红掌R2R3-MYB转录因子基因AaMYB6调控花青素苷合成

doi:10.16420/j.issn.0513-353x.2021-0476

园艺学报 ›› 2021, Vol. 48 ›› Issue (10): 1859-1872.doi: 10.16420/j.issn.0513-353x.2021-0476

红掌R2R3-MYB转录因子基因AaMYB6调控花青素苷合成

李崇晖¹^,³, 杨光穗¹^,³, 张志群¹^,³, 尹俊梅²^,³^,^*()

¹中国热带农业科学院热带作物品种资源研究所农业部华南作物基因资源与种质创制重点实验室,海口 571101
²中国热带农业科学院海口实验站,海口 571101
³海南省热带观赏植物种质创新利用工程技术研究中心,海南儋州 571737

收稿日期:2021-07-19 修回日期:2021-09-02 出版日期:2021-10-25 发布日期:2021-11-01
通讯作者: 尹俊梅 E-mail:yinjunmei2004@163.com
基金资助:
海南省自然科学基金创新研究团队项目(2018CXTD344);中国热带农业科学院基本科研业务费专项资金项目(1630032017024)

A Novel R2R3-MYB Transcription Factor Gene AaMYB6 Involved in Anthocyanin Biosynthesis in Anthurium andraeanum

LI Chonghui¹^,³, YANG Guangsui¹^,³, ZHANG Zhiqun¹^,³, YIN Junmei²^,³^,^*()

¹Tropical Crops Genetic Resources Institute,Chinese Academy of Tropical Agricultural Sciences;Key Laboratory of Crop Gene Resources and Germplasm Enhancement in Southern China,Ministry of Agriculture,Haikou 571101,China
²Haikou Experimental Station,Chinese Academy of Tropical Agricultural Sciences,Haikou 571101,China
³The Engineering Technology Research Center of Tropical Ornamental Plant Germplasm Innovation and Utilization,Danzhou,Hainan 571737,China

Received:2021-07-19 Revised:2021-09-02 Online:2021-10-25 Published:2021-11-01
Contact: YIN Junmei E-mail:yinjunmei2004@163.com

摘要/Abstract

摘要：

为了挖掘调控红掌肉穗花序花青素苷合成的转录因子基因,基于前期的转录组数据,以‘粉冠军’红掌（Anthurium andraeanum‘Pink Champion’）肉穗花序为材料,克隆了1个R2R3-MYB家族基因AaMYB6（GenBank序列号MZ171064）。通过氨基酸序列比对和系统进化树分析、亚细胞定位、在烟草中的过表达分析、qRT-PCR、酵母双杂交（yeast two-hybrid assay,Y2H）和双分子荧光互补（bimolecular fluorescence complementation,BiFC）试验,研究了AaMYB6的功能。结果表明AaMYB6与已报道的AaMYB1具有较高的同源性,属于以玉米ZmC1为代表的,调控单子叶植物花青素苷合成的R2R3-MYB家族C1亚组。AaMYB6定位于细胞核。在烟草中过表达AaMYB6,NtF3H表达量下调,但却激活了T₁代转基因植株花丝中花青素苷生物合成途径关键酶基因NtF3′H、NtDFR、NtANS、NtUFGT的表达,产生了花丝中积累花青素苷的表型。AaMYB6主要在红掌‘紫公主’的肉穗花序中表达,且在不同品种肉穗花序中其表达量与花青素苷含量显著正相关。Y2H和BiFC试验表明AaMYB6与前期报道的参与花青素苷和原花青素合成调控的bHLH转录因子AabHLH1有相互作用,进一步验证了AaMYB6调控花青素苷合成的功能。

关键词: 红掌, MYB转录因子, 花青素苷, 异源过表达, 转录调控, 基因功能

Abstract:

A new R2R3-MYB gene AaMYB6 with GenBank accession number MZ171064 was isolated from the spadix of Anthurium andraeanum‘Pink Champion’based on the information from previous transcriptome data for identification of transcription factor genes associated with anthocyanin biosynthesis. The function of AaMYB6 was analyzed through the assays such as amino acid sequence alignment,phylogenetic tree analysis,subcellular localization,overexpression in tobacco,qRT-PCR,yeast two-hybrid assay（Y2H）,and bimolecular fluorescence complementation（BiFC）. The results suggested that AaMYB6 showed homology with previously identified AaMYB1 from anthurium and belong to C1 subgroup R2R3-MYBs,represented by Zea may C1,that regulate anthocyanin biosynthesis in monocots. AaMYB6 was proved to be nucleus-localized protein. Overexpression of AaMYB6 in tobacco markedly activated the expression of the enzyme genes NtF3′H,NtDFR,NtANS,NtUFGT,specifically involved in anthocyanin biosynthesis in filaments and promoted the accumulation of anthocyanin pigments. AaMYB6 was mainly expressed in the spadix of‘Rapido’. And the expression of AaMYB6 was closely linked to anthocyanin accumulation in the spadix of various cultivars. The interaction between AaMYB6 and the AabHLH1 protein,which was identified previously to regulate the biosynthesis of anthocyanin and proanthocyanidin in anthurium,was confirmed by Y2H and BiFC assays. The results indicated that AaMYB6 play a role in the regulation of anthocyanin biosynthesis in anthurium.

