甘蔗 P 型细胞周期蛋白基因家族的鉴定及 ScCycP3;1-1 基因功能研究

Identification of the sugarcane P-type cyclin gene family and functional study of the ScCycP3;1-1 gene

摘要

[目的]本研究旨在系统鉴定甘蔗 P 型细胞周期蛋白(CYCP)基因家族，并重点解析关键成员 ScCYCP3;1-1 在激素及黑穗病、花叶病等生物胁迫下的表达模式与功能，揭示其在甘蔗抗病调控网络中的潜在功能。[方法]在甘蔗基因组和转录组数据的基础上，通过生物信息学方法系统鉴定 CYCP 基因家族。对关键基因 ScCYCP3;1-1 进行基因克隆、RT-qPCR 表达分析和亚细胞定位，并构建其共表达调控网络。[结果]生物信息学分析结果显示，甘蔗 CYCP 基因家族包括 71 个 CYCP 基因，共线性分析揭示了 287 对共线基因对，表明片段复制是该基因家族扩张的主要驱动力；进化树分析显示，ScCYCP 可分为 4 个亚家族，同一亚家族内的成员具有相似的内含子/外显子模式和保守基序；蛋白理化性质预测显示，ScCYCP 的蛋白长度为 211~356aa，蛋白质相对分子质量为(22.55~38.52)×10³，多数为不稳定的酸性蛋白，预测其主要定位于细胞核和细胞质；基因表达分析发现，亚家族 3 的成员在甘蔗苗期茎组织中表达水平较高，其表达受多种激素调控，在脱落酸(ABA)、乙烯(ETH)和赤霉素(GA)胁迫后下调，而在生长素(IAA)胁迫后上调。实时荧光定量 PCR 结果显示：亚家族 3 的一个典型基因 ScCYCP3;1-1 在蔗髓中的表达量最高；在生物胁迫下，ScCYCP3;1-1 的表达呈现显著的品种特异性响应：在感黑穗病品种中受病菌诱导而显著上调，但在抗病品种中则下调表达；在感高粱花叶病毒的感病品种中，其表达被显著抑制。共表达分析预测 ScCYCP3;1-1 与多个转录因子(NAC、MYB)及功能蛋白(CKRMPHR、DnaJ、PTF4)存在互作关系。通过在本氏烟草中进行的亚细胞定位试验，证实 ScCYCP3;1-1 蛋白主要定位于细胞核。[结论]本研究系统解析了甘蔗 CYCP 基因家族，并鉴定出 1 个关键基因 ScCYCP3;1-1。该基因不仅响应多种激素信号，而且在甘蔗响应黑穗病和花叶病毒胁迫时表现出显著的转录调控，且其表达模式与品种的抗病性紧密相关，揭示了 CYCP 在植物生物胁迫响应中的新功能，表明了 ScCYCP3;1-1 可能是连接细胞周期调控与植物免疫信号的潜在枢纽。

Abstract

[Objectives] This study aimed to systematically identify the P-type cyclin (CYCP) gene family in sugarcane (Saccharum officinarum) and to elucidate the expression patterns and functions of a key member, ScCYCP3;1-1, under hormonal and biotic stresses (smut and mosaic disease), thereby revealing its potential role in the disease-resistance regulatory network. [Methods] The CYCP gene family was systematically identified from sugarcane genomic and transcriptomic data using bioinformatic approaches. For the key gene ScCYCP3;1-1, gene cloning, RT-qPCR expression analysis, and subcellular localization were performed, and its co-expression regulatory network was constructed. [Results] Bioinformatic analysis identified 71 CYCP gene family members in sugarcane. Collinearity analysis revealed 287 syntenic gene pairs, indicating that segmental duplication was the primary driver of this gene family's expansion. Phylogenetic analysis classified the ScCYCP into four subfamilies, with members within the same subfamily sharing similar intron-exon structures and conserved motifs. Physicochemical property predictions showed that ScCYCP proteins range from 211 to 356 aa in length, with molecular weights between (22.55-38.52) × 10³. Most were unstable acidic proteins predicted to localize to the nucleus and cytoplasm. Gene expression analysis revealed that members of subfamily 3 exhibited higher expression levels in sugarcane seedling stems. Their expression was regulated by multiple hormones, being downregulated under abscisic acid (ABA), ethylene (ETH), and gibberellin (GA) stresses, but upregulated by indole-3-acetic acid (IAA). RT-qPCR results showed that the ScCYCP3;1-1 gene had the highest expression in the pith. Critically, under biotic stress, ScCYCP3;1-1 displayed a significant variety-specific response: it was significantly upregulated by pathogen induction in smut-susceptible varieties but downregulated in resistant ones. Its expression was significantly suppressed in susceptible varieties infected with Sorghum mosaic virus. Co-expression analysis suggested that ScCYCP3;1-1 was functionally associated with several transcription factors (NAC, MYB) and other functional proteins (CKRMPHR, DnaJ, PTF4). Subcellular localization experiments in Nicotiana benthamiana confirmed that the ScCYCP3;1-1 protein was predominantly localized in the nucleus. [Conclusions] This study provided systematic analysis of the CYCP gene family in sugarcane and identified a key gene, ScCYCP3;1-1. This gene not only responded to multiple hormonal signals, but also exhibited significant transcriptional regulation in response to smut and mosaic virus stresses. Its expression pattern was closely linked to the host's susceptibility to smut disease. These findings unveil a novel function for P-type cyclins in plant biotic stress responses, suggesting that ScCYCP3;1-1 might act as a potential hub linking cell cycle regulation with plant immune signaling. It therefore represents a promising candidate gene for the molecular breeding of disease-resistant sugarcane.

