马铃薯SUMO E3连接酶基因家族分析及StSIZ1基因的克隆与表达模式分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-1252

摘要/Abstract

摘要：

目的对马铃薯StSIZ1基因进行克隆、亚细胞定位及组织表达特异性分析，为阐明StSIZ1基因的功能提供理论依据。方法利用拟南芥SUMO E3连接酶基因序列进行同源检索，获得马铃薯SUMO E3连接酶基因家族成员，并对其进行生物信息学分析；采用RT-PCR从马铃薯品种Atlantic克隆StSIZ1基因；通过RT-qPCR探究StSIZ1基因在马铃薯组织特异性及响应非生物胁迫的表达模式。构建pEGFP-StSIZ1亚细胞定位载体，通过农杆菌介导的烟草瞬时转化系统检测StSIZ1在细胞中的定位。结果 StSIZ1基因位于第11号染色体，CDS区长2 634 bp。RT-qPCR结果显示StSIZ1基因在茎中相对表达量最低，块茎中相对表达量最高；StSIZ1基因广泛响应多种胁迫处理，如渗透、盐、高温胁迫。融合蛋白荧光检测确定StSIZ1在细胞核中发挥功能。结论马铃薯StSIZ1响应多种非生物胁迫，且在细胞核中发挥功能。

关键词: 马铃薯, StSIZ1, 基因克隆, 亚细胞定位, 表达模式分析

Abstract:

Objective This study is aimed to clone, characterize, and analyze the subcellular localization and tissue-specific expression of the StSIZ1 in potato, providing theoretical foundation for elucidating its functional role. Method Homology search was conducted using the Arabidopsis thaliana SUMO E3 ligase gene sequence to obtain members of the potato SUMO E3 ligase gene family, and have bioinformatics analysis of them. The StSIZ1 was cloned from the potato variety Atlantic using reverse transcription PCR (RT-PCR). The expression patterns of StSIZ1 in various potato tissues and its responses to abiotic stresses and hormone were analyzed using quantitative real-time PCR (RT-qPCR). A pCEGFP-StSIZ1 subcellular localization vector was constructed, and the localization of StSIZ1 was confirmed via Agrobacterium-mediated transient expression in tobacco. Result The coding sequence (CDS) of the StSIZ1 is 2 634 base pairs (bp) in length and is located on chromosome 11. RT-qPCR results revealed that StSIZ1 has the lowest relative expression in stems and the highest in tubers. The gene presents significant responsiveness to various abiotic stress treatments, including osmotic, salt, and high-temperature stress. Subcellular localization analysis using fluorescence detection indicated that StSIZ1 primarily functions within the nucleus. Conclusion The StSIZ1 is responsive to a wide range of abiotic stresses and functions in the nucleus, highlighting its potential role in tolerance to stress.

Key words: potato, StSIZ1, gene cloning, subcellular localization, expression pattern analysis

许慧珍, SHANTWANA Ghimire, RAJU Kharel, 岳云, 司怀军, 唐勋. 马铃薯SUMO E3连接酶基因家族分析及StSIZ1基因的克隆与表达模式分析[J]. 生物技术通报, 2025, 41(6): 119-129.

XU Hui-zhen, SHANTWANA Ghimire, RAJU Kharel, YUE Yun, SI Huai-jun, TANG Xun. Analysis of the Potato SUMO E3 Ligase Gene Family and Cloning and Expression Pattern of StSIZ1[J]. Biotechnology Bulletin, 2025, 41(6): 119-129.

图/表 9

图1 SUMO化和去SUMO化过程

Fig. 1 Sumoylation and de-sumoylation processes

表1 引物信息

Table 1 Information about the primers

引物名称 Primer name	引物序列 Primer sequence （5′-3′）	用途 Purpose
pCAMBIA1300-StSIZ1-F	ggtacccggggatcctctagaATGGATTTGGTTGCTAGCTGCA	基因克隆
pCAMBIA1300-StSIZ1-R	gcccttgctcaccatgtcgacCTATTCAGAATCCGAATCAATACTTAGAT	基因克隆
qPCR-StSIZ1-F	TTAGACTTGTGAAGCGGCGT	RT-qPCR
qPCR-StSIZ1-R	CCACCAACACAACGACGAAC	RT-qPCR
Eflα-F	GATGGTCAGACCCGTGAACA	内参基因
Eflα-R	CCTTGGAGTACTTCGGGGTC	内参基因

