduction of (+)-dihydroflavonols to flavan-3, 4-cis-diols with flower extracts from Matthiola incana and its role in anthocyanin biosynthesis [J]. Planta, 1985, 165(2): 284−287. doi: 10.1007/BF00395052
[5]	MARTENS S, TEERI T, FORKMANN G. Heterologous expression of dihydroflavonol 4-reductases from various plants [J]. FEBS Letters, 2002, 531(3): 453−458. doi: 10.1016/S0014-5793(02)03583-4
[6]	TANAKA Y, BRUGLIERA F, CHANDLER S. Recent progress of flower colour modification by biotechnology [J]. International Journal of Molecular Sciences, 2009, 10(12): 5350−5369. doi: 10.3390/ijms10125350
[7]	GUTTERSON N. Anthocyanin biosynthetic genes and their application to flower color modification through sense suppression [J]. HortScience, 1995, 30(5): 964−966. doi: 10.21273/HORTSCI.30.5.964
[8]	AIDA R, YOSHIDA K, KONDO T, et al. Copigmentation gives bluer flowers on transgenic torenia plants with the antisense dihydroflavonol-4-reductase gene [J]. Plant Science, 2000, 160(1): 49−56. doi: 10.1016/S0168-9452(00)00364-2
[9]	孙蓉, 刘姗, 高静雷, 等. 三角梅CHS基因的克隆及表达分析 [J]. 西北农业学报, 2021, 30(10):1565−1572. doi: 10.7606/j.issn.1004-1389.2021.10.014 SUN R, LIU S, GAO J L, et al. Cloning and expression analysis of CHS gene from Bougainvillea spectabilis [J]. Acta Agriculturae Boreali-Occidentalis Sinica, 2021, 30(10): 1565−1572.(in Chinese) doi: 10.7606/j.issn.1004-1389.2021.10.014
[10]	孙蓉, 刘姗, 高静雷. 三角梅黄烷酮3-羟化酶基因的克隆及表达分析 [J]. 生物技术通报, 2022, 38(11):122−128. doi: 10.13560/j.cnki.biotech.bull.1985.2022-0048 SUN R, LIU S, GAO J L. Cloning and expression analysis of flavanone 3-hydroxylase gene from Bougainvillea spectabilis [J]. Biotechnology Bulletin, 2022, 38(11): 122−128.(in Chinese) doi: 10.13560/j.cnki.biotech.bull.1985.2022-0048
[11]	于婷婷, 倪秀珍, 高立宏, 等. 高等植物二氢黄酮醇4-还原酶基因研究进展 [J]. 植物研究, 2018, 38(4):632−640. doi: 10.7525/j.issn.1673-5102.2018.04.020 YU T T, NI X Z, GAO L H, et al. Advances in study of dihydroflavonol 4-reductase(DFR) genes of higher plants [J]. Bulletin of Botanical Research, 2018, 38(4): 632−640.(in Chinese) doi: 10.7525/j.issn.1673-5102.2018.04.020
[12]	STAFFORD H A. Anthocyanins and betalains: Evolution of the mutually exclusive pathways [J]. Plant Science, 1994, 101(2): 91−98. doi: 10.1016/0168-9452(94)90244-5
[13]	胡可, 孟丽, 韩科厅, 等. 瓜叶菊花青素合成关键结构基因的分离及表达分析 [J]. 园艺学报, 2009, 36(7):1013−1022. doi: 10.3321/j.issn:0513-353X.2009.07.011 HU K, MENG L, HAN K T, et al. Isolation and expression analysis of key genes involved in anthocyanin biosynthesis of Cineraria [J]. Acta Horticulturae Sinica, 2009, 36(7): 1013−1022.(in Chinese) doi: 10.3321/j.issn:0513-353X.2009.07.011
[14]	NIELSEN K, DEROLES S C, MARKHAM K R, et al. Antisense flavonol synthase alters copigmentation and flower color in lisianthus [J]. Molecular Breeding, 2002, 9(4): 217−229. doi: 10.1023/A:1020320809654
[15]	DAVIES K M, SCHWINN K E, DEROLES S C, et al. Enhancing anthocyanin production by altering competition for substrate between flavonol synthase and dihydroflavonol 4-reductase [J]. Euphytica, 2003, 131(3): 259−268. doi: 10.1023/A:1024018729349
[16]	JOHNSON E T, YI H, SHIN B, et al. Cymbidium hybrida dihydroflavonol 4-reductase does not efficiently reduce dihydrokaempferol to produce orange pelargonidin-type anthocyanins [J]. The Plant Journal:for Cell and Molecular Biology, 1999, 19(1): 81−85. doi: 10.1046/j.1365-313X.1999.00502.x
[17]	钟晓缘, 刘姗, 孙蓉, 等. 三角梅苞叶色彩参数和色素含量分析及蓝色转基因受体的筛选 [J]. 西北植物学报, 2022, 42(4):646−655. doi: 10.7606/j.issn.1000-4025.2022.04.0646 ZHONG X Y, LIU S, SUN R, et al. Screening of blue transgenic receptor varieties in Bougainvillea spectabilis based on the analysis of color parameters and pigment content [J]. Acta Botanica Boreali-Occidentalia Sinica, 2022, 42(4): 646−655.(in Chinese) doi: 10.7606/j.issn.1000-4025.2022.04.0646
[18]	JOHNSON E T, RYU S, YI H, et al. Alteration of a single amino acid changes the substrate specificity of dihydroflavonol 4-reductase [J]. The Plant Journal:for Cell and Molecular Biology, 2001, 25(3): 325−333. doi: 10.1046/j.1365-313x.2001.00962.x