PpyERF060-PpyABF3-PpyMADS71调控乙烯信号通路介导的梨芽休眠进程

doi:10.16420/j.issn.0513-353x.2022-0585

摘要/Abstract

摘要：

为明确DAM（Dormancy-Associated MADS-box）基因参与休眠过程调控的分子机制,以'砀山酥梨'花芽为研究材料,对前期鉴定出的梨DAM基因PpyMADS71的上游调控因子开展挖掘与初步分析。结果表明：（1）芽休眠过程中乙烯响应基因PpyERF060（ethylene responsive factor 060）与PpyMADS71共表达,酵母点突变实验及双荧光素酶实验确定PpyERF060结合在PpyMADS71启动子的DRE1元件处并激活其表达;过表达PpyERF060的'茄梨'愈伤组织中PpyMADS71表达上调,进一步证明转录激活作用。（2）对PpyERF060的上游基因展开探索,发现可对其转录产生激活作用的ABA响应因子PpyABF3（ABRE BINDING FACTOR 3）,并初步确定PpyABF3在PpyERF060启动子上的结合位点。（3）外源乙烯可诱导'砀山酥梨'芽中PpyERF060表达,同时抑制PpyABF3转录;且在'茄梨'愈伤组织中过表达PpyERF060对PpyABF3的转录起到抑制作用。通过上述结果,初步发现由PpyERF060、PpyABF3和PpyMADS71构成的互作网络可整合乙烯与脱落酸的信号通路进而调控梨芽休眠进程。

关键词: 梨, 芽, 休眠, 乙烯, 转录调控, DAM

Abstract:

In order to clarify the specific regulation mode of DAM gene in dormancy,'Dangshan Suli'pear floral buds were used as the material to explore the upstream transcriptional factors of a DAM gene PpyMADS71 identified in previous studies. The results are as follows：(1) PpyERF060（Ethylene responsive factor 060）and PpyMADS71 were co-expressed during bud dormancy cycle. The yeast binding site mutation assays and the dual-luciferase assays were used to confirm that PpyERF060 could bind to the DRE1 element of the PpyMADS71 promoter and activates PpyMADS71's expression,which was further proved by the upregulation of PpyMADS71 expression in PpyERF060 transgenic pear calli. (2) To explore the upstream genes of PpyERF060,we found that the transcription factor PpyABF3（ABRE BINDING FACTOR 3）could activate the expression of PpyERF060 and the binding site of PpyABF3 on the promoter of PpyERF060 was primarily determined. (3) Furthermore,ethylene treatment on'Dangshan Suli'buds caused up-regulation of PpyERF060 and down-regulation of PpyABF3. In addition,overexpression of PpyERF060 in pear calli caused inhibition of the transcription of PpyABF3. Based on the above results,we found that there is a regulatory network among PpyERF060,PpyABF3,and PpyMADS71,which could combine ethylene and ABA signaling pathway,and ultimately regulates the dormancy process.

Key words: pear, bud, dormancy, ethylene, transcriptional regulation, DAM

中图分类号:

S661.1

杨博, 魏佳, 李坤峰, 王程亮, 倪隽蓓, 滕元文, 白松龄. PpyERF060-PpyABF3-PpyMADS71调控乙烯信号通路介导的梨芽休眠进程[J]. 园艺学报, 2022, 49(10): 2249-2262.

YANG Bo, WEI Jia, LI Kunfeng, WANG Chengliang, NI Junbei, TENG Yuanwen, and BAI Songling. PpyERF060-PpyABF3-PpyMADS71 Regulates Ethylene Signaling Pathway- Mediated Pear Bud Dormancy Process[J]. Acta Horticulturae Sinica, 2022, 49(10): 2249-2262.