图2 SUMO E3连接酶基因家族系统发育树

Fig. 2 Phylogenetic tree of SUMO E3 ligase gene family

图3 马铃薯SUMO E3连接酶基因家族成员的染色体定位（A），Motif、保守结构域和基因结构分析（B）

Fig. 3 Chromosomal localization (A), Motif, conserved structural domains and gene structure (B) of potato SUMO E3 ligase gene family members

图4 StSIZ1和StSIZ2同源基因氨基酸序列比对

Fig. 4 Homologous amino acid sequence alignment of StSIZ1 and StSIZ2

图5 StSIZ1和StSIZ2蛋白结构分析及StSIZ1基因克隆A： StSIZ1和StSIZ2蛋白保守结构域； B-C： StSIZ1和StSIZ2的三级结构； D：StSIZ1基因PCR产物

Fig. 5 Protein structure analysis of StSIZ1 and StSIZ2 and cloning of StSIZ1 geneA: Conserved structural domains of the StSIZ1 and StSIZ2 protein. B-C: Tertiary structure of StSIZ1 and StSIZ2. D: PCR production of gene StSIZ1

表2 马铃薯SUMO E3连接酶基因家族成员的蛋白理化性质

Table 2 Physicochemical properties of protein members in the potato SUMO E3 ligase gene family

基因ID Gene ID	基因名 Gene name	理论分子量 Molecular weight （Da）	等电点 Point isoelectric	不稳定指数 Instability index	脂肪族氨基酸指数 Aliphatic index	亲水度 Grand average of hydropathicity
Soltu.DM.11G022540.5	StSIZ1	95 449.87	4.96	44.28	76.34	-0.453
Soltu.DM.06G005690.1	StSIZ2	93 832.16	5.44	47.05	80.67	-0.373
Soltu.DM.07G023970.1	StMMS21	28 532.31	5.10	58.06	77.34	-0.533
Soltu.DM.08G004520.1	StPIAS1	96 099.04	6.01	48.14	73.54	-0.390
Soltu.DM.06G005850.1	StPIAS2	163 534.42	6.74	41.34	88.45	-0.296
Soltu.DM.05G011900.1	StRanBP2a	30 458.93	9.24	58.23	29.93	-0.922
Soltu.DM.12G024390.1	StRanBP2b	26 349.84	8.17	52.64	58.67	-0.482
Soltu.DM.09G019740.1	StRanGAP1a	58 094.47	4.58	37.95	94.34	-0.283
Soltu.DM.01G024550.1	StRanGAP1b	60 594.10	4.64	41.86	90.05	-0.386
Soltu.DM.05G007610.1	StRanGAP1c	64 532.83	8.78	30.85	99.40	-0.067

图6 马铃薯StSIZ1在烟草细胞的亚细胞定位pEGFP：空白对照（pCAMBIA1300-35S-EGFP空载体）；pEGFP-StSIZ1：pEGFP-StSIZ1融合蛋白；比例尺=20 μm

Fig. 6 Subcellular localization of potato StSIZ1 in the tobacco cellspEGFP: Control (pCAMBIA1300-35S-EGFP vector); pEGFP-StSIZ1: pEGFP-StSIZ1 fusion protein. Scale bar=20 μm

图7 马铃薯StSIZ1基因的表达模式A：不同组织中StSIZ1基因的表达；B： 20% PEG、C： 150 mmol/L NaCl、D： 50 μmol/L ABA、E： 50 μmol/L GA3、F：高温胁迫（35 ℃）处理下StSIZ1的相对表达量，不同字母代表差异显著（P<0.05）

Fig. 7 Expression pattern of StSIZ1 in the potatoA: Expression of StSIZ1 gene in different tissues. Relative expressions of the StSIZ1 under treatment with 20% PEG (B), 150 mmol/L NaCl (C), 50 μmol/LABA (D), 50 μmol/L GA3 (E), and heat stress (35 ℃) (F). Different letters indcate significant differences (P<0.05)

