梅花PmMYB21在花丝伸长过程中的功能与表达调控分析

doi:10.16420/j.issn.0513-353x.2022-0119

摘要/Abstract

摘要：

MYB21是调控雄蕊花丝伸长的关键转录因子。以梅花（Prunus mume）早花品种‘粉红朱砂’与晚花品种‘早绿萼’的花芽为材料，分别克隆得到两个序列完全一致的PmMYB21。序列分析表明PmMYB21蛋白含有MYB转录因子的R2R3保守结构域，系统进化分析表明PmMYB21与其他物种的MYB21有较高的同源性，亚细胞定位结果显示其位于细胞核内。PmMYB21的表达水平在两个梅花品种开花过程中随花丝伸长而升高；相对于晚花品种，早花品种表达上调时间显著提前。在两个梅花品种的PmMYB21启动子-1 529 ~-1 243 bp区域中均检测到CG序列型的甲基化水平下调，该区域甲基化水平与PmMYB21的表达量呈负相关；并在此区域检测到脱落酸、赤霉素和光响应元件，推测这些调控过程可能受到甲基化修饰的影响。加权基因共表达网络分析（WGCNA）结果显示，5 296个差异表达基因与PmMYB21具有连通性，显著富集于激素响应等生物过程。结合WGCNA筛选结果和表达趋势分析，预测PmMYB21介导生长素（IAA）、赤霉素（GA）和茉莉酸（JA）等激素的信号转导，进而参与梅花花丝的伸长。

关键词: 梅花, 花丝伸长, PmMYB21, 甲基化修饰, WGCNA, 激素响应

Abstract:

MYB21 is the key transcription factor regulating filament elongation. The flower buds from two Prunus mume cultivars，‘Fenhong Zhusha’（early-flowering）and‘Zaolü’e’（late-flowering），were used as experimental materials. The full length cDNA sequences of PmMYB21 from two cultivars were cloned and found that they have the same sequence. Sequence analysis showed that PmMYB21 protein contains the R2R3 conserved domain of MYB transcription factor. Phylogenetic analysis showed high homology between PmMYB21 and MYB21 protein from other plant species. The subcellular localization experiment showed that PmMYB21 was located in the cell nucleus. The expression level of PmMYB21 in both cultivars increased as the filament elongate during the flower opening process. The up-regulated timing of PmMYB21 expression level in early-flowering cultivar was significantly earlier than in late-flowering cultivar. Additionally，Hypomethylation region of CG-context were detected within-1 529 to-1 243 bp of PmMYB21 promoter region during flowering process in both two cultivars. The methylation level of this region was negatively correlated with the expression level of PmMYB21. The cis-acting elements including elements responsive to abscisic acid，gibberellin，and light were also detected within the hypomethylation region，suggesting that these regulatory processes might be affected by methylation modifications. The results of weighted gene co-expression network analysis（WGCNA）showed that 5 296 DEGs had highest connectivity with PmMYB21，significantly enriched in response to hormone. Combining the candidate genes screened by WGCNA and the expression pattern of candidate genes，PmMYB21 was predicted participated the signal transduction of hormones such as auxin（IAA），gibberellin（GA）and jasmonic acid（JA），and resulting in the filament elongation of P. mume.

Key words: Prunus mume, filament elongation, PmMYB21, methylation modification, WGCNA, response to hormone

中图分类号:

S685.17

袁茜, 张曼, 马开峰, 王佳, 张启翔. 梅花PmMYB21在花丝伸长过程中的功能与表达调控分析[J]. 园艺学报, 2023, 50(5): 1048-1062.

Yuan Xi, Zhang Man, Ma Kaifeng, Wang Jia, Zhang Qixiang. Function and Expression Regulation Analysis of Prunus mume PmMYB21 During Filament Elongation[J]. Acta Horticulturae Sinica, 2023, 50(5): 1048-1062.

图/表 12

表1 ‘粉红朱砂’和‘早绿萼’梅花样品信息

Table 1 Samples information of flower buds of Prunus mume‘Fenhong Zhusha’and‘Zaolü’e’

采样日期/（M-D） Sampling date	温度/℃ Temperature	开花阶段 Flowering stage
采样日期/（M-D） Sampling date	温度/℃ Temperature	粉红朱砂 Fenhong Zhusha	早绿萼 Zaolü’e
01-09	0	S2（花芽开始膨大 Bud swelling）	S2（花芽开始膨大 Bud swelling）
02-09	0	S4（萼片全露 Sepals clearly visible）	S2（花芽开始膨大Bud swelling）
03-02	15 ~ 18	S6（花瓣全露 Petal clearly visible）	S4（萼片全露 Sepals clearly visible）
03-13	19	S8（花盛开 Full blooming）	S6（花瓣全露 Petal clearly visible）
03-19	23		S8（花盛开 Full blooming）