关键词

细胞周期蛋白 / 甘蔗 / 生物信息学 / 胁迫响应 / 基因家族 / 功能分析

Key words

cyclin / sugarcane / bioinformatics / stress response / gene family / functional analysis

参考文献

[1] Pinto SC, Stojilkovic B, Zhang XY, et al. Plant cell size: links to cell cycle, differentiation and ploidy[J]. Current Opinion in Plant Biology, 2024, 78: 102527.

[2] Zheng ZL. Cyclin-dependent kinases and CTD phosphatases in cell cycle transcriptional control: conservation across eukaryotic Kingdoms and uniqueness to plants[J]. Cells, 2022, 11(2): 279.

[3] Mizukami Y. A matter of size: developmental control of organ size in plants[J]. Current Opinion in Plant Biology, 2001, 4(6): 533-539.

[4] Yang WB, Wightman R, Meyerowitz EM. Cell cycle control by nuclear sequestration of CDC20 and CDH1 mRNA in plant stem cells[J]. Molecular Cell, 2017, 68(6): 1108-1119.

[5] Lu KK, Song RF, Guo JX, et al. CycC1;1-WRKY75 complex-mediated transcriptional regulation of SOS1 controls salt stress tolerance in Arabidopsis[J]. The Plant Cell, 2023, 35(7): 2570-2591.

[6] Zhang N, Wang ZX, Bao ZL, et al. MOS1 functions closely with TCP transcription factors to modulate immunity and cell cycle in Arabidopsis[J]. The Plant Journal, 2018, 93(1): 66-78.

[7] Wang GF, Kong HZ, Sun YJ, et al. Genome-wide analysis of the cyclin family in Arabidopsis and comparative phylogenetic analysis of plant cyclin-like proteins[J]. Plant Physiology, 2004, 135(2): 1084-1099.

[8] Torres-Acosta JA, de Almeida Engler J, Raes J, et al. Molecular characterization of Arabidopsis PHO80-like proteins, a novel class of CDKA;1-interacting cyclins[J]. Cellular and Molecular Life Sciences CMLS, 2004, 61(12): 1485-1497.

[9] Deng MJ, Hu B, Xu L, et al. OsCYCP1;1, a PHO80 homologous protein, negatively regulates phosphate starvation signaling in the roots of rice (Oryza sativa L.)[J]. Plant Molecular Biology, 2014, 86(6): 655-669.

[10] Zulfiqar S, Zhao TT, Liu YM, et al. Genome-wide identification, characterization, and transcriptomic analysis of the cyclin gene family in Brassica rapa[J]. International Journal of Molecular Sciences, 2022, 23(22): 14017.

[11] Chen SY, Feng XX, Zhang Z, et al. ScDB:a comprehensive database dedicated to Saccharum, facilitating functional genomics and molecular biology studies in sugarcane[J]. Plant Biotechnology Journal, 2024, 22(12): 3386-3388.

[12] 张庭婷， 汪沛， 黄建洪， 等. 滇西典型甘蔗种植地土壤环境质量及风险评估[J]. 土壤通报， 2023， 54(4): 920-929.

[13] Zhang TT, Wang P, Huang JH, et al. Soil environmental quality and risk assessment of typical sugarcane plantation in western Yunnan[J]. Chinese Journal of Soil Science, 2023, 54(4): 920-929 (in Chinese with English abstract).

[14] 陶新， 夏世斌， 刘秋梅， 等. 甘蔗黑穗病的微生物防治研究进展[J]. 应用生态学报， 2023， 34(3): 846-852.

[15] Tao X, Xia SB, Liu QM, et al. Research progress on microbial control of sugarcane smut[J]. Chinese Journal of Applied Ecology, 2023, 34(3): 846-852 (in Chinese with English abstract).

[16] Viswanathan R, Balamuralkrishnan M. Impact of mosaic infection on growth and yield of sugarcane[J]. Sugar Tech, 2005, 7(1): 61-65.

[17] Wu QB, Pan YB, Su YC, et al. WGCNA identifies a comprehensive and dynamic gene co-expression network that associates with smut resistance in sugarcane[J]. International Journal of Molecular Sciences, 2022, 23(18): 10770.

[18] Rice JL, Hoy JW. Recovery from mosaic caused by sorghum mosaic virus in sugarcane and impact on yield[J]. Plant Disease, 2020, 104(12): 3166-3172.

[19] 潘洁明， 田绍锐， 梁艳兰， 等. 甘蔗 PIN-LIKES 基因家族的鉴定与表达分析[J]. 作物学报， 2023， 49(2): 414-425.

