黄瓜 CsTON1 基因的鉴定及分析

Identification and Analysis of Cucumber CsTON1 Gene

摘要

黄瓜作为一种全球范围内广泛栽培的蔬菜作物，不仅在饮食领域占据重要地位，其独特的生长特性和丰富的营养价值也使它成为农业研究的热点之一。TON1（TONNEAU1）是一种关键的细胞骨架结合蛋白，在植物细胞分裂平面确定、形态建成及器官大小形状调控中发挥核心作用，但其在黄瓜中的同源基因 CsTON1的功能尚属未知。本研究对黄瓜 CsTON1 基因进行了鉴定与分析，结果显示，黄瓜中仅存在一个 CsTON1 基因，定位于5 号染色体上，基因全长为 6 989 bp，其CDS区域长度为798 bp，基因结构包含8个外显子。CsTON1蛋白由265个氨基酸残基组成，其分子量约为 29.7 kDa。通过生物信息学分析，发现CsTON1蛋白结构不稳定，具有多个磷酸化修饰位点，并且在二级结构组成中，无规则卷曲占据主要比例。系统进化树和多序列比对表明，CsTON1 与冬瓜BhTON1 的亲缘关系最近，与小立碗藓 PpTON1的亲缘关系最远。CsTON1高度保守，含有M1-M8保守基序和TOF、LisH、PLL三个保守结构域。RNA-seq分析和qRT-PCR结果显示， CsTON1在黄瓜各组织中均有表达且在果实中的表达量最高。此外， CsTON1可响应盐胁迫和高温胁迫，同时也响应白粉病原菌和霜霉病菌的侵染。该研究结果为后续开展 CsTON1基因在黄瓜中的功能解析提供参考依据。

Abstract

Cucumber, as a widely cultivated vegetable crop globally, holds an important position not only in the field of diet but also becomes one of the hotspots in agricultural research due to its unique growth characteristics and rich nutritional value. TON1 (TONNEAU1) is a critical cytoskeleton-associated protein that plays a central role in determining the cell division plane, morphogenesis, and regulating the organ size and shape. However, the function of its homolog, CsTON1 in cucumber remains largely unknown. This study This study identifies and analyzes the cucumber CsTON1 gene, revealing that there is only one CsTON1 gene in cucumber, located on chromosome 5; the gene possesses a genomic sequence of 6 989 bp, with a coding sequence (CDS) of 798 bp that is organized into 8 exons. The CsTON1 protein is composed of 265 amino acid residues with a molecular weight of approximately 29.7 kDa. Bioinformatics analysis reveals that the CsTON1 protein has an unstable structure, contains multiple phosphorylation sites, and random coils dominate its secondary structure. Phylogenetic tree and multiple sequence alignment analyses indicate that CsTON1 is most closely related to BhTON1 from Benincasa hispida and most distantly related to PpTON1 from Physcomitrella patens. CsTON1 is highly conserved， harboring eight conserved motifs （M1-M8） and three conserved domains， namely TOF， LisH， and PLL. RNA-seq and qRT-PCR results demonstrate that CsTON1 is ubiquitously expressed in all cucumber tissues， with the highest expression level in the fruit. Furthermore， CsTON1 responds to salt stress and high temperature stress， as well as infections by powdery mildew and downy mildew pathogens. The findings of this study provide a reference basis for further functional analysis of the CsTON1 gene in cucumber.

关键词

黄瓜 / TON1基因 / 生物信息学分析 / 非生物胁迫 / 生物胁迫

Key words

Cucumber / TON1 gene / bioinformatics analysis / abiotic stress / biotic stress

参考文献

[1] 薛秀花, 任海云. 细胞骨架在植物细胞周期进程中的动态变化及其调控[J]. 北京师范大学学报(自然科学版), 2016 (06): 687-695.

[2] Traas J, Bellini C, Nacry P, et al. Normal differentiation patterns in plants lacking microtubular preprophase bands [J]. Nature, 1995, 375: 676-677.

[3] Mineyuki Y. The preprophase band of microtubules: its function as a cytokinetic apparatus in higher plants [J]. International Review of Cytology, 1999, 187: 1-49.

[4] Azimzadeh J, Nacry P, Chriodoulidou A, et al. Arabidopsis TONNEAU1 proteins are essential for preprophase band formation and interact with centrin [J]. The Plant Cell, 2008, 20(8): 2146-2159.

[5] Camilleri C, Azimzadeh J, Pastuglia M, et al. The Arabidopsis TONNEAU2 gene encodes a putative novel protein phosphatase 2A regulatory subunit essential for the control of the cortical cytoskeleton [J]. The Plant Cell, 2002, 14 (4): 833-845.

[6] Drevensek S, Goussot M, Duroc Y, et al. The Arabidopsis TRM1-TON1 interaction reveals a recruitment network common to plant cortical microtubule arrays and eukaryotic centrosomes [J]. The Plant Cell, 2012, 24 (1): 178-191.

