PpMADS2与PpMADS3协同调控黄肉桃果实类胡萝卜素积累机制的研究

doi:10.16420/j.issn.0513-353x.2022-0221

摘要/Abstract

摘要：

为探究黄肉桃果实中MADS-box家族转录因子对类胡萝卜素代谢的调控作用，从黄肉桃果实中克隆得到两个MADS基因，分别命名为PpMADS2（PRUPE_5G208500）和PpMADS3（PRUPE_5G208400）。PpMADS2开放阅读框为768 bp，编码255个氨基酸；PpMADS3开放阅读框为756 bp，编码251个氨基酸。生物信息分析显示二者蛋白的N端均含有MADS-box家族典型的特征结构域。亚细胞定位分析显示PpMADS2和PpMADS3定位于细胞核。荧光定量PCR结果表明PpMADS2、PpMADS3以及类胡萝卜素合成基因PpPSY和PpCHYB在黄肉桃果实成熟过程中表达量呈上升趋势，与果实成熟期间类胡萝卜素的含量增加一致。酵母双杂交试验结果表明PpMADS2和PpMADS3蛋白存在相互作用。双荧光素酶试验结果表明PpMADS2和PpMADS3可以协同激活PpPSY和PpCHYB启动子。因此推测PpMADS2和PpMADS3可通过转录激活PpPSY和PpCHYB促进桃果实类胡萝卜素的积累。

关键词: 桃, 黄肉, 类胡萝卜素, MADS-box, 蛋白互作, 转录激活

Abstract:

To explore the regulation of MADS-box family transcription factors on carotenoid metabolism in yellow fleshed peach, two MADS genes named as PpMADS2（PRUPE_5G208500）and PpMADS3（PRUPE_5G208400）were cloned. The open reading frames of PpMADS2 and PpMADS3 were 768 bp and 756 bp，encoding 255 and 251 amino acids, respectively. Bioinformatics analysis revealed that the N-terminus of both proteins contained typical characteristic domains of MADS-box family. Subcellular localization showed that both PpMADS2 and PpMADS3 were located at the nucleus. The results showed the gene expression levels of PpMADS2，PpMADS3 as well as carotenoid biosynthetic genes such as PpPSY and PpCHYB increased during fruit ripening，which was consistent with the carotenoid accumulation in yellow fleshed peach. Furthermore, the results demonstrated that PpMADS2 could interact with PpMADS3 then synergistically activate the promoters of PpPSY and PpCHYB genes. Therefore，it is speculated that PpMADS2 and PpMADS3 promoted the accumulation of carotenoids in yellow fleshed peach fruit cooperatively through transcriptional activation of PpPSY and PpCHYB. These research provided a theoretical basis for further exploring the regulatory mechanism of MADS-box transcription factors on carotenoid accumulation in peach fruit.

Key words: peach, yellow flesh, carotenoids, MADS-box, protein interaction, transcriptional activation

中图分类号:

S662.1

肖翔, 周储江, 金舒婉, 施丽愉, 杨震峰, 曹士锋, 陈伟. PpMADS2与PpMADS3协同调控黄肉桃果实类胡萝卜素积累机制的研究[J]. 园艺学报, 2023, 50(6): 1173-1186.

XIAO Xiang, ZHOU Chujiang, JIN Shuwan, SHI Liyu, YANG Zhenfeng, CAO Shifeng, CHEN Wei. Mechanism of PpMADS2 and PpMADS3 Synergistically Regulating Carotenoids Accumulation in Peach Fruit[J]. Acta Horticulturae Sinica, 2023, 50(6): 1173-1186.

