南瓜NAC转录因子家族鉴定及生物信息学分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-1069

摘要/Abstract

摘要：

目的探究南瓜（Cucurbita moschata Duch.）NAC基因结构特征和表达模式，为阐明CmoNAC基因的生物学功能及进一步培育抗逆性强的砧用南瓜品种及裸仁南瓜品种提供参考。方法基于南瓜全基因组信息，使用生物信息学方法对NAC基因家族进行鉴定，系统分析该家族成员的理化性质、亚细胞定位、染色体定位、基因结构及保守基序等信息。结合转录组数据及实时荧光定量PCR（RT-qPCR）方法，分析CmoNAC的组织表达特征及对逆境胁迫应答和种皮发育的影响。结果 CmoNAC基因家族有133个成员，其编码蛋白主要定位于细胞核，均含有典型的NAM结构域。这些成员不均匀分布于20条染色体上，4号染色体分布最多。系统进化分析将其划分为15个亚家族，其中NAM亚家族成员数量最多。保守基序和基因结构分析显示同一亚族内成员具有相似Motif组成和外显子‒内含子结构，Motif 2/3/4广泛存在，部分Motif具有亚家族特异性。顺式作用元件分析表明CmoNAC基因启动子区域包含光、激素、逆境响应及发育相关等多种调控元件。组织表达分析表明，CmoNAC家族成员在根中表达量最高。100个CmoNAC基因在盐胁迫下显著上调，64个CmoNAC基因在有种皮和裸仁南瓜种皮差异表达。RT-qPCR分析表明，15个CmoNAC基因在盐胁迫和不同类型种皮发育下的表达模式与转录组数据基本一致。结论在南瓜全基因组中共鉴定出133个NAC基因，可分为15个亚家族，其基因结构与保守基序在亚家族内高度保守。CmoNAC108、CmoNAC102和CmoNAC013分别与根部发育、茎部发育与逆境响应密切相关，CmoNAC026、CmoNAC062和CmoNAC121在裸仁种子中特异性上调，可能参与裸仁种皮的形成。

关键词: 南瓜, NAC基因家族, 系统进化分析, 保守基序, 顺式作用元件, 盐胁迫响应, 种皮发育, 组织表达特异性

Abstract:

Objective We investigated the structural characteristics and expression patterns of CmoNAC genes in Cucurbita moschata,which provides insights into the biological functions of CmoNAC genes and facilitates the breeding of stress-resistant rootstock and hull-less seeded C. moschata. Method Based on the whole genome information of C. moschata, CmoNAC gene family members were identified using bioinformatic approaches. Systematic analyses were conducted on the physicochemical properties, subcellular localization, chromosomal localization, gene structure, and conserved motifs of its members. By integrating transcriptome data and Quantitative Real-time PCR (RT-qPCR), the tissue-specific expression patterns of CmoNAC genes, as well as their responses to stress and their roles in seed coat development, were investigated. Result The CmoNAC gene family comprises 133 members, all of which encode proteins predominantly localized in the nucleus and contain a typical NAM domain. These members are unevenly distributed across 20 chromosomes, with chromosome 4 harboring the highest number. Phylogenetic analysis classified them into 15 distinct subfamilies, among which the NAM subfamily is the largest. Conserved Motif and gene structure analyses revealed that members within the same subfamily share similar motif compositions and exon-intron structures. Motifs 2/3/4 were widely present across most members, while some Motifs presented subfamily-specific distribution. Cis-acting element analysis indicated that the promoter regions of CmoNACs contain various regulatory elements related to light responsiveness, hormone signaling, stress response, and developmental processes. Tissue-specific expression analysis revealed that CmoNAC family members had the highest expressions in the roots. Among the CmoNAC genes in C. moschata, 100 ones were significantly upregulated under salt stress. Furthermore, 64 members showed differential expression between hull and hull-less seeded C. moschata. RT-qPCR showed the expression patterns of CmoNAC genes under salt stress and during seed coat development were consistent with the transcriptome data. Conclusion A total of 133 CmoNAC genes are identified in the C. moschata genome, which could be classified into 15 subfamilies. The gene structure and conserved motifs are highly conserved within each subfamily. CmoNAC108, CmoNAC102, and CmoNAC013 are respectively closely associated with root, stem development and stress responses while CmoNAC026, CmoNAC062 and CmoNAC121 are specifically upregulated in hull-less seeds, suggesting their potential roles in the formation of the seed coat.

Key words: Cucurbita moschata, NAC gene family, phylogenetic analysis, conserved motif, cis-acting element, salt stress response, seed coat development, tissue-specific expression

孟洪玉, 刘亚楠, 武峻新, 申琼. 南瓜NAC转录因子家族鉴定及生物信息学分析[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1069.

MENG Hong-yu, LIU Ya-nan, WU Jun-xin, SHEN Qiong. Identification and Analysis of the NAC Transcription Factors Family in Cucurbita moschata Duch.[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1069.

图/表 9

图1 南瓜NAC基因家族在染色体上的分布

Fig. 1 Chromosomal distribution of NAC gene family in C. moschata

图2 南瓜（133个）和拟南芥（105个）NAC蛋白的系统发育进化树

Fig. 2 Phylogenetic tree of NAC protein in C. moschata (133) and A. thaliana (105)

图3 南瓜NAC基因家族的结构分布模式和结构域预测A：蛋白保守基序（Motif）分析；B：基因结构分析；C：基因保守结构域分析

Fig. 3 Structure distribution patterns and domains prediction of the NAC gene family in C. moschata.A: Protein conserved motif analysis. B: Gene structure analysis. C: Gene conserved domain analysis

图4 南瓜NAC基因家族顺式作用元件

Fig. 4 Prediction of cis-acting elements of the NAC gene family in C. moschata

图5 南瓜NAC基因家族的蛋白质互作网络分析

Fig. 5 Analysis of the protein interaction network of the NAC gene family in C. moschata

图6 南瓜NAC基因家族在各组织中的表达模式及响应盐胁迫的表达分析L、S、F、R分别代表叶、茎、果、根

Fig. 6 Expression patterns in various tissues of the NAC gene family in C. moschata and its expression analysis in response to salt stressL, S, F, and R indicates leaves, stems, fruits and roots, respectively. 0: 0 mmol/L NaCl; 75: 75 mmol/L NaCl

图7 NAC基因家族不同时期有种皮和裸仁南瓜种皮的表达分析HS-A-15、HS-A-20、HS-A-30：基因在有种皮南瓜HS-A的15、20和30 DPA种皮中的表达量；HLS-B-15、HLS-B-20、HLS-B-30：基因在裸仁南瓜HLS-B的15、20和30 DPA种皮中的表达量。下同

Fig. 7 Expression analysis of NAC gene family in seed coat of hull and hull-less seeded C. moschata at different developmental stagesHS-A-15, HS-A-20, HS-A-30: The expressions of the gene in the seed coat of seeded pumpkin HS-A at 15, 20 and 30 DPA. HLS-B-15, HLS-B-20, HLS-B-30: The expressions of the gene in the seed coat of hull-less pumpkin HLS-B at 15, 20 and 30 DPA. The same below

图8 15个CmoNACs在盐胁迫中的相对表达量不同小写字母表示在P<0.05水平差异显著，下同

Fig. 8 Relative expressions of 15 genes under salt stress of C. moschataDifferent lower letters indicate significant difference at P<0.05 level. The same below

图9 南瓜种子发育过程中15个CmoNACs的相对表达量

Fig. 9 Relative expressions of 15 CmoNACs during the development of C. moschata seeds

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