Key words: Anthurium andraeanum, MYB transcription factor, anthocyanin, heterologous overexpression, transcriptional regulation, gene function

中图分类号:

S682.14

李崇晖, 杨光穗, 张志群, 尹俊梅. 红掌R2R3-MYB转录因子基因AaMYB6调控花青素苷合成[J]. 园艺学报, 2021, 48(10): 1859-1872.

LI Chonghui, YANG Guangsui, ZHANG Zhiqun, YIN Junmei. A Novel R2R3-MYB Transcription Factor Gene AaMYB6 Involved in Anthocyanin Biosynthesis in Anthurium andraeanum[J]. Acta Horticulturae Sinica, 2021, 48(10): 1859-1872.

图/表 7

图1 红掌AaMYB6与其他植物参与花青素苷合成的R2R3-MYB的氨基酸序列比对分析红色框:bHLH互作motif;绿色框:单子叶植物调控花青素苷合成的R2R3MYB保守序列“DNEI”;蓝色框:单子叶植物调控花青素苷合成MYB转录因子C-末端保守序列KAX[K/R]C[S/T];Aa:红掌;Zm:玉米;Pe:蝴蝶兰;Fh:小苍兰。

Fig. 1 Sequences alignment of AaMYB6 from anthurium with known R2R3-MYBs involved in anthocyanin biosynthesis A:The red box indicates the bHLH interacting motif;the green box indicates the different conserved“DNEI”motif for monocots R2R3-MYB;The blue box indicates the C-terminal-conserved motif KAX[K/R]C[S/T] for anthocyanin-regulating MYBs of monocots. Aa:Anthurium andraeanum;Zm:Zea mays;Pe:Phalaenopsis equestris;Fh:Freesia hybrid.

图2 红掌AaMYB6与其他植物参与花青素苷合成的R2R3-MYB的系统进化树分析 *既与花青素苷相关又与原花青素相关。

Fig. 2 Phylogenic tree of AaMYB6 from anthurium with known R2R3-MYBs involved in anthocyanin biosynthesis * Both correlated with anthocyanin and proanthocyanidin.

图3 AaMYB6在拟南芥原生质体中的亚细胞定位

Fig. 3 Subcellular location of AaMYB6 in protoplasts of Arabidopsis

图4 AaMYB6在红掌中的表达情况和花青素苷含量

Fig. 4 The expression of AaMYB6 and the anthocyanin content in anthurium P < 0.05.

图5 转AaMYB6烟草的表型（A）和花青素苷含量（B）及目标基因检测（C、D） WT:野生型植株;L1 ~ L3:过表达AaMYB6的3个T1代株系,每个株系2株（-1、-2）。t检验[与野生型WT（A、D）或不同组织间（B、C）相比],** α = 0.01。

Fig. 5 The phenotypes and the content of anthocyanin of AaMYB6 transgenic tobacco（A）and detection of gene expression（B,C） WT:Wild type tobacco plants;L1-L3:Three AaMYB6 overexpression T1 tobacco lines,and there were two plants of each line（-1,-2）.t-test [Comparison between transgenic lines and wild type plants（A,D）or between different tissue（B,C）],** α = 0.01.

图6 转基因烟草花瓣和花丝中AaMYB6可能的靶基因的相对表达量 FC:相对于野生型的变化倍数;L1 ~ L3:过表达AaMYB6的3个T1代烟草株系,每个株系2株（-1、-2）。

Fig. 6 The relative expression of enzyme genes in petals and filaments of transgenic tobacco plants FC:Fold changes against the WT;L1-L3:Three AaMYB6 overexpression T1 tobacco lines,there were two plants of each line（-1,-2）.

图7 利用Y2H和BiFC技术验证AaMYB6和AabHLH1的体内互作 A:在2种氨基酸缺陷型和4种氨基酸缺陷型酵母培养基上筛选包含质粒的Y2H Gold酵母细胞,X-α-gal用来验证互作;BD:pGBKT7空质粒;AD:pGADT7空质粒;AD-AaMYB6:pGAD-AaMYB6重组质粒;BD-bHLH:pGBK-AabHLH1重组质粒。B:在拟南芥原生质体中AaMYB6和AabHLH1的双分子荧光互补。

Fig. 7 Interactions between AaMYB6 and AabHLH1 detected using the Y2H and BiFC assays A:Y2H Gold yeast cells containing plasmids were grown on double- and quadruple-selection SD media. The X-α-gal assay was performed to confirm the positive interactions;BD:pGBKT7 empty plasmid;AD:pGADT7 empty plasmid;AD-AaMYB6:pGAD-AaMYB6 recombinant plasmid;BD-AabHLH1:pGBK-AabHLH1 recombinant plasmid;B:Bimolecular fluorescence complementation visualization of the AaMYB6 and AabHLH1 interaction in the in protoplasts of Arabidopsis.

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红掌R2R3-MYB转录因子基因AaMYB6调控花青素苷合成

A Novel R2R3-MYB Transcription Factor Gene AaMYB6 Involved in Anthocyanin Biosynthesis in Anthurium andraeanum

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红掌R2R3-MYB转录因子基因AaMYB6调控花青素苷合成

A Novel R2R3-MYB Transcription Factor Gene AaMYB6 Involved in Anthocyanin Biosynthesis in Anthurium andraeanum

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