甜樱桃TCP家族全基因组鉴定及在非生物胁迫下的表达分析

Genome-wide identification of the TCP gene family in sweet cherry and expression profiling in Gisela 17 rootstock in response to abiotic stresses

摘要

【目的】深入解析TCP家族基因在甜樱桃中的表达模式，探究其在Gisela 17砧木响应非生物胁迫过程中的功能，以期为分子育种提供候选基因资源。【方法】利用生物信息学对甜樱桃TCP基因家族进行全基因组鉴定，并结合qRT-PCR分析甜樱桃砧木Gisela 17在不同非生物胁迫下 TCP基因的表达特征。【结果】在甜樱桃基因组中鉴定出19个 PaTCP基因，分布在7条染色体上，所有蛋白均含有完整的TCP保守结构域。编码区长度268~601 aa，外显子数1~2，亲水指数均为正值；亚细胞预测显示， PaTCP1~ 17均定位于细胞核。系统发育与序列特征联合分析将19个 PaTCP成员分为Class Ⅰ（PCF）与Class Ⅱ（CIN、CYC/TB1）两大支。共线性分析发现6对片段重复事件，提示扩增主要源于片段复制。甜樱桃与其近缘核果类果树（桃、李、杏）间共线性对较多，与单子叶植物水稻间最少。 PaTCP启动子区富集逆境与激素响应顺式元件。qRT-PCR结果显示，在干旱、盐、缺铁三种胁迫处理下， PaTCP1/3/4/7/9/10/15/17/18/19在24与48 h均显著上调； PaTCP2/8/11/12/13/16整体下调； PaTCP14对干旱无显著应答，但对盐与缺铁表现出特异性诱导。【结论】通过分析甜樱桃Gisela17砧木在响应不同逆境下的特异性分子应答机制与协同抗逆调控规律，系统阐释其抵御干旱损伤、盐离子毒害、缺铁失绿的内在分子机制，旨在为Gisela17砧木抗逆种质鉴定、关键抗逆基因的挖掘与功能验证、抗逆分子机制深度解析提供坚实的理论依据。

Abstract

Abstract:【Objective】TCP transcription factors (TFs) are a class of plant-specific TFs that play a crucial regulatory role in processes such as seed germination, vegetative growth, flowering, fruit development, senescence, and stress response in plants. To date, although the TCP family has been extensively studied in model plants such as Arabidopsis thaliana and rice, there have been no systematic reports on this gene family in Prunus avium L., a globally important economic fruit crop. This study aims to conduct a comprehensive genome-wide identification and systematic expression analysis of the TCP family in sweet cherries, with a focus on exploring its stress (drought, salt and iron deficiency) resistance functions.【Methods】The latest genome sequences, protein sequences, and GFF3 annotation files of the sweet cherry cultivar Tieton v2.0 were retrieved from the Rosaceae Genome Database (https://www.rosaceae.org/), a widely recognized repository for genomic resources of species in Rosaceae. A series of bioinformatics tools and pipelines were employed to characterize the TCP family members. Then, ExPASy ProtParam was used to analyze physicochemical properties (such as molecular weight, isoelectric point, instability index); MEGA 11 was utilized to construct a neighbor-joining phylogenetic tree with 1000 bootstrap replicates; Gene Structure Display Server (GSDS 2.0) was used to visualize gene structures (exons, introns, and UTRs); MEME Suite (v5.5.3) was conducted to identify conserved motifs. In addition, TBtools was used to map genes to chromosomes and analyze syntenic relationships; and PlantCARE was used to predict cis-acting elements in the 2000 bp upstream promoter regions. For expression analysis, in vitro rooted plantlets of Gisela 17, a commonly used rootstock with good adaptability, were subjected to three abiotic stress treatments: drought, salt, and iron deficiency. Total RNA was extracted from leaves using TRIzol reagent at 0, 12, 24, 48 and 72 hours after treatment, and the first strand cDNA was synthesized by reverse transcription using PrimeScript RT kit. Quantitative real-time PCR (qRT-PCR) was performed to analyze the gene expression levels.【Results】Candidate genes were further verified by domain confirmation via the SMART and NCBI CDD databases to exclude sequences with incomplete or truncated domains, resulting in the final identification of 19 TCP family members, designated as PaTCP1-PaTCP19 based on their chromosomal locations. These genes were unevenly distributed across 8 of the 16 sweet cherry chromosomes. The coding sequences of PaTCP genes ranged from 268 to 601 amino acids (aa), with corresponding molecular weights of 29.87-66.73 kDa. The isoelectric points (pI) varied from 6.14 to 9.51, including 10 alkaline proteins (pI＞7) and 9 acidic proteins (pI＜7). The instability index ranged from 48.14 to 78.73, indicating all PaTCP proteins are unstable (instability index＞40), while the aliphatic index (52.28-87.44) and negative hydrophobicity values confirmed their hydrophilic nature, consistent with their predicted nuclear (PaTCP1-17), mitochondrial (PaTCP18), and cytoplasmic (PaTCP19) localization. Motif analysis revealed 10 conserved motifs, among which motif 1 (corresponding to the core TCP domain) was present in all 19 PaTCP proteins, highlighting its high conservation. Gene structure analysis indicated 8 PaTCP genes contained a single exon, 4 had two or more exons, and 7 lacked untranslated regions (UTRs), with intron numbers ranging from 1 to 5. Phylogenetic analysis of 19 sweet cherry TCP proteins and 24 Arabidopsis TCP proteins classified PaTCP members into two major clades: ClassⅠ(PCF subfamily, 10 genes) and Class Ⅱ(9 genes total, including 7 in the CIN subfamily and 2 in the CYC/TB1 subfamily). Synteny analysis identified 6 pairs of homologous PaTCPs ( PaTCP1-PaTCP9, PaTCP2-PaTCP15, PaTCP4-PaTCP5, PaTCP5-PaTCP14, PaTCP4-PaTCP14), all derived from segmental duplication (no tandem duplication events were detected), indicating large-fragment duplication as the primary mechanism driving the expansion of PaTCP family. The Ka/Ks ratios of all homologous gene pairs ranged from 0.13 to 0.29 (all＜1), suggesting strong purifying selection during evolution to maintain functional stability. The TCP genes in sweet cherries show significant selective conservation in evolution, which maintain a high degree of linearity with dicotyledonous model plants and closely related stone fruit trees. Among them, 32 pairs and 22 pairs of homologous genes were identified in Arabidopsis thaliana and tomato, respectively, while the collinear logarithms with peach, plum and apricot reached 38 to 40 pairs, confirming the closer evolutionary distance and more complete preservation of chromosomal segments within stone fruit trees. In contrast, the collinear relationship with the monocotyledonous plant rice is only 13 pairs, and the evolutionary distance between the two is relatively far. Promoter cis-acting element analysis identified 7 types of functional elements in the 2000 bp upstream regions of PaTCP genes, including abiotic stress-responsive elements (low temperature, drought, defense and stress) and hormone (gibberel-

关键词

甜樱桃砧木 / TCP蛋白 / 基因家族 / 生物信息学

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基金资助

中央财政林业草原科技推广示范项目(甘[2025]ZYTG 18号)

甘肃省农业科学院重点研发计划项目(2024GAAS15)

天水市科技支撑计划项目(TS-STK-2024A-241)