紫丁香SoF3′H对花瓣中花青苷合成的功能解析

doi:10.16420/j.issn.0513-353x.2023-0587

园艺学报 ›› 2024, Vol. 51 ›› Issue (8): 1833-1843.doi: 10.16420/j.issn.0513-353x.2023-0587

• 遗传育种·种质资源·分子生物学 • 上一篇下一篇

紫丁香SoF3′H对花瓣中花青苷合成的功能解析

马波¹^,², 李雷¹, 胡增辉¹, 冷平生¹, 汪进萱¹, 冷卓¹, 杨艺慧¹, 贾黎明², 吴静¹^,^*()

¹ 北京农学院园林学院，国家林业草原古树健康与古树文化工程技术研究中心，林木分子设计育种高精尖创新中心，北京 102206
² 北京林业大学林学院，林木分子设计育种高精尖创新中心，林木资源高效生产全国重点实验室，省部共建森林培育与保护教育部重点实验室，北京 100083

收稿日期:2024-02-26 修回日期:2024-04-12 出版日期:2024-08-25 发布日期:2024-08-21
通讯作者:
^*E-mail：wjmxy1988@126.com
基金资助:
国家重点研发计划项目(2018YFD1000406); 北京市教委—基金委重点项目(KZ201810020028); 北京市教委“生态修复工程学”高精尖学科项目(GJJXK210102); 中央高校基本科研业务费专项(BFUKF202211)

Functional Analysis of SoF3′H in the Synthesis of Anthocyanidin in Syringa oblata Petals

MA Bo¹^,², LI Lei¹, HU Zenghui¹, LENG Pingsheng¹, WANG Jinxuan¹, LENG Zhuo¹, YANG Yihui¹, JIA Liming², WU Jing¹^,^*()

¹ Ancient Tree Health and Culture Engineering Technology Research Center，National Forestry and Grassland Administration，Beijing Advanced Innovation Center for Tree Breeding by Molecular Design，College of Landscape Architecture，Beijing University of Agriculture，Beijing 102206，China
² Beijing Advanced Innovation Center for Tree Breeding by Molecular Design，State Key Laboratory of Efficient Production of Forest Resources，Key Laboratory of Silviculture and Conservation of the Ministry of Education，College of Forestry，Beijing Forestry University，Beijing 100083，China

Received:2024-02-26 Revised:2024-04-12 Published:2024-08-25 Online:2024-08-21

摘要/Abstract

摘要：

作为花青苷合成通路中的关键酶，类黄酮3′-羟化酶（flavonoid-3′-hydroxylase，F3′H）对植物花的呈色非常重要。为探究SoF3′H在紫丁香（Syringa oblata）花青苷合成中的作用，克隆SoF3'H基因，并进行蛋白同源序列比对和进化树分析，通过qRT-PCR分析该基因在不同花发育时期与器官中的表达模式，在紫丁香花瓣中瞬时沉默和过表达SoF3'H进行功能验证，并对其启动子进行克隆与分析，利用酵母单杂交试验验证SoAN2与SoF3′H的结合关系，对SoAN2的表达模式进行初步探索。结果显示，SoF3′H的CDS序列长1 587 bp，推测编码528个氨基酸；SoF3′H具有保守的p450 superfamily结构域，属于细胞色素P450家族，与油橄榄（Olea europaea）相似性最高（82.46%）。随着花发育，紫丁香花瓣褪色，SoF3′H的表达水平逐渐降低；SoF3′H在盛花期花冠裂片中表达最高，在同时期根、雄蕊和茎中相对较低。沉默SoF3′H后花瓣显著褪色，花青苷含量显著降低；过表达SoF3′H的花瓣着色明显，花青苷含量显著升高。克隆得到2 000 bp的SoF3′H启动子序列具有MYB识别位点，并发现MYB家族成员SoAN2可以与该启动子直接结合，且SoAN2在紫丁香花不同发育阶段的表达呈降低趋势，与SoF3′H在不同开花时期的表达趋势基本一致，表明SoAN2可能促进SoF3′H的表达，从而调控花青苷含量变化，影响花瓣着色。

关键词: 紫丁香, 花青苷, 类黄酮3′-羟化酶（F3′H）, SoAN2, 功能解析

Abstract:

Flavonoid-3′-hydroxylase（F3′H），a key enzyme in the anthocyanidin synthesis pathway，plays an important role in plant flower coloration. In this study，in order to further explore the role of F3'H in flower pigment synthesis in lilac（Syringa oblata），the SoF3'H was cloned，and the protein homology sequence comparison and phylogenetic tree were examined. qRT-PCR in different flower developmental stages and organs was used to analyze the SoF3'H expression patterns. Functional validation was performed by transient silencing and overexpression of the SoF3'H gene in the lilac petals. The promoter of SoF3′H was cloned and analyzed，the yeast one-hybrid assay was performed to verify the binding of SoAN2 and SoF3'H promoter，and the expression patterns of SoAN2 was performed. The results showed that the coding sequence of SoF3′H was 1 587 bp，putatively encoding for 528 amino acids. SoF3′H has a conserved domain of p450 superfamily，belonging to the cytochrome P450 family，and has the highest similarity（82.46%）with Olea europaea. Accompanied with flower development，the petals were discolored and the expression levels of SoF3'H was decreased gradually. In different organs at flower full blossom stage，the highest expression level of SoF3'H was found in corolla lobes，and relatively lower expression occurred in roots，stamens and stems. The silencing of SoF3'H resulted in significant fading of petals and remarkable reduction of anthocyanidin content. Contrarily the overexpression of SoF3'H led to significant coloration of petals and obvious increased of anthocyanidin content. Then，the 2 000 bp long promoter sequence of SoF3'H was cloned，which has MYB binding site. SoAN2，a member of MYB family，was confirmed to bind to this promoter directly and the expression of SoAN2 at different flower development stages tended to decrease. The results further suggested that SoAN2 can promote the expression level of SoF3'H to increase anthocyanidin content regulating petal coloration.

