杏果实成熟过程中SPLs基因的ABA响应特征与调控网络解析

doi:10.13560/j.cnki.biotech.bull.1985.2026-0040

摘要/Abstract

摘要：

目的探究PaSPLs在普通杏（Prunus armeniaca）果实成熟中的表达模式及响应外源脱落酸（abscisic acid, ABA）处理的分子机理，鉴定调控PaSPLs的miRNA，为后续深入研究miR156-PaSPLs介导ABA调控果实成熟奠定基础。方法以‘Sungold’为供试材料，利用生物信息学方法鉴定PaSPL基因家族成员，分析PaSPLs基本理化性质、保守基序、系统进化关系及启动子顺式作用元件；通过实时荧光定量PCR（RT-qPCR）分析外源ABA处理后果实PaSPLs基因的表达特征。通过瞬时转化烟草叶片验证PaSPLs蛋白的亚细胞定位。根据全转录组数据，构建miR156-PaSPLs调控网络，分析miR156和PaSPLs在普通杏果实成熟中的表达情况；利用STRING预测SPL互作蛋白。结果普通杏PaSPL基因家族共鉴定到15个成员，分为8个亚家族。启动子序列分析表明，存在4类参与生长发育调控的元件。表达分析结果显示，杏果实成熟过程中，PaSPL1和PaSPL2随着果实成熟表达量升高，且外源ABA处理后表达量增加，脱落酸抑制剂处理后表达量降低，而PaSPL9表达变化与之相反。PaSPL1、PaSPL2和PaSPL9均定位于细胞核。通过转录调控和蛋白互作预测分析，构建了miR156-PaSPLs表达调控网络及PaSPLs蛋白互作网络。结论共鉴定出15个普通杏的PaSPLs基因，其中PaSPL1和PaSPL2对果实成熟可能具有正向促进作用，而PaSPL9对果实成熟可能具有抑制作用，且均可响应ABA信号；同时miR156c可能通过负调控PaSPL9表达调控果实成熟，为miR156-SPLs与ABA协同调控果实成熟的分子机制提供理论依据。

关键词: 普通杏, 果实成熟, SPL基因家族, 脱落酸, miR156, 生物信息学, 基因表达, 调控网络

Abstract:

Objective This study aimed to investigate the expression patterns of PaSPLs during fruit ripening in apricot (Prunus armeniaca) and their molecular responses to exogenous abscisic acid (ABA) treatment. Additionally, we sought to identify the miRNAs that regulate PaSPLs, thereby providing a foundation for further exploring the mechanism by which ABA regulates fruit ripening through the miR156-PaSPL module. Method Using ‘Sungold’ as the experimental material, we identified members of the PaSPL gene family through bioinformatics approaches and analyzed their basic physicochemical properties, conserved motifs, phylogenetic relationships, and promoter cis-elements. The expression features of PaSPLs to exogenous ABA treatment were examined by quantitative real-time PCR (RT-qPCR). Subcellular localization of PaSPL proteins was verified via transient transformation of Nicotiana benthamiana leaves. Based on whole-transcriptome data, we constructed the miR156-PaSPL regulatory network and analyzed the expression patterns of miR156 and PaSPLs during apricot fruit ripening. In addition, the STRING database was used to predict PaSPL-interacting proteins. Result A total of 15 members of the PaSPL gene family were identified in apricot and classified into eight subfamilies. Promoter sequence analysis revealed four major categories of cis-elements involved in growth and development regulation. Expression analysis showed that during apricot fruit ripening, the transcript levels of PaSPL1 and PaSPL2 increased as ripening progressed. Their expression was further upregulated after exogenous ABA treatment and downregulated following NGDA (ABA biosynthesis inhibitor) treatment. In contrast, PaSPL9 exhibited the opposite expression pattern. Subcellular localization assays confirmed that PaSPL1, PaSPL2, and PaSPL9 were all localized in the nucleus. Using transcriptional and protein interaction prediction analyses, we constructed the miR156-PaSPL expression regulatory network and a PaSPL protein-protein interaction network. Conclusion A total of 15 PaSPLs were identified in apricot. Among them, PaSPL1 and PaSPL2 may positively promote fruit ripening, whereas PaSPL9 may exert an inhibitory effect. All three genes are responsive to ABA signals. Furthermore, miR156c may regulate fruit ripening by negatively modulating PaSPL9 expression. These findings provide a theoretical basis for elucidating the molecular mechanism underlying the synergistic regulation of fruit ripening by the miR156-SPL module and ABA.

