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

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

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

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

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 Online:2024-08-25 Published:2024-08-21
Contact: WU Jing

Abstract

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

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.

Figures/Tables 8

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

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.

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

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.

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.

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.

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

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

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