图/表 14

表1 试验中所需引物

Table 1 Primers for experiments

用途 Function	名称 Name	基因ID Gene ID	引物序列（5′-3′） Primer sequence
高保真PCR High fidelity PCR	gPpMADS2	PRUPE_5G208500	F：ATGGGGAGGGGAAGGGTGCAGCTGAAGAG R：CTATTCATTAAGGTGGCGGAGCATCCATGGG
	gPpMADS3	PRUPE_5G208400	F：ATGGGAAGAGGTAGAGTTGAGCTGAAGAGGAT R：TCAAAGCATCCACCCAGGAATGAATCCAT
荧光定量PCR qRT-PCR	PpMADS2	PRUPE_5G208500	F：TGGCAAAGAAGGTAAAGGAGAAG R：GAAGGGTGGAGGAGCAGTC
	PpMADS3	PRUPE_5G208400	F：CCAGAACAAGGAACAAATGC R：AGTGAGGATGATGAGGAAGG
	PpPSY	PRUPE_3G013200	F：TATTATGTTGCTGGGACTG R：GTGTTTGTGAGCTGATTCG
	PpCHYB	PRUPE_2G300800	F：AGGTTGCTGCTGCTCATC R：CGCTTTGATTCTCCTTTC
	PpPDS	PRUPE_1G174100	F：CCGTTGAAGGTCGTGATTG R：CTTTCCGCCCAGAACATC
	PpLCYB	PRUPE_7G046100	F：CGTGGCACAGCAAGTCTCAG R：CAGGTAGTGTCCAGGCAATCAAG
	PpZEP	PRUPE_7G133100	F：AGAAGCAGAACAAGAAGTG R：AACCAGTCCTCCAATTCC
	PpZDS	PRUPE_6G340000	F：AGAAGCAGAACAAGAAGTG R：AACCAGTCCTCCAATTCC
载体构建 Carrier construction	PpMADS2-GFP	PRUPE_5G208500	F：ACGGGGGACTCTTGACCATGGCCATGGGGAGGGGAAGGGTG R：GCCCTTGCTCACCATACTAGTTTCATTAAGGTGGCGGAGCA
	PpMADS3-GFP	PRUPE_5G208400	F：ACGGGGGACTCTTGACCATGGCCATGGGAAGAGGTAGAGTTGAGCTG R：GCCCTTGCTCACCATACTAGTAAGCATCCACCCAGGAATGA
	PpMADS2-AD	PRUPE_5G208500	F：GCCATGGAGGCCAGTGAATTCATGGGGAGGGGAAGGGTG R：CAGCTCGAGCTCGATGGATCCCTATTCATTAAGGTGGCGGAGC
	PpMADS3-AD	PRUPE_5G208400	F：GCCATGGAGGCCAGTGAATTCATGGGAAGAGGTAGAGTTGAGCTG R：CAGCTCGAGCTCGATGGATCCTCAAAGCATCCACCCAGGAA
	PpMADS2-BD	PRUPE_5G208500	F：ATGGCCATGGAGGCCGAATTCATGGGGAGGGGAAGGGTG R：CTAGTTATGCGGCCGCTGCAGCTATTCATTAAGGTGGCGGAGC
	PpMADS3-BD	PRUPE_5G208400	F：ATGGCCATGGAGGCCGAATTCATGGGAAGAGGTAGAGTTGAGCTG R：CTAGTTATGCGGCCGCTGCAGTCAAAGCATCCACCCAGGAA
	PpMADS2-62-sk	PRUPE_5G208500	F：CGCTCTAGAACTAGTGGATCCATGGGGAGGGGAAGGGTG R：GTCGACGGTATCGATAAGCTTCTATTCATTAAGGTGGCGGAGC
	PpMADS3-62-sk	PRUPE_5G208400	F：CGCTCTAGAACTAGTGGATCCATGGGAAGAGGTAGAGTTGAGCTG R：GTCGACGGTATCGATAAGCTTTCAAAGCATCCACCCAGGAA
载体构建（启动子克隆） Carrier construction	PpPSYpro-LUC	PRUPE_3G013200	F：ATCCCGGGTTGCTCAGGATCTGCCACGTGTA R：ATCCATGGGTTGTTTGAACTTGAAACCTCAGCC
（Promoter cloning）	PpCHYBpro-LUC	PRUPE_2G300800	F：GCGGATCCTCTGGAAGTGAAGGAATTGTCGTAC R：GCCCATGGAAAAGATGCCTGGTTTGTGGAC

