Function and Expression Regulation Analysis of Prunus mume PmMYB21 During Filament Elongation

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

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (5): 1048-1062.doi: 10.16420/j.issn.0513-353x.2022-0119

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

Function and Expression Regulation Analysis of Prunus mume PmMYB21 During Filament Elongation

Yuan Xi, Zhang Man, Ma Kaifeng(), Wang Jia, Zhang Qixiang

Beijing Key Laboratory of Ornamental Plants Cermplasm Innovation & Molecular Breeding，National Engineering Research Center for Floriculture，Beijing Laboratory of Urban and Rural Ecological Environment，Engineering Research Center of Landscape Environment Ministry of Education，Key Laboratory of Genetics and Breeding in Forest trees and Ornamental Plants of Ministry of Education，School of Landscape Architecture，Beijing Forestry University，Beijing 100083，China

Received:2022-12-05 Revised:2023-02-07 Online:2023-05-25 Published:2023-05-31
Contact: Ma Kaifeng, Zhang Qixiang E-mail:makaifeng@bjfu.edu.cn

Abstract

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

CLC Number:

S685.17

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.

Figures/Tables 12

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）

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

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.

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

Fig. 3 The subcellular localization of PmMYB21 of Prunus mume

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.

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

Fig. 6 Soft-thresholding of WGCNA

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.

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）

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）

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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Function and Expression Regulation Analysis of Prunus mume PmMYB21 During Filament Elongation

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