Study on the Regulation of Anthocyanin Metabolism by miR156-PhSPL3 in Pericallis hybrida

doi:10.13560/j.cnki.biotech.bull.1985.2024-0325

Abstract

Abstract:

【Objective】Pericallis hybrida is an important potted ornamental flower with a variety of colors and anthocyanin metabolic branches, which is an ideal material to study the molecular mechanism of flower color regulation. miR156-PhSPL3 module is differently expressed in different varieties of P. hybrida, exploring the mechanism of this module in the formation of flower color would provide a theoretical basis for the regulation mechanism of flower anthocyanin metabolism.【Method】PhSPL3 and two Ph-miR156 precursor genes（Ph-MIR156a and Ph-MIR156b）were cloned from P. hybrida for bioinformatics analysis and tissue expression analysis. Experimental studies using VIGS, yeast one-hybrid（Y1H）and two-hybrid（Y2H）system, and dual-luciferase assays elucidated the function and regulatory role of miR156-PhSPL3 in anthocyanin metabolism.【Result】The open reading frame of PhSPL3 gene was 1 119 bp long, encoded a protein of 372 amino acids and belonged to Clade VII branch of the SPL family. Ph-MIR156a and Ph-MIR156b precursor stem-loop sequences were 108 bp and 104 bp long respectively, both containing mature miR156 sequences. Expression analysis revealed the high expression of PhSPL3 in carmine and blue varieties of P. hybrida ray florets, inversely correlated with Ph-miR156 expression. Dual-luciferase assays demonstrated that Ph-miR156 targeted to PhSPL3. Virus-induced gene silencing（VIGS）of PhSPL3 reduced anthocyanin content and downregulated expressions of anthocyanin metabolic genes PhCHS2, PhF3H1, PhANS, and regulatory gene PhbHLH17. Y2H and Y1H revealed PhSPL3 interacted with PhMYB5 and PhMYB7 and specifically bound to the promoters of PhCHS2 and PhbHLH17, thus regulating their expressions. 【Conclusion】The miR156-PhSPL3 pathway in P. hybrida may play a role in the biosynthesis of anthocyanin substances by directly regulating structural genes on the anthocyanin metabolism branch or indirectly regulating genes regulating anthocyanin metabolism.

Key words: Pericallis hybrida, miR156, PhSPL3, anthocyanin metabolism

SUN Dan-ni, LI Hao, CUI Yu-meng, HUANG He. Study on the Regulation of Anthocyanin Metabolism by miR156-PhSPL3 in Pericallis hybrida[J]. Biotechnology Bulletin, 2024, 40(12): 113-123.

Figures/Tables 8

Fig. 1 Cloning of PhSPL3 gene and alignment of SPL amino acid sequences with those from other species A: Cloning of PhSPL3 gene; M: DL2000 DNA Marker; 1: amplification using cDNA from PeC ray florets as template; 2: amplification using cDNA from PeB ray florets as template. B: Amino acid sequence alignment of PhSPL3 protein; PhSPL3: Pericallis hybrida（Unigene0015241）; CcSPL13: Cynara cardunculus var. scolymus（XP_024977060.1）; CmSPL3: Chrysanthemum ×morifolium（ALF46633.1）; HaSPL3: Helianthus annuus（XP_021978557.1）; AtSPL13: Arabidopsis thaliana（AT5G50570）. The red lines in the figure B indicate the positions of the SBP domain

Fig. 2 Cloning and sequence alignment of Ph-miR156 precursor genes A: Cloning of Ph-miR156 precursor gene; M: DL2000 DNA Marker; 1: cloning of Ph-MIR156a gene; 2: cloning of Ph-MIR156b gene. B: Sequence alignment of Ph-MIR156a and Ph-MIR156b. The red lines in the figure B indicate mature miR156 sequences

Fig. 3 Phylogenetic analysis of PhSPL3 protein family in comparison with SPL protein from A. thaliana and C. ×morifolium At: Arabidopsis thaliana; Cm: Chrysanthemum ×morifolium; Ph: Pericallis hybrida

Fig. 4 RT-qPCR expression analysis of PhSPL3 and Ph-miR156 in ray florets of PeW, PeC and PeB at stage S1-S3 PeW: White Pericallis hybrida, PeC: Carmine Pericallis hybrida, PeB: Blue Pericallis hybrida. * and ** indicate significant differences at 0.05 and 0.01 levels

Fig. 5 Dual-luciferase assay to verify the targeting of PhSPL3 by Ph-MIR156a and Ph-MIR156b

Fig. 6 Transient silencing of PhSPL3 by VIGS in the leaves of PeC A: Phenotypes of CK and PhPSL3-silenced Pericallis hybrida leaves. B:Detection of pTRV1 and pTRV2-PhSPL3 in the silenced tissue. C: Quantitative analysis of anthocyanins in PhSPL3-silenced leaves and the control. D: Expressions of genes in the anthocyanin biosynthesis pathway in PhSPL3-silenced and the control. CK: Leaf samples infiltrated with pTRV1 and pTRV2::00. PhSPL3-SL: Leaf samples infiltrated with pTRV1 and pTRV2::PhSPL3. * and ** indicate significant differences at 0.05 and 0.01 levels respectively

Fig. 7 Protein interaction analysis between PhSPL3 and PhMYB1-PhMYB7 by Y2H assay

Fig. 8 Yeast one-hybrid results of PhSPL3 with the promoter of PhCHS2, PhANS and PhbHLH17 by Y1H

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