表1 试验中所需引物

Table 1 Primers for experiments

用途 Function	名称 Name	基因ID Gene ID	引物序列（5′-3′） Primer sequence
高保真PCR High fidelity PCR	gPpMADS2	PRUPE_5G208500	F：ATGGGGAGGGGAAGGGTGCAGCTGAAGAG R：CTATTCATTAAGGTGGCGGAGCATCCATGGG
	gPpMADS3	PRUPE_5G208400	F：ATGGGAAGAGGTAGAGTTGAGCTGAAGAGGAT R：TCAAAGCATCCACCCAGGAATGAATCCAT
荧光定量PCR qRT-PCR	PpMADS2	PRUPE_5G208500	F：TGGCAAAGAAGGTAAAGGAGAAG R：GAAGGGTGGAGGAGCAGTC
	PpMADS3	PRUPE_5G208400	F：CCAGAACAAGGAACAAATGC R：AGTGAGGATGATGAGGAAGG
	PpPSY	PRUPE_3G013200	F：TATTATGTTGCTGGGACTG R：GTGTTTGTGAGCTGATTCG
	PpCHYB	PRUPE_2G300800	F：AGGTTGCTGCTGCTCATC R：CGCTTTGATTCTCCTTTC
	PpPDS	PRUPE_1G174100	F：CCGTTGAAGGTCGTGATTG R：CTTTCCGCCCAGAACATC
	PpLCYB	PRUPE_7G046100	F：CGTGGCACAGCAAGTCTCAG R：CAGGTAGTGTCCAGGCAATCAAG
	PpZEP	PRUPE_7G133100	F：AGAAGCAGAACAAGAAGTG R：AACCAGTCCTCCAATTCC
	PpZDS	PRUPE_6G340000	F：AGAAGCAGAACAAGAAGTG R：AACCAGTCCTCCAATTCC
载体构建 Carrier construction	PpMADS2-GFP	PRUPE_5G208500	F：ACGGGGGACTCTTGACCATGGCCATGGGGAGGGGAAGGGTG R：GCCCTTGCTCACCATACTAGTTTCATTAAGGTGGCGGAGCA
	PpMADS3-GFP	PRUPE_5G208400	F：ACGGGGGACTCTTGACCATGGCCATGGGAAGAGGTAGAGTTGAGCTG R：GCCCTTGCTCACCATACTAGTAAGCATCCACCCAGGAATGA
	PpMADS2-AD	PRUPE_5G208500	F：GCCATGGAGGCCAGTGAATTCATGGGGAGGGGAAGGGTG R：CAGCTCGAGCTCGATGGATCCCTATTCATTAAGGTGGCGGAGC
	PpMADS3-AD	PRUPE_5G208400	F：GCCATGGAGGCCAGTGAATTCATGGGAAGAGGTAGAGTTGAGCTG R：CAGCTCGAGCTCGATGGATCCTCAAAGCATCCACCCAGGAA
	PpMADS2-BD	PRUPE_5G208500	F：ATGGCCATGGAGGCCGAATTCATGGGGAGGGGAAGGGTG R：CTAGTTATGCGGCCGCTGCAGCTATTCATTAAGGTGGCGGAGC
	PpMADS3-BD	PRUPE_5G208400	F：ATGGCCATGGAGGCCGAATTCATGGGAAGAGGTAGAGTTGAGCTG R：CTAGTTATGCGGCCGCTGCAGTCAAAGCATCCACCCAGGAA
	PpMADS2-62-sk	PRUPE_5G208500	F：CGCTCTAGAACTAGTGGATCCATGGGGAGGGGAAGGGTG R：GTCGACGGTATCGATAAGCTTCTATTCATTAAGGTGGCGGAGC
	PpMADS3-62-sk	PRUPE_5G208400	F：CGCTCTAGAACTAGTGGATCCATGGGAAGAGGTAGAGTTGAGCTG R：GTCGACGGTATCGATAAGCTTTCAAAGCATCCACCCAGGAA
载体构建（启动子克隆） Carrier construction	PpPSYpro-LUC	PRUPE_3G013200	F：ATCCCGGGTTGCTCAGGATCTGCCACGTGTA R：ATCCATGGGTTGTTTGAACTTGAAACCTCAGCC
（Promoter cloning）	PpCHYBpro-LUC	PRUPE_2G300800	F：GCGGATCCTCTGGAAGTGAAGGAATTGTCGTAC R：GCCCATGGAAAAGATGCCTGGTTTGTGGAC

图1 双荧光素酶试验载体构建原理图

Fig. 1 Construction principle of double luciferase experimental vector

图2 黄肉桃‘锦绣’果实成熟期间总类胡萝卜素含量变化 S1：第1次膨大软核期；S2：硬核期；S3：第2次膨大期；S4：硬熟期；S5：完熟期。不同小写字母表示在0.05水平上差异显著（P < 0.05）。下同。

Fig. 2 Changes of total carotenoid content different maturity in yellow fleshed peach‘Jinxiu’ S1：The first soft-nucleus stage of enlargement；S2：Hardcore stage；S3：Second stage of enlargement；S4：Hard ripening stage；S5：Ripening stage. Different lowercase letters indicate significant differences at the 0.05 level. The same below.

