PpC3H37参与ALA提高桃花期子房抗寒性的分子机制

doi:10.16420/j.issn.0513-353x.2025-0703

摘要/Abstract

摘要：

前期研究发现，5-氨基乙酰丙酸（5-aminolevulinic acid，ALA）能够上调PpC3H37促进PpWRKY表达提升桃花抗寒性。以此为基础，进一步研究完善其提高抗寒性的分子机制，明确了50 mg · L^-1 ALA预处理能够提高低温下桃花抗氧化酶超氧化物歧化酶（superoxide dismutase，SOD）、过氧化物酶（peroxidase，POD）和过氧化氢酶（catalase，CAT）活性以及脯氨酸、可溶性糖和可溶性蛋白含量，降低丙二醛和活性氧物质含量，从而增强桃花抗寒性。以PpC3H37启动子为诱饵进行酵母单杂交筛库，获得响应ALA上调表达转录因子PpC2H2。GUS染色和双荧光素酶实验表明ALA能够上调PpC2H2表达，增加PpC3H37启动子活性。瞬时过表达PpC2H2能够显著提升桃花器官抗寒性。

关键词: 桃, 花器官, ALA, 低温胁迫, 抗寒性, PpC3H37, PpC2H2

Abstract:

Previous studies have found that ALA（5-aminolevulinic acid）can up-regulate PpC3H37 to promote the expression of PpWRKY and enhance the cold resistance of peach blossoms. Building on previous research，the result demonstrated that 50 mg · L^-1 ALA pretreatment enhanced cold tolerance by increasing antioxidant enzyme superoxide dismutase（SOD），peroxidase catalase（POD），catalase（CAT）activities，proline content，and soluble sugar/protein levels while reducing malondialdehyde（MDA）and reactive oxygen species（ROS）accumulation in peach flowers. Yeast one-hybrid screening using the PpC3H37 promoter as bait identified the ALA-responsive transcription factor PpC2H2. GUS staining and dual-luciferase assays revealed that ALA upregulates PpC2H2 expression and activates the PpC3H37 promoter. Transient overexpression of PpC2H2 significantly improved cold tolerance in floral organs.

Key words: peach, floral organ, ALA, low-temperature stress, cold resistance, PpC3H37, PpC2H2

陈政, 王晓俊, 严娟, 蔡志翔, 张斌斌, 许建兰, 马瑞娟, 俞明亮, 沈志军. PpC3H37参与ALA提高桃花期子房抗寒性的分子机制[J]. 园艺学报, 2026, 53(1): 41-55.

CHEN Zheng, WANG Xiaojun, YAN Juan, CAI Zhixiang, ZHANG Binbin, XU Jianlan, MA Ruijuan, YU Mingliang, SHEN Zhijun. PpC3H37 Involvement in ALA-Enhanced Cold Tolerance of Peach Ovary During Bloom Stage[J]. Acta Horticulturae Sinica, 2026, 53(1): 41-55.

图/表 8

表1 本研究中用到的引物

Table 1 The primer used in this study

用途 Usage	引物名称 Primer name	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer
实时荧光定量 qRT-PCR	PpTEF	ggtgtgacgatgaagagtgatg	tgaaggagagggaaggtgaaag
	Prupe.6G278600	atggagggacatcatcgtcatcatc	tcaccgtgcttgccttatt
	Prupe.1G313300	atggctgctgtcacaaagagtgcat	tgttcttcctggcaaatgctgtg
	Prupe.2G205700	atggggtgcaaggaacaacaccaca	tcgttctcgccgcctccatcac
	Prupe.2G135000	atggaaataggtgggaacatgaaca	gctctttcgtcttcgctca
	Prupe.1G508100	atgttacaagagagaatgagctatc	ctccagttgtgccagtttt
	Prupe.2G058400	atggggatctttgaagaaatgggtt	gctgttgcctctgggtcat
	PpC3H37	ggggaatgtaggtttggcga	cgtttgcttcccctccagat
转录活性验证 Transcriptional activity verification	pGBKT7-PpC2H2	catatggccatggaggccgaattcatggggtgcaaggaacaaca	cgctgcaggtcgacggatccttagtaatgacaatccaccagaatttgt
亚细胞定位 Subcellular localization	1302-PpC2H2	actcttgaccatggtagatctatggggtgcaaggaacaaca	aaagttcttctcctttactagtgtaatgacaatccaccagaatttgtgtt
Y1H验证 Y1H verification	pHIS2-proPpC3H37	gactcactatagggcgaattcccccccccccccccacaaacttt	ttcgcgaacgcgtgagctctgattgttatgagatgtaaatcccaaatt
Y1H验证 Y1H verification	pGADT7-PpC2H2	gaattcactggcctccatggcatggggtgcaaggaacaacac	gctcgagctcgatggatccttagtaatgacaatccaccagaatttgt
GUS活性验证 GUS activity verification	PBI121-proPpC3H37	gagaacacgggggactctagaccccccccccccccacaaact	ctgaccacccggggatcctgattgttatgagatgtaaatcccaaat
双荧光素酶报告实验 Luciferase complementation assay	proC3H37-LUC	tatcgaattcctgcagcccgggccccccccccccccacaaactt	cgctctagaactagtggatcctgattgttatgagatgtaaatccc

