SnRK2.6对芍药远缘杂交花粉管生长的影响及对ABA的响应

doi:10.3864/j.issn.0578-1752.2025.15.011

摘要/Abstract

摘要：

【目的】从芍药转录组筛选SnRK2基因，探究芍药SnRK2介导ABA通路调控芍药属远缘杂交花粉萌发和花粉管生长的分子机制，为克服芍药属远缘杂交不亲和提供理论依据。【方法】基于芍药柱头转录组数据（NCBI登录号：PRJNA592882），筛选ABA信号通路基因和第Ⅲ亚组SnRK2基因PlSnRK2.6；克隆PlSnRK2.6，并进行生物信息学分析和亚细胞定位；采用酶联免疫吸附法（ELISA）测定芍药自交、杂交后4、8、12、24和36 h的柱头内ABA激素含量，以及目的基因相对表达量。外施ABA及其抑制剂后，测定芍药自交与杂交不同时期柱头中PlSnRK2.6的相对表达量；通过Y2H试验筛选PlSnRK2.6的互作蛋白。【结果】通过体外花粉培养发现，与正常培养基相比，ABA培养基中花粉的萌发率和花粉管长度显著降低，表明ABA对牡丹花粉的萌发与花粉管生长具有抑制作用。通过芍药转录组筛选ABA信号通路中调控离子运输的KAT1、调控ROS产生的RBOHF、蛋白激酶SnRK2、蛋白磷酸酶PP2C、ABFs和ABI家族基因。qPCR分析结果显示，在ABA信号通路中，与芍药自交相比，PlSnRK2.6和PlRBOHF的表达量显著升高；PlKAT1表达量下降；PlABF2、PlABI5表达量呈先上升后下降的趋势，该趋势与转录组中FPKM值趋势一致。经转录组筛选克隆的PlSnRK2.6 CDS全长为1 092 bp，编码363个氨基酸，经基因结构分析比对，发现其具有显著的STKc_SnRK2-3保守结构域；多物种系统进化关系表明，SnRK2家族可分为3个亚家族，PlSnRK2.6属于第Ⅲ亚组，其中，PlSnRK2.6与牡丹、拟南芥的SnRK2.6聚类到同一分支。PlSnRK2.6与蛇莓SnRK2.6亲缘关系最近，同源比对发现SnRK2-3结构域较为保守。亚细胞定位显示，PlSnRK2.6蛋白定位于细胞核。芍药杂交组合中的ABA含量在大部分时期高于自交，PlSnRK2.6表达量同样高于自交，在24和36 h较为显著，其杂交时期的表达量分别是自交的8.94和5.07倍。ABA响应试验发现PlSnRK2.6对ABA具有明显的响应性，其表达趋势在授粉后期更为明显。酵母双杂交试验显示，PlSnRK2.6不具有自激活特性，与PlACP1、PlHMGB3有相互作用。【结论】筛选出响应ABA诱导的蛋白激酶PlSnRK2.6，其在芍药远缘杂交柱头中高表达，与PlACP1和PlHMGB3相互作用。推测PlSnRK2.6及其互作蛋白通过ABA信号通路调控花粉萌发和花粉管生长，可能在调节芍药远缘杂交不亲和性过程中发挥关键作用。

关键词: 芍药, 远缘杂交不亲和, ABA, SnRK2, 激素响应

Abstract:

【Objective】 This study aims to identify SnRK2 genes from the Paeonia transcriptome to investigate the molecular mechanism of SnRK2 mediates the ABA signaling pathway in regulating pollen germination and pollen tube growth during distant hybridization of Paeonia lactiflora (P. lactiflora). The goal is to provide a theoretical basis for overcoming hybridization incompatibility in P. lactiflora.【Method】 Based on the transcriptome data of P. lactiflora stigmas (NCBI accession number: PRJNA592882), ABA signaling pathway genes and group Ⅲ SnRK2 genes (PlSnRK2.6) were screened and identified. The PlSnRK2.6 gene was cloned and analyzed for bioinformatics analysis and subcellular localization analysis. ABA content in stigmas at 4, 8, 12, 24, and 36 hours post-pollination (self- and cross-pollination) was measured using enzyme-linked immunosorbent assay (ELISA), along with relative gene expression using quantitative real-time PCR (qPCR). The relative expression levels of PlSnRK2.6 in stigmas under exogenous ABA and its inhibitor treatments were analyzed, and yeast two-hybrid (Y2H) assays were conducted to identify PlSnRK2.6-interacting proteins.【Result】 In vitro pollen culture revealed that pollen germination rates and pollen tube lengths were significantly reduced in ABA-supplemented media compared to normal media, indicating that ABA inhibits pollen germination and pollen tube growth in P. lactiflora. Transcriptome analysis identified key genes in the ABA signaling pathway, including KAT1 (ion transport), RBOHF (ROS production), SnRK2 (protein kinase), PP2C (protein phosphatase), ABFs, and ABI family genes. qPCR analysis showed that, compared to self-pollination, the expression of PlSnRK2.6 and PlRBOHF was significantly upregulated, while PlKAT1 expression was downregulated. PlABF2 and PlABI5 exhibited a rise-then-fall expression trend, consistent with transcriptome FPKM trends. The cloned PlSnRK2.6 CDS was 1 092 bp in length, encoding a protein of 363 amino acids. Gene structure analysis revealed a conserved STKc_SnRK2-3 domain. Phylogenetic analysis showed that the SnRK2 family is divided into three subgroups, with PlSnRK2.6 clustering into group Ⅲ alongside SnRK2.6 from Paeonia suffruticosa and Arabidopsis thaliana. PlSnRK2.6 was most closely related to SnRK2.6 in Potentilla indica, with a highly conserved SnRK2-3 domain. Subcellular localization indicated that PlSnRK2.6 is localized in the nucleus. The ABA content in hybrid stigmas was generally higher than in self-pollination. Similarly, PlSnRK2.6 expression was significantly higher during hybridization, particularly at 24 and 36 hours post-pollination, with expression levels 8.94- and 5.07-fold higher than in self-pollination, respectively. ABA responsiveness assays confirmed that PlSnRK2.6 is strongly responsive to ABA, with a more pronounced expression trend in the late stages of pollination. Yeast two-hybrid assays showed that PlSnRK2.6 lacks self-activation activity and interacts with PlACP1 and PlHMGB3.【Conclusion】 An ABA-induced protein kinase, PlSnRK2.6, was identified to be highly expressed in the stigmas of P. lactiflora during distant hybridization and interacts with PlACP1 and PlHMGB3. It is proposed that PlSnRK2.6 and its interacting proteins regulate pollen germination and pollen tube growth via the ABA signaling pathway, thereby playing a key role in modulating hybrid incompatibility in P. lactiflora.

Key words: Paeonia lactiflora, distant hybridization incompatibility, ABA, SnRK2.6, hormone response

周平西, 王警琨, 尤啸龙, 华超, 郭浩楠, 张明星, 刘艺平, 贺丹, 何松林. SnRK2.6对芍药远缘杂交花粉管生长的影响及对ABA的响应[J]. 中国农业科学, 2025, 58(15): 3081-3096.

ZHOU PingXi, WANG JingKun, YOU XiaoLong, HUA Chao, GUO HaoNan, ZHANG MingXing, LIU YiPing, HE Dan, HE SongLin. SnRK2.6 Regulates Pollen Tube Growth and ABA Response in Distant Hybridization in Paeonia lactiflora[J]. Scientia Agricultura Sinica, 2025, 58(15): 3081-3096.

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https://www.chinaagrisci.com/CN/Y2025/V58/I15/3081