图/表 11

表1 本研究中所用的引物序列

Table 1 Primer sequences

用途 Use	引物 Primer	序列 Sequence
定量表达	Q-PpyERF060-F	TCGGATTCTCAGTGGGACGA
qRT-PCR	Q-PpyERF060-R	TTACAGCATCACCACGCCTT
	Q-PpyMADS71-F	AACAACCAGCTAAGCCAGAAG
	Q-PpyMADS71-R	TCAAGGGTTGGAGAAGGTGG
	Q-PpyABF3-F	CCGGTTCGAATTTGCCACAG
	Q-PpyABF3-R	CGGTTGTTGAAAGCCGGAAG
亚细胞定位	PpyERF060-GFP-F	AGCTCGGTACCCGGGATGGCAACATCCATAGATCTTTAC
Subcellular localization	PpyERF060-GFP-R	CATGTCGACTCTAGAGATAGCAGACCAATCAATCTCCA
酵母单杂	pMADS71-pAbAi-F	GAAAAGCTTGAATTCGAGCTCGGGTGACTGGTTGCTGGTTGTT
Y1H assay	pMADS71-mutDRE1-pAbAi-R	ACGTGGAGTTTTTCACACCT
	pMADS71-mutDRE1-pAbAi-F	AGGTGTGAAAAACTCCACGT
	pMADS71-mutDRE2-pAbAi-R	GTTTATTGTTTTTTTAGCCT
	pMADS71-mutDRE2-pAbAi-F	AGGCTAAAAAAACAATAAAC
	pMADS71-pAbAi-R	AGCACATGCCTCGAGGTCGACCAACGGTGATCGATGCGGTTGG
双荧光素酶	pMADS71-all-LUC-F	TCGAGGTCGACGGTATCGATACCTAAAGCGCAACCGTAACTG
Dual-Luciferase assay	pMADS71-all-LUC-R	CCGCTCTAGAACTAGTGGATCCTTCTCTTTCTTCCTACCCCCT
	PpyERF060-SK-F	GCGGCCGCTCTAGAACTAGTGATGGCAACATCCATAGATCTTTAC
	PpyERF060-SK-R	GGTCGACGGTATCGATAAGCTTTAGATAGCAGACCAATCAATCTC
	pMADS71-delDRE1-LUC-F	TCGAGGTCGACGGTATCGATATCCACGTGAACTCGGATAAACACC
	pMADS71-mutDRE1-LUC-R	ACGTGGAGTTTTTCACACCT
	pMADS71-mutDRE1-LUC-F	AGGTGTGAAAAACTCCACGT
	pERF060-LUC-F	TCGAGGTCGACGGTATCGATATAAAAAAGTTAGGGCTAAAATTGT
	pERF060-LUC-R	CCGCTCTAGAACTAGTGGATCTTAGATTTAGTCACAAAAGATAAA
	PpyABF3-SK-F	GCGGCCGCTCTAGAACTAGTGATGGGGTCTAATTTCAACTTCAA
	PpyABF3-SK-R	GGTCGACGGTATCGATAAGCTTTACCAAGGGCCTGTCAATGT
PpyERF060过表达	PpyERF060-1301-F	AGAACACGGGGGACTCTTGACATGGCAACATCCATAGATCTTTAC
PpyERF060 overexpression	PpyERF060-1301-R	GGGGAAATTCGAGCTGGTCACTTAGATAGCAGACCAATCAATCTC

图1 PpyERF060在休眠期间的表达模式（a）及其亚细胞定位（b） Free GFP蛋白作为对照。红色荧光表示细胞核所在位置,绿色荧光表示融合蛋白的位置,若两种荧光信号在同一处,叠加后荧光信号变为黄色。

Fig. 1 The expression patterns of PpyERF060 during dormancy cycle（a）and subcellular localization of PpyERF060（b） Free GFP is used as the control. The red fluorescence indicates the location of the nucleus,and the green fluorescence indicates the location of the fusion protein. If the two fluorescence signals are in the same place,the fluorescence signal will be yellow after superposition.

图2 PpyERF060对PpyMADS71表达的调控（a）及其转录激活活性验证（b）

Fig. 2 The regulation pattern of PpyERF060 to PpyMADS71 expression（a）and yeast transcriptional activation assay of PpyERF060（b） Student's t-test,**P < 0.01.

图3 PpyERF060在PpyMADS71启动子上潜在的结合位点

Fig. 3 The potential binding site of PpyERF060 on the promoter of PpyMADS71

图4 酵母单杂（Y1H）实验验证PpyEFR060与PpyMADS71启动子互作

Fig. 4 Y1H assay confirmed the interaction between PpyERF060 and the promoter of PpyMADS71

图5 双荧光素酶试验验证PpyEFR060与PpyMADS71启动子结合

Fig. 5 Dual luciferase assay confirmed the interaction between PpyERF060 and the promoter of PpyMADS71 Student's t-test,**P < 0.01.

图6 PpyERF060转基因愈伤组织中PpyERF060和PpyMADS71的表达情况

Fig. 6 Expression of PpyERF060 and PpyMADS71 in PpyERF060 transgenic callus Student's t-test,**P < 0.01.

图7 PpyABF3激活PpyERF060的表达

Fig. 7 PpyABF3 can activates PpyERF060 expression Student's t-test,**P < 0.01.

图8 PpyABF3在PpyERF060启动子上的潜在结合位点（a）及酵母单杂交确认其结合（b）

Fig. 8 The potential binding site of PpyABF3 on the promoter of PpyERF060（a）and Y1H confirmed the interaction between PpyABF3 and the promoter of PpyERF060（b）

图9 '砀山酥梨'芽乙烯处理后PpyERF060和PpyABF3的表达情况

Fig. 9 The expression patterns of PpyERF060 and PpyABF3 in'Dangshan Suli'pear buds after ethylene treatment Student's t-test,**P < 0.01,***P < 0.001.

图10 '茄梨'PpyERF060转基因愈伤组织中PpyERF060和PpyABF3的表达情况

Fig. 10 Expression of PpyERF060 and PpyABF3 in PpyERF060 transgenic callus of'Qieli'pear Student's t-test,**P < 0.01.

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