[7] Spinner L, Gadeyne A, Belcram K, et al. A protein phosphatase 2A complex spatially controls plant cell division [J]. Nature Communications, 2013, 4: 1863.

[8] Schaefer E, Belcram K, Uyttewaal M, et al. The preprophase band of microtubules controls the robustness of division orientation in plants [J]. Science, 2017, 356 (6334): 186-189.

[9] Yang Y Q, Chen B Q, Dang X, et al. Arabidopsis IPGA1 is a microtubule-associated protein essential for cell expansion during petal morphogenesis [J]. Journal of Experimental Botany, 2019, 70 (19): 5231-5243.

[10] Wang S K, Li S, Liu Q, et al. The OsSPL16-GW7 regulatory module determines grain shape and simultaneously improves rice yield and grain quality [J]. Nature Genetics, 2015, 47 (8): 949-954.

[11] Wang Y X, Xiong G S, Hu J, et al. Copy number variation at the GL7 locus contributes to grain size diversity in rice [J]. Nature Genetics, 2015, 47 (8): 944-948.

[12] Wu Y Z, Zhao Y, Yu J P, et al. Heterotrimeric G protein γ subunit DEP1 synergistically regulates grain quality and yield by modulating the TTP (TON1-TRM-PP2A) complex in rice [J]. Journal of Genetics and Genomics, 2023, 50 (7): 528-531.

[13] Wang Q, Fan J J, Cong J, et al. Natural variation of ZmLNG1 alters organ shapes in maize [J]. New Phytologist, 2023, 237 (2): 471-482.

[14] 葛 倩. 黄瓜果型候选基因的克隆及功能探究[D]. 泰安: 山东农业大学, 2018.

[15] Wang X F, Li H, Gao H, et al. Localization of quantitative trait loci for cucumber fruit shape by a population of chromosome segment substitution lines [J]. Scientific Reports, 2020, 10 (1): 11030.

[16] 李 豪. 黄瓜果形主效 QTL Fl1.2 和 Fl2.1近等基因系的构建及其候选基因的功能分析[D]. 泰安: 山东农业大学, 2020.

[17] 虎亚静. CsTRM5近等基因系表型鉴定及基因功能初步研究[D]. 泰安: 山东农业大学, 2022.

[18] Chen C, Chen H, Zhang Y, et al. TBtools: An integrative toolkit developed for interactive analyses of big biological data [J]. Molecular Plant, 2020, 13: 1194-1202.

[19] Li Z, Zhang Z, Yan P, et al. RNA-Seq improves annotation of protein-coding genes in the cucumber genome [J]. BMC Genomics, 2011, 12: 540.

[20] 陈琳杰, 常婧姝,张 宇, 等. 黄瓜FtsZs基因家族鉴定及分析[J]. 山东农业大学学报(自然科学版), 2024, 55(05): 774-781.

[21] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C (T)) Method [J]. Methods, 2001, 25 (4): 402-408.

[22] Zhu Y, Yin J, Liang Y, et al. Transcriptomic dynamics provide an insight into the mechanism for silicon-mediated alleviation of salt stress in cucumber plants [J]. Ecotoxicology Environmental Safety, 2019 , 174: 245-254.

[23] Xu Q, Xu X, Shi Y, et al. Elucidation of the molecular responses of a cucumber segment substitution line carrying Pm5.1 and its recurrent parent triggered by powdery mildew by comparative transcriptome profiling [J]. BMC Genomics, 2017 , 18(1): 21.

[24] Traas J, Bellini C, Nacry P, et al. Normal differentiation patterns in plants lacking microtubular preprophase bands [J]. Nature , 1995 , 375: 676-677.

[25] Spinner L, Pastuglia M, Belcram K, et al. The function of TONNEAU1 in moss reveals ancient mechanisms of division plane specification and cell elongation in land plants [J]. Development, 2010, 137 (16): 2733-2742.

[26] Popovici C, Zhang B, Gregoire MJ, et al. The t(6;8) (q27; p11) translocation in a stem cell myeloproliferative disorder fuses a novel gene, FOP, to fibroblast growth factor receptor 1 [J]. Blood, The Journal of the American Society of Hematology, 1999, 93(4): 1381-1389.

[27] Emes R D, Ponting C P. A new sequence motif linking lissencephaly, Treacher Collins and oral-facial-digital type 1 syndromes, microtubule dynamics and cell migration [J]. Human Molecular Genetics, 2001, 10(24): 2813-2820.

[28] Mikolajka A, Yan X, Popowicz G M, et al. Structure of the N-terminal domain of the FOP (FGFR1OP) protein and implications for its dimerization and centrosomal localization [J]. Journal of Molecular Biology, 2006, 359: 863-875.

基金资助

山东省重点研发计划（农业良种工程）项目(2022LZGCQY001)

国家自然科学基金(31972419)

国家自然科学基金(31672170)

山东“双一流”专项建设基金(SYL2017YSTD06)

山东省泰山学者建设工程(ts20130932)

山东省重点研发计划（竞争性创新平台）项目(2025CXPT170)