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引物名称 Gene names	引物序列 Primer sequence (5′-3′)	作用 Function
PlSnRK2.6-F PlSnRK2.6-R	ATGGATCGATCGACCATTAC AGGTAGTGTATGCAATGTGA	克隆PlSnRK2.6 Clone PlSnRK2.6
PlSnRK2.6-2300-F	ACGGGGGACGAGCTCGGTACCATGGATCGATCGACCATTACGGT	2300载体构建 2300 vector construction
PlSnRK2.6-2300-R	CTTGCTCACCATGGTGTCGAC CATTGCATACACTACCTCTCCA	2300载体构建 2300 vector construction
β-Tubulin-F	TGAGCACCAAAGAAGTGGACGAAC	β内参基因 β reference gene
β-Tubulin-R	CACACGCCTGAACATCTCCTGAA	β内参基因 β reference gene
PlSnRK2.6-BD-F	CATGGAGGCCGAATTCATGGATCGATCGACCATTACGGT	酵母双杂交 Yeast two-hybrid (Y2H)
PlSnRK2.6-BD-R	GCAGGTCGACGGATCCCATTGCATACACTACCTCTCCA	酵母双杂交 Yeast two-hybrid (Y2H)
qPCR-PlSnRK2.6-F	TTTCAGCGAGGATGAGGCAC	PlSnRK2.6相对表达量测定 PlSnRK2.6 relative expression measurement
qPCR-PlSnRK2.6-R	CGGGCTTCCATCCAACAATG
qPCR-PlACP1-F	ACGGTGGACAAGGTTTGTGA	PlACP1相对表达量测定 PlACP1 relative expression measurement
qPCR-PlACP1-R	TCAGCTCCAAGAGACGCAAA	PlACP1相对表达量测定 PlACP1 relative expression measurement
qPCR-PlHMGB3-F	ACAAACCAAAGAGACCCGCA	PlHMGB3相对表达量测定 PlHMGB3 relative expression measurement
qPCR-PlHMGB3-R	CAACCACCGCAACACCTTTT	PlHMGB3相对表达量测定 PlHMGB3 relative expression measurement
qPCR-PlPP2C-F	GCATATCTCAAGAGGACGGAGTTC	PlPP2C相对表达量测定 PlPP2C relative expression measurement
qPCR-PlPP2C-R	TTGTGTATTGTTACGGATGGCTCAG	PlPP2C相对表达量测定 PlPP2C relative expression measurement
qPCR-PlABI5-F	CACCGCCACCGCCTCAG	PlABI5相对表达量测定 PlABI5 relative expression measurement
qPCR-PlABI5-R	GCCACCACCATCTACTCCAATAATG	PlABI5相对表达量测定 PlABI5 relative expression measurement
qPCR-PlPYR-F	CACGACCCTGCCGATAATCAATG	PlPYR相对表达量测定 PlPYR relative expression measurement
qPCR-PlPYR-R	TTCCCTGCACTACACACCTACTG	PlPYR相对表达量测定 PlPYR relative expression measurement
qPCR-RBOHF-F	TCTCGGATAGGTGTCTTCTACTGTG	PlRBOHF相对表达量测定 PlRBOHF relative expression measurement
qPCR-RBOHF-R	TGGAATTGGAAGCGGGTTGATG	PlRBOHF相对表达量测定 PlRBOHF relative expression measurement

编号 Number	蛋白 Description	基因长度 Gene length (bp)	分子功能 Molecular function
1	酰基载体蛋白1 Acyl carrier protein 1 (ACP1)	424	激活酰基载体活性、参与脂肪酸生物合成过程 Activates acyl carrier activity and participates in fatty acid biosynthesis
2	高迁移率族蛋白B3 High mobility group B protein 3 (HMGB3)	490	参与DNA结合转录因子活性、染色质的结构组成 Involved in DNA-binding transcription factor activity and chromatin structure composition
3	胚胎晚期丰富蛋白18 Late embryogenesis abundant protein 18 (LEA18)	645	涉及脱水耐受性、胚胎发育、花粉管发育、参与对渗透压、失水的反应 Involved in desiccation tolerance, embryo development, and pollen tube development; responds to osmotic stress and water loss
4	胚胎晚期丰富蛋白29 Late embryogenesis abundant protein 29 (LEA29)	896	涉及脱水耐受性 Involved in desiccation tolerance
5	胚胎晚期丰富蛋白76 Late embryogenesis abundant protein 76 (LEA76)	996	涉及脱水耐受性、在胚胎发生晚期和干燥种子中表达 Involved in desiccation tolerance, expressed during late embryogenesis and in dry seeds
6	ABA诱导蛋白PHV A1 ABA-inducible protein PHV A1 (HVA1; LEA4)	496	涉及脱水耐受性、胚胎发育、花粉管发育、参与对渗透压、失水的反应 Involved in desiccation tolerance, embryo development, and pollen tube development; responds to osmotic stress and water loss
7	铁氧还蛋白Ⅲ FerredoxinⅢ (Fd Ⅲ)	430	叶绿体基质来源、在多种代谢反应中转移电子 Chloroplast stroma-derived; transfers electrons in various metabolic reactions