参考文献 39

1	Scaramagli S, Biondi S, Leone A, et al. Acclimation to low water potential in potato cell suspension cultures leads to changes in putrescine metabolism [J]. Plant Physiol Biochem, 2000, 38(4): 345-351.
2	贾明飞, 樊建英, 封志明, 等. 氮素不同形态对早熟马铃薯产量和氮素积累的影响 [J]. 中国土壤与肥料, 2024, (2): 146-151.
	Jia MF, Fan JY, Feng ZM, et al. Effects of different forms of nitrogen on yield and nitrogen accumulation in early maturing potatoes [J]. Soil and Fertiliser China, 2024, (2): 146-151.
3	汪昕, 杨德秋, 李洋, 等. 马铃薯机械化收获技术进展与展望 [J]. 农机化研究, 2024, 46(9): 1-7.
	Wang X, Yang DQ, Li Y, et al. Progress and prospect of potato mechanized harvesting technology [J]. J Agric Mech Res, 2024, 46(9): 1-7.
4	Seo J, Lee KJ. Post-translational modifications and their biological functions: proteomic analysis and systematic approaches [J]. J Biochem Mol Biol, 2004, 37(1): 35-44.
5	Yeh ET, Gong L, Kamitani T. Ubiquitin-like proteins: new wines in new bottles [J]. Gene, 2000, 248(1/2): 1-14.
6	Kurepa J, Walker JM, Smalle J, et al. The small ubiquitin-like modifier (SUMO) protein modification system in Arabidopsis. Accumulation of SUMO1 and-2 conjugates is increased by stress [J]. J Biol Chem, 2003, 278(9): 6862-6872.
7	Elrouby N. Analysis of small ubiquitin-like modifier (SUMO) targets reflects the essential nature of protein SUMOylation and provides insight to elucidate the role of SUMO in plant development [J]. Plant Physiol, 2015, 169(2): 1006-1017.
8	Park HJ, Yun DJ. SUMO proteins grapple with biotic and abiotic stresses in Arabidopsis [J]. J Plant Biol, 2013, 56(2): 77-84.
9	Novatchkova M, Tomanov K, Hofmann K, et al. Update on sumoylation: defining core components of the plant SUMO conjugation system by phylogenetic comparison [J]. New Phytol, 2012, 195(1): 23-31.
10	Han YF, Zhao QY, Dang LL, et al. The SUMO E3 ligase-like proteins PIAL1 and PIAL2 interact with MOM1 and form a novel complex required for transcriptional silencing [J]. Plant Cell, 2016, 28(5): 1215-1229.
11	Ritterhoff T, Das H, Hofhaus G, et al. The RanBP2/RanGAP1*SUMO1/Ubc9 SUMO E3 ligase is a disassembly machine for Crm1-dependent nuclear export complexes [J]. Nat Commun, 2016, 7: 11482.
12	Zheng Z, Liu D. SIZ1 regulates phosphate deficiency-induced inhibition of primary root growth of Arabidopsis by modulating Fe accumulation and ROS production in its roots [J]. Plant Signal Behav, 2021, 16(10): 1946921.
13	Rawat SS, Sandhya S, Laxmi A. Complex genetic interaction between glucose sensor HXK1 and E3 SUMO ligase SIZ1 in regulating plant morphogenesis [J]. Plant Signal Behav, 2024, 19(1): 2341506.
14	Gao SJ, Zeng XQ, Wang JH, et al. Arabidopsis SUMO E3 ligase SIZ1 interacts with HDA6 and negatively regulates HDA6 function during flowering [J]. Cells, 2021, 10(11): 3001.
15	Castro PH, Couto D, Santos MÂ, et al. SUMO E3 ligase SIZ1 connects sumoylation and reactive oxygen species homeostasis processes in Arabidopsis [J]. Plant Physiol, 2022, 189(2): 934-954.