表2 梅花MYB21的亚细胞定位和qRT-PCR引物

Table 2 Primers for subcellular localization and qRT-PCR of PmMYB21

引物名称 Primer name	引物序列（5′-3′） Primer sequence
PmMYB21-1300F	CCAAATCGACTCTAGTCTAGA ATGGATAAAAAGCCATGCAATAGTT
PmMYB21-1300R	ATGGTACCGGATCCACTAGT TTACTCTCCATTTAGTAACTGCATAGACC
PmMYB21-1300R-TTA	ATGGTACCGGATCCACTAGT CTCTCCATTTAGTAACTGCATAGACC
SUPER-C-F	AGCAAGAACGGAATGCGCGTGAC
SUPER-C-R	TTGCCGGTGGTGCAGATGAACTTC
PmMYB21-qPCR-F	TGAGAAAAGGGCCATGGACC
PmMYB21-qPCR-R	CCTAACATCAGGCCGCAGAT

图1 梅花PmMYB21与其他植物MYB21氨基酸序列的进化树（A）和5个梅花MYB同源基因的表达量热图（B） Pm：梅花；At：拟南芥；Rc：月季；Os：水稻；Zm：玉米；Sl：番茄；Tc：可可。

Fig. 1 Phylogenetic tree（A）of MYB21 protein from Prunus mume and other speciesand heat map（B）of the expression of five MYB homologous genes in Prunus mume Pm：Prunus mume；At：Arabidopsis thaliana；Rc：Rosa chinensis；Os：Oryza sativa；Zm：Zea mays；Sl：Solanum lycopersicum；Tc：Theobroma cacao.

图2 梅花PmMYB21与其他物种MYB21氨基酸序列比对 Pm：梅花；At：拟南芥；Rc：月季。

Fig. 2 Multiple sequence alignment of MYB21 protein from Prunus mume and other species Pm：Prunus mume；At：Arabidopsis thaliana；Rc：Rosa chinensis.

图3 梅花PmMYB21的亚细胞定位

Fig. 3 The subcellular localization of PmMYB21 of Prunus mume

图4 梅花‘粉红朱砂’和‘早绿萼’开花过程中PmMYB21启动子区的差异甲基化区域（A）和顺式作用元件预测（B） S4：萼片全露；S6：花瓣全露；S8：花盛开。

Fig. 4 Different methylation region（A）and predict cis-acting element on the promoter region（B）of PmMYB21 during flowering process of two Prunus mume cultivars S4：Sepals clearly；S6：Petal clearly visible；S8：Full blooming.

图5 梅花‘粉红朱砂’和‘早绿萼’开花过程中PmMYB21的表达（FPKM）及其启动子的加权甲基化水平（MLw）

Fig. 5 The fpkm value of PmMYB21 and its weighted methylation level（MLw）in the R3 area of PmMYB21 promoter region during flowering of two Prunus mume cultivars

图6 WGCNA的软阈值

Fig. 6 Soft-thresholding of WGCNA

图7 梅花PmMYB21共表达基因的KEGG富集（A）和网络图（B）菱形代表转录因子（TF）。蓝色：MYB；浅红：生长素相关基因；浅紫：B3类基因。

Fig. 7 KEGG enrichment result（A）nd Network diagram（B）of PmMYB21 co-expression genes in Prunus mume The diamonds represent transcription factors（TF）. Blue：MYB；Light red：Auxin-related genes；Light purple：B3 class genes.

表3 与PmMYB21高权重关联的共表达基因中的开花相关基因功能注释

Table 3 Functional annotation of flowering-related genes among high-weight co-expressed genes associated with PmMYB21

基因编号 Gene ID	拟南芥参考基因 Match tair ID	基因功能注释 Gene function annotions
Pm010066*	AT1G28300.1	推定的转录因子B3域家族 Putative transcription factor B3-Domain family
Pm022570*	AT1G76890.2	三螺旋转录因子GT-2 like Trihelix transcription factor GT-2-like
Pm027133*	AT5G20820.1	生长素响应蛋白SAUR 50-like Auxin-responsive protein SAUR 50-like
Pm020852*	AT4G38620.1	MYB相关蛋白308-like MYB-related protein 308-like
Pm020227*	AT5G43700.1	AUX/IAA 转录调节家族蛋白（AtAUX2-11） AUX/IAA transcriptional regulator family protein（AtAUX2-11）
Pm018533	AT4G30080.1	生长素响应因子ARF16 Auxin response factor 16
Pm021891	AT4G38840.1	SAUR类生长素反应蛋白家族 SAUR-like auxin-responsive protein family
Pm003647	AT1G08550.1	非光化学淬灭1（NPQ1） Non-photochemical quenching 1（NPQ1）

图8 雄蕊花丝伸长调控通路（A）和梅花开花过程中PmMYB21上游调控基因的表达量热图（B）

Fig. 8 The regulation pathway of filament elongation（A）and the expression of PmMYB21 and its upstream regulatory genes during flowering of two Prunus mume cultivars（B）

图9 梅花‘粉红朱砂’和‘早绿萼’开花过程中PmMYB21表达量的qRT-PCR验证

Fig. 9 The qRT-PCR verification of PmMYB21 expression during flowering of two Prunus mume cultivars ‘Fenhong Zhusha’and‘Zaolü’e’

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