瓜叶菊miR156-PhSPL3调控花青素代谢的研究

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

摘要/Abstract

摘要：

【目的】瓜叶菊（Pericallis hybrida）是重要的盆栽观赏花卉，具有多种花色和花青素代谢支路，是研究花色调控分子机制的理想材料。miR156-PhSPL3模块在瓜叶菊不同花色品种中差异表达，探究该模块在花色形成中的作用机制，为花卉花青素代谢调控机制提供理论依据。【方法】从瓜叶菊中克隆获得一个PhSPL3基因和两个Ph-miR156前体基因（Ph-MIR156a和Ph-MIR156b），对其进行生物信息学分析及表达模式分析，利用VIGS、酵母杂交实验和双荧光素酶实验等方法研究miR156-PhSPL3参与花青素代谢的功能和调控机制。【结果】PhSPL3基因开放阅读框长1 119 bp，编码372个氨基酸，属于SPL家族的Clade VII分支。Ph-MIR156a和Ph-MIR156b前体茎环序列长度分别为108 bp和104 bp，均含miR156成熟序列。表达分析显示PhSPL3在玫色和蓝色品种的瓜叶菊舌状花中高表达，与Ph-miR156表达呈负相关。双荧光素酶实验表明，Ph-miR156可靶向PhSPL3。PhSPL3的VIGS沉默株系中花青素含量降低，花青素代谢结构基因PhCHS2、PhF3H1、PhANS及调节基因PhbHLH17的表达下调。酵母杂交结果显示，PhSPL3可与PhMYB5和PhMYB7互作，并特异性结合PhCHS2和PhbHLH17的启动子，从而调控其表达。【结论】瓜叶菊PhSPL3可通过直接调控花青素代谢分支上结构基因，或间接调控花青素代谢调节基因的方式，在瓜叶菊花青素物质的生物合成中发挥作用。

关键词: 瓜叶菊, miR156, PhSPL3, 花青素代谢

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

孙丹旎, 李好, 崔宇萌, 黄河. 瓜叶菊miR156-PhSPL3调控花青素代谢的研究[J]. 生物技术通报, 2024, 40(12): 113-123.

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.

图/表 8

图1 PhSPL3基因克隆以及与其他物种SPL氨基酸序列比对 A：PhSPL3基因克隆；M：DL2000 DNA Marker；1：以PeC舌状花cDNA为模板扩增；2：以PeB舌状花cDNA为模板扩增。B：PhSPL3氨基酸序列比对；PhSPL3：瓜叶菊；CcSPL13：朝鲜蓟（XP_024977060.1）；CmSPL3：菊花（ALF46633.1）；HaSPL3：向日葵（XP_021978557.1）；AtSPL13：拟南芥（AT5G50570）。图B中红线部分为SBP结构域位置

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

图2 Ph-miR156前体基因克隆与基因序列比对 A：Ph-miR156前体基因克隆；M：DL2000 DNA Marker；1：Ph-MIR156a基因克隆；2：Ph-MIR156b基因克隆。B: Ph-MIR156a和Ph-MIR156b序列比对，红线部分为成熟的miR156序列

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

图3 PhSPL3与拟南芥和菊花的SPL系统进化分析 At：拟南芥；Cm：菊花；Ph：瓜叶菊

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

图4 S1-S3阶段PeW、PeC和PeB舌状花PhSPL3和Ph-miR156的RT-qPCR表达分析 PeW：白色瓜叶菊，PeC：洋红色瓜叶菊，PeB：蓝色瓜叶菊。*表示在0.05水平上差异显著，**表示在0.01水平上差异显著

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

图5 双荧光素酶实验验证Ph-MIR156a、Ph-MIR156b靶向PhSPL3

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

图6 用VIGS技术在PeC叶片中短暂沉默PhSPL3 A：CK和PhSPL3沉默后的瓜叶菊叶片的表型；B：沉默组织中pTRV1和pTRV2-PhSPL3的确认；C：CK和PhPL3沉默的瓜叶菊花青素提取及含量检测；D：CK和PhSPL3沉默的瓜叶菊中类黄酮生物合成相关基因（PhSPL3、PhCHS2、PhF3H1、PhDFR3、PhANS和PhbHLH17）的RT-qPCR表达分析。CK：只注射了pTRV1和pTRV2空载的瓜叶菊叶片；PhPSL3-SL：PhSPL3沉默后的瓜叶菊叶片。*表示在0.05水平上差异显著，**表示在0.01水平上差异显著

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

图7 PhSPL3与PhMYB1-PhMYB7之间的酵母双杂交结果

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

图8 PhSPL3与PhCHS2、PhANS和PhbHLH17的启动子之间的酵母单杂交结果

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

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