表2 转录组测序结果

Table 2 Transcriptome sequencing results

名称 Name	基因ID Gene ID	FPKM
名称 Name	基因ID Gene ID	第2次膨大期 Second stage of enlargement（S3）	完熟期 Ripening stage（S5）
PpMADS2	PRUPE_5G208500	38.45 ± 1.14 b	63.50 ± 2.05 a
PpMADS3	PRUPE_5G208400	51.14 ± 1.33 b	127.24 ± 2.17 a

图3 桃果实PpMADS2和PpMADS3的电泳检测

Fig. 3 Electrophoresis detection of PpMADS2 and PpMADS3 in peach fruit

图4 桃果实PpMADS2（A）和PpMADS3（B）的氨基酸同源序列分析 Pp：桃；Pd：扁桃；Pm：日本杏；Pa：甜樱桃；Ppvc：梨；Md：苹果；Lc：荔枝；Nt：烟草；Rc：月季；Cc：克莱门柚。

Fig. 4 The alignment of PpMADS2（A）and PpMADS3（B）amino acid sequences Pp：Prunus persica；Pd：Prunus dulcis；Pm：Prunus mume；Pa：Prunus avium；Ppvc：Pyrus pyrifolia；Md：Malus domestica；Lc：Litchi chinensis；Nt：Nicotiana tabacum；Rc：Ricinus communis；Cc：Citrus clementina.

图5 PpMADS2（A）和PpMADS3（B）系统发育树

Fig. 5 Phylogenetic trees of PpMADS2（A）and PpMADS3（B）

图6 PpMADS2和PpMADS3蛋白在烟草表皮细胞中的亚细胞定位

Fig. 6 Subcellular localization of PpMADS2 and PpMADS3 protein in tobacco leaf epidermal cells

图7 桃果实成熟期间类胡萝卜素合成基因表达变化

Fig. 7 Changes of carotenoid synthesis gene expression during fruit ripening

图8 桃果实成熟期间类胡萝卜素降解基因PpCCD4表达变化

Fig. 8 Changes of carotenoid degradation gene PpCCD4 expression during fruit ripening

图9 桃果实成熟期间PpMADS2和PpMADS3的表达变化

Fig. 9 Changes of PpMADS2 and PpMADS3 gene expression during fruit ripening

图10 PpMADS2和PpMADS3的自主激活活性分析 pGBKT7-P53与pGADT7-T-antigen为阳性对照组，pGBKT7为阴性对照。

Fig. 10 Transcriptional activation of PpMADS2 and PpMADS3 was analyzed by yeast two-hybrid system pGBKT7-P53 and pGADT7-T-antigen were used as positive control group, pGBKT7 was negative control group.

图11 酵母双杂系统分析PpMADS2和PpMADS3的互作 pGBKT7-P53与pGADT7-T-antigen为阳性对照组，pGBKT7-Lamin与pGADT7-T-antigen为阴性对照组。

Fig. 11 Interaction between PpMADS2 and PpMADS3 was analyzed by yeast two-hybrid system pGBKT7-P53 and pGADT7-T-antigen were used as positive control group, pGBKT7-Lamin and pGADT7-T-antigen were negative control group.

图12 PpMADS2和PpMADS3转录因子对PpPSY、PpCHYB和PpCCD4基因启动子的转录调控荧光强度LUC/REN可反映报告基因的表达量，与转录因子的作用强度呈正比。

Fig. 12 Transcriptional regulation of PpMADS2 and PpMADS3 transcription factors on promoters of PpPSY，PpCHYB and PpCCD4 genes The fluorescence intensity of LUC/REN can reflect the expression of reporter genes, and is directly proportional to the intensity of transcription factors to reporter genes.

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