图1 ALA预处理对低温处理下桃花花器官表型（A）及生理生化指标（B）的影响对照：清水培养 + 常温；LT：低温处理，清水培养 +-3 ℃低温6 h；ALA + LT：50 mg · L-1 ALA预处理 + -3 ℃低温6 h。不同小写字母表示在P < 0.05水平差异显著。下同

Fig. 1 Effects of ALA pretreatment on phenotype（A）and physiological-biochemical indices（B） of peach floral organs under low-temperature stress Control：Cultured in distilled water at normal temperature；LT：Low-temperature treatment，cultured in distilled water and subjected to-3 ℃ for 6 h；ALA + LT：Pretreatment with 50 mg · L-1 ALA followed by the-3 ℃ low-temperature treatment for 6 h. Different lowercase letters indicate significant differences at P < 0.05 level. The same below

表2 与PpC3H37启动子互作的候选蛋白

Table 2 Screened proteins that may interact with the PpC3H37 promoter

编号 Number	序列号 Accession No.	注释 Description	相似度/% Similarity
1	Prupe.6G278600	Trihelix transcription factor GT-3b-like	90.94
2	Prupe.1G313300	Transcription factor MYB62	96.96
3	Prupe.2G205700	PpC2H2_zinc finger protein ZAT5	99.69
4	Prupe.2G135000	Squamosa promoter-binding-like protein 7	90.89
5	Prupe.1G508100	Transcription factor TGA7	99.46
6	Prupe.2G058400	ETHYLENE INSENSITIVE 3-like 1	99.77

图2 低温条件下ALA处理对候选基因在桃花子房中相对表达的影响

Fig. 2 The influence of ALA treatment on the relative expression of candidate genes in peach gynoecium under low-temperature treatment

图3 PpC2H2转录活性检测和亚细胞定位 A：PpC2H2的功能域预测；B：PpC2H2转录活性鉴定。BD-53加AD-T作为阳性对照，单独的BD-empty作为阴性对照，其中BD为pGBKT7载体；C：PpC2H2亚细胞定位

Fig. 3 PpC2H2 transcriptional activity assay and subcellular localization A：The functional domain predication of PpC2H2；B：the PpC2H2 transcriptional activity assay. The combination of BD-53 plus AD-T was used as a positive control，and BD-empty was used as a negative control，where BD was pGBKT7 vector；C：The subcellular localization of PpC2H2 in tobacco leaves

图4 酵母单杂交验证PpC2H2与PpC3H37启动子之间的互作 A：PpC3H37启动子自激活实验；B：酵母单杂交一对一验证。SD-TL：-trp、-leu；SD-TLH：-trp、-leu、-his；3AT：3-氨基-1,2,4-三唑

Fig. 4 Yeast One-Hybrid Assay show that PpC2H2 interacted with the promoter of the PpC3H37 in yeast A：PpC3H37 promoter auto-activation assay；B：Yeast one-hybrid pairwise validation. SD-TL：-trp，-leu；SD-TLH：-trp，-leu，-his；3-AT：3-Amino-1,2,4-triazole

图5 ALA正调控PpC2H2提升PpC3H37启动子活性 A：GUS染色；B：GUS活性；C：双荧光素酶报告实验；D：烟草叶片共转化“效应子”“报告基因”后的LUC和REN的比率（用于标准化启动子激活水平）

Fig. 5 ALA promotes PpC2H2-mediated activation of the PpC3H37 promoter activity A：GUS staining；B：GUS activity；C：Dual-luciferase reporter assay；D：Ratio of LUC to REN in tobacco leaves after co-transfection with“effector”and“reporter”constructs（for standardizing the activation level of the promoter）

图6 PpC2H2瞬时表达提高桃花的抗寒性 A：低温处理下桃花表型；B：PpC2H2荧光定量；C：PpC3H37荧光定量；D：低温处理下桃花的理化指标

Fig. 6 Transient expression of PpC2H2 enhances cold resistance in peach flowers A：Phenotype of peach flowers under low-temperature treatment；B：Quantitative real-time PCR（qRT-PCR）of PpC2H2； C：Quantitative real-time PCR of PpC3H37；D：Determination of physicochemical parameters in peach flowers under low-temperature treatment

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