[20] Pan JM, Tian SR, Liang YL, et al. Identification and expression analysis of PIN-LIKES gene family in sugarcane[J]. Acta Agronomica Sinica, 2023, 49(2): 414-425 (in Chinese with English abstract).

[21] Ling H, Fu XQ, Huang N, et al. A sugarcane smut fungus effector simulates the host endogenous elicitor peptide to suppress plant immunity[J]. New Phytologist, 2022, 233(2): 919-933.

[22] Ling H, Huang N, Wu QB, et al. Transcriptional insights into the sugarcane-sorghum mosaic virus interaction[J]. Tropical Plant Biology, 2018, 11(3): 163-176.

[23] La HG, Li J, Ji ZD, et al. Genome-wide analysis of cyclin family in rice (Oryza sativa L.)[J]. Molecular Genetics and Genomics, 2006, 275(4): 374-386.

[24] Chen CJ, Chen H, Zhang Y, et al. TBtools:an integrative toolkit developed for interactive analyses of big biological data[J]. Molecular Plant, 2020, 13(8): 1194-1202.

[25] Liu F, Huang N, Wang L, et al. A novel L-ascorbate peroxidase 6 gene, ScAPX6, plays an important role in the regulation of response to biotic and abiotic stresses in sugarcane[J]. Frontiers in Plant Science, 2017, 8: 2262.

[26] Livak KJ, Schmittgen TD. 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.

[27] 黄宁， 惠乾龙， 方振名， 等. 甘蔗 β-胡萝卜素异构酶基因家族的鉴定、定位和表达分析[J]. 作物学报， 2021， 47(5): 882-893.

[28] Huang N, Hui QL, Fang ZM, et al. Identification, localization and expression analysis of beta-carotene isomerase gene family in sugarcane[J]. Acta Agronomica Sinica, 2021, 47(5): 882-893 (in Chinese with English abstract).

[29] Hartig K, Beck E. Endogenous cytokinin oscillations control cell cycle progression of tobacco BY-2 cells[J]. Plant Biology, 2005, 7(1): 33-40.

[30] Ciardo D, Goldar A, Marheineke K. On the interplay of the DNA replication program and the intra-S phase checkpoint pathway[J]. Genes, 2019, 10(2): 94.

[31] Wang K, Xue BP, He Y, et al. Evolution, gene duplication, and expression pattern analysis of CrRLK1L gene family in Zea mays (L.)[J]. International Journal of Molecular Sciences, 2024, 25(19): 10487.

[32] 石玉， 沈诗雅， 张倩茹， 等. LBD 基因家族研究进展[J]. 中国细胞生物学学报， 2019， 41(4): 738-745.

[33] Shi Y, Shen SY, Zhang QR, et al. Research progress of LBD gene family[J]. Chinese Journal of Cell Biology, 2019, 41(4): 738-745 (in Chinese with English abstract).

[34] Chen YX, Sun SY, Wang XL. The epidermis-specific cyclin CYCP3;1 is involved in the excess brassinosteroids signaling-inhibited root meristem cell division[J]. Journal of Integrative Plant Biology, 2020, 62(11): 1674-1687.

[35] Mroue S, Simeunovic A, Robert HS. Auxin production as an integrator of environmental cues for developmental growth regulation[J]. Journal of Experimental Botany, 2018, 69(2): 201-212.

[36] Zhang ZQ, Li Q, Li ZM, et al. Dual regulation role of GH3.5 in salicylic acid and auxin signaling during Arabidopsis-Pseudomonas syringae interaction[J]. Plant Physiology, 2007, 145(2): 450-464.

[37] Li YG, Yang YH, Hu YL, et al. DELLA and EDS1 form a feedback regulatory module to fine-tune plant growth-defense tradeoff in Arabidopsis[J]. Molecular Plant, 2019, 12(11): 1485-1498.

[38] 李皎琪， 李美琪， 王玉婷， 等. 果蔬逆境胁迫应答中 AP2/ERF 转录因子的激素调控研究进展[J]. 食品科学， 2024， 45(12): 315-323.

[39] Li JQ, Li MQ, Wang YT, et al. Research progress in hormonal regulation of AP2/ERF transcription factors in fruits and vegetables in response to stress[J]. Food Science, 2024, 45(12): 315-323 (in Chinese with English abstract).

[40] Yang YB, An B, Guo YF, et al. A novel effector, FSE1, regulates the pathogenicity of Fusarium oxysporum f. sp. cubense tropical race 4 to banana by targeting the myb transcription factor MaEFM-like[J]. Journal of Fungi, 2023, 9(4): 472.

[41] Qian H, Lin L, Zhang ZC, et al. A MYB-related transcription factor regulates effector gene expression in an oomycete pathogen[J]. Molecular Plant Pathology, 2024, 25(6): e13468.

基金资助

广西自然科学基金项目(2023GXNSFAA026467, 2022GXNSFBA035535, 2021GXNSFBA196046)

湖南省自然科学基金项目(2024JJ5141)

国家自然科学基金项目(32360647, 32260777)