Key words: Syringa oblata, anthocyanidin, flavonoid 3′-hydroxylase（F3′H）, SoAN2, function analysis

马波, 李雷, 胡增辉, 冷平生, 汪进萱, 冷卓, 杨艺慧, 贾黎明, 吴静. 紫丁香SoF3′H对花瓣中花青苷合成的功能解析[J]. 园艺学报, 2024, 51(8): 1833-1843.

MA Bo, LI Lei, HU Zenghui, LENG Pingsheng, WANG Jinxuan, LENG Zhuo, YANG Yihui, JIA Liming, WU Jing. Functional Analysis of SoF3′H in the Synthesis of Anthocyanidin in Syringa oblata Petals[J]. Acta Horticulturae Sinica, 2024, 51(8): 1833-1843.

图/表 8

表1 本研究中用到的引物序列

Table 1 Primer sequences used in this study

引物用途 Primer function	引物名称 Primer name	引物序列（5′-3′） Primer sequence
基因克隆Gene clone	SoF3'H	F：ATGTTCACTTCGAATTCCAGTACC；R：TTATAAGTTTAATACATGAGGAGCT
基因克隆Gene clone	SoAN2	F：ATGTTAAGTACACAAGTTGAAG；R：TCATATGATATTTTCAGAACTCAGA
qRT-PCR	qSoActin	F：TGGAATGTGCTGAGAGATGC；R：TGCTGACCGTATGAGCAAAG
	qSoF3'H	F：TCAACGTTTGCACCACCAAT；R：ACCATCAACTCCACCACCAT
	qSoAN2	F：ATTCCTGGAAGAACCGGCAA；R：TCCCCTCTTCTCGACCCAAT
基因沉默Gene silencing	TRV2-SoF3'H	F：TAAGGTTACCGAATTCATGTTCACTTCGAATTCCAGTACCC；
基因沉默Gene silencing		R：GCTCGGTACCGGATCCGGTTTCGGGCCAATGTGGG
基因过表达 Gene overexpression	PRI101-SoF3'H	F：ATGCCCGTCGACCCCGGGATGTTCACTTCGAATTCCAGTACCC；
基因过表达 Gene overexpression		R：TGCTCACCAT GGATCCTAAGTTTAATACATGAGGAGCTAATCTGGGC
启动子克隆Promoter clone	SoF3'H-promoter	F：GAGCAATTAAAGTCTTGCCC；R：TTTGAAGTTGGAAGGTTATACATAT

图1 SoF3′H与其他物种F3′H蛋白序列比对分析红色框为P450保守基序，motif Ⅰ（PPGPKPWP），motif Ⅱ（FGAGRRICAG）。绿色框为F3′H特有基序，Ⅰ（VVVAAS），Ⅱ（GGEK）。Oe：油橄榄；Ce：咖啡；Eg：草原龙胆；Lf：枫香树。

Fig. 1 Alignment and analysis of SoF3′H with F3′H protein sequences of other species Red boxes are P450 conserved motifs，motif Ⅰ（PPGPKPWP），motif Ⅱ（FGAGRRICAG），and green boxes are F3′H characteristic motifs，Ⅰ（VVVAAS），Ⅱ（GGEK）. Oe：Olea europaea；Ce：Coffea eugenioides；Eg：Eustoma grandiflorum；Lf：Liquidambar formosana.

图2 紫丁香SoF3′H与其他20个物种F3′H系统进化树

Fig. 2 Phylogenetic tree of SoF3′H and F3′H from other 20 plant species

图3 紫丁香不同器官组织和不同花发育时期花瓣中SoF3'H的表达水平不同小写字母表示在P < 0.05水平差异显著。

Fig. 3 The relative expression levels of SoF3'H in different organs，tissues and flowers in different development stages Different lowercase letters indicate significant differences at 0.05 level.

图4 紫丁香沉默SoF3'H植株的基因表达水平（A）、花色（B）和花青苷含量（C）的变化 *** 表示P < 0.001，**** 表示P < 0.0001。

Fig. 4 Change of silencing lilac SoF3'H on gene expression level（A），flower color（B）and flower anthocyanin content（C） *** indicate P < 0.001，**** indicate P < 0.0001.

图5 紫丁香过表达SoF3'H植株的基因表达水平（A）、花青苷含量（B）和花色（C）的变化 **表示P < 0.01。

Fig. 5 Change of over expression lilac SoF3'H on gene expression level（A），flower anthocyanin content（B）and flower color（C） ** indicate P < 0.01.

图6 酵母单杂系统分析SoAN2和SoF3'H启动子的结合

Fig. 6 Yeast one-hybrid system analysis of SoAN2 and SoF3'H promoter binding

图7 紫丁香不同器官组织和不同花发育时期花瓣中SoAN2的表达水平不同小写字母表示在P < 0.05水平差异显著。

Fig. 7 The relative expression levels of SoAN2 in different flower development stages and different organs in lilac Different lowercase letters indicate significant differences at 0.05 level.

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紫丁香SoF3′H对花瓣中花青苷合成的功能解析

Functional Analysis of SoF3′H in the Synthesis of Anthocyanidin in Syringa oblata Petals

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紫丁香SoF3′H对花瓣中花青苷合成的功能解析

Functional Analysis of SoF3′H in the Synthesis of Anthocyanidin in Syringa oblata Petals

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