Key words: apricot, fruit ripening, SPL gene family, ABA, miR156, bioinformatics, gene expression, regulatory network

张钰婧, 陈晨, 苟宁宁, 姜楠, 乌云塔娜. 杏果实成熟过程中SPLs基因的ABA响应特征与调控网络解析[J]. 生物技术通报, 2026, 42(11): 174-185.

ZHANG Yu-jing, CHEN Chen, GOU Ning-ning, JIANG Nan, WUYUN Ta-na. ABA Response Characteristics and the Regulatory Network of SPLs during Prunus armeniaca Fruit Ripening[J]. Biotechnology Bulletin, 2026, 42(11): 174-185.

图/表 8

表1 SPL转录因子在不同物种中的功能

Table 1 Function of SPL transcription factors in various plant species

物种 Species	基因 Gene	功能 Function	拟南芥同源基因 Arabidopsis ortholog gene	杏同源基因 Apricot ortholog gene
香蕉 Musa acuminata	MaSPL1	果实成熟	AtSPL3/4/5	PaSPL11/12
	MaSPL16	果实成熟	AtSPL13	PaSPL4/9
蓝莓 Vaccinium spp.	VvSPL2	果实成熟	AtSPL2/10/11	PaSPL5
苹果 Malus domestica	MdSPL6	株型	AtSPL6	PaSPL6
	MdSPL13B	单性结实	AtSPL13	PaSPL4/9
柑橘 Citrus reticulata	CiSPL2	果实成熟	AtSPL2	PaSPL5
	CiSPL3	株型	AtSPL3/4/5	PaSPL11/12
	CiSPL6	株型	AtSPL6	PaSPL6

图1 普通杏‘Sungold’、西伯利亚杏‘F106’、大扁杏‘LWM’、拟南芥、番茄、樱桃和桃SPL家族蛋白质系统进化树

Fig. 1 Phylogenetic tree of SPL family proteins in Prunus armeniaca 'Sungold', P. sibirica 'F106', P. armeniaca × P. sibirica 'LWM', Arabidopsis thaliana, Solanum lycopersicum, P. avium and P. persica

图2 普通杏PaSPLs基因家族保守基序分析

Fig. 2 Conserved motif analysis of the PaSPLs gene family in P. armeniaca

图3 普通杏PaSPLs基因种内（A）与物种间（B）共线性分析

Fig. 3 Collinearity analysis of PaSPLs genes within species (A) and between species (B) in P. armeniaca

图4 普通杏PaSPL基因家族启动子顺式元件分析

Fig. 4 Analysis of the cis-elements in the promoter region of the PaSPL gene family in P. armeniaca

图5 普通杏PaSPLs在不同发育时期（A）及响应外源ABA处理（B）的表达量变化GF-1：63 DAFB，绿果期-1；GF-2：70 DAFB，绿果期-2；TF：74 DAFB，转色期；RF：84 DAFB，成熟期。不同字母表示在0.05水平上差异显著（P<0.05）。下同

Fig. 5 Changes in the expression of PaSPLs in P. armeniaca at different developmental stages (A) and in response to exogenous ABA treatment (B)GF-1: 63 DAFB, green fruit stage-1; GF-2: 70 DAFB, green fruit stage-2; TF: 74 DAFB, turning fruit stage; RF: 84 DAFB, ripening fruit stage. Different letters indicate significant differences at 0.05 level (P < 0.05). The same below

图6 普通杏PaSPLs的亚细胞定位（A）及蛋白质相互作用分析（B）

Fig. 6 Subcellular localization (A) and protein interaction analysis (B) of PaSPL in P. armeniaca

图7 普通杏miR156结合PaSPLs基因的靶位点及调控网络分析A：7个miR156家族成员成熟序列比对；B：PaSPL4/5/6/8/9/14/15基因中miR156靶位点；黄色方框代表PaSPLs基因编码区，紫色方框代表SBP结构域，蓝色方框代表miR156靶位点；C：miRNAs与PaSPLs基因的调控网络；D：miRNAs在普通杏不同发育时期的表达量变化

Fig. 7 Analysis of target site prediction and regulatory network of miR156-PaSPLs in P. armeniacaA: Alignment of mature sequences of seven miR156 family members. B: miR156 target sites in PaSPL4/5/6/8/9/14/15 genes. Yellow boxes represent the coding sequences (CDS) of PaSPLs genes, purple boxes represent the SBP domain, and blue boxes represent the miR156 target sites. C: Regulatory network between miRNAs and PaSPLs genes. D: Expression variation of miRNAs in common apricot at different developmental stages

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