16	Miura K, Nozawa R. Overexpression of SIZ1 enhances tolerance to cold and salt stresses and attenuates response to abscisic acid in Arabidopsis thaliana [J]. Plant Biotechnol, 2014, 31(2): 167-172.
17	Kim JY, Song JT, Seo HS. Post-translational modifications of Arabidopsis E3 SUMO ligase AtSIZ1 are controlled by environmental conditions [J]. FEBS Open Bio, 2017, 7(10): 1622-1634.
18	Yoo CY, Miura K, Jin JB, et al. SIZ1 small ubiquitin-like modifier E3 ligase facilitates basal thermotolerance in Arabidopsis independent of salicylic acid [J]. Plant Physiol, 2006, 142(4): 1548-1558.
19	Miura K, Sato A, Ohta M, et al. Increased tolerance to salt stress in the phosphate-accumulating Arabidopsis mutants siz1 and pho2 [J]. Planta, 2011, 234(6): 1191-1199.
20	Jiang H, Zhou LJ, Gao HN, et al. The transcription factor MdMYB2 influences cold tolerance and anthocyanin accumulation by activating SUMO E3 ligase MdSIZ1 in apple [J]. Plant Physiol, 2022, 189(4): 2044-2060.
21	张亚丽. 苹果SUMO E3连接酶MdSIZ1蛋白SUMO化MdMYB30调控蜡质形成的机理 [D]. 泰安: 山东农业大学, 2023.
	Zhang YL. Mechanism of regulating wax formation by sumoylation of apple SUMO E3 ligase MdSIZ1 protein MdMYB30 [D]. Tai'an: Shandong Agricultural University, 2023.
22	Zhang S, Wang SJ, Lv JL, et al. SUMO E3 ligase SlSIZ1 facilitates heat tolerance in tomato [J]. Plant Cell Physiol, 2018, 59(1): 58-71.
23	Zhang S, Zhuang KY, Wang SJ, et al. A novel tomato SUMO E3 ligase, SlSIZ1, confers drought tolerance in transgenic tobacco [J]. J Integr Plant Biol, 2017, 59(2): 102-117.
24	Cai B, Kong XX, Zhong C, et al. SUMO E3 Ligases GmSIZ1a and GmSIZ1b regulate vegetative growth in soybean [J]. J Integr Plant Biol, 2017, 59(1): 2-14.
25	Li YJ, Wang GX, Xu ZQ, et al. Organization and regulation of soybean SUMOylation system under abiotic stress conditions [J]. Front Plant Sci, 2017, 8: 1458.
26	Lei SK, Wang QZ, Chen Y, et al. Capsicum SIZ1 contributes to ABA-induced SUMOylation in pepper [J]. Plant Sci, 2022, 314: 111099.
27	Lai RQ, Jiang JM, Wang J, et al. Functional characterization of three maize SIZ/PIAS-type SUMO E3 ligases [J]. J Plant Physiol, 2022, 268: 153588.
28	汪永平. 梭梭SUMO E3连接酶基因HaSIZ1的克隆和功能分析 [D]. 兰州: 兰州大学, 2020.
	Wang YP. Cloning and functional analysis of HaSIZ1 ligase gene of Haloxylon ammodendron SUMO E3 [D]. Lanzhou: Lanzhou University, 2020.
29	Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets [J]. Mol Biol Evol, 2016, 33(7): 1870-1874.
30	Chen CJ, Wu Y, Li JW, et al. TBtools-II: a "one for all, all for one" bioinformatics platform for biological big-data mining [J]. Mol Plant, 2023, 16(11): 1733-1742.
31	Srivastava M, Verma V, Srivastava AK. The converging path of protein SUMOylation in phytohormone signalling: highlights and new frontiers [J]. Plant Cell Rep, 2021, 40(11): 2047-2061.
32	Crozet P, Margalha L, Butowt R, et al. SUMOylation represses SnRK1 signaling in Arabidopsis [J]. Plant J, 2016, 85(1): 120-133.
33	Xiong JW, Yang FB, Wei F, et al. Inhibition of SIZ1-mediated SUMOylation of HOOKLESS1 promotes light-induced apical hook opening in Arabidopsis [J]. Plant Cell, 2023, 35(6): 2027-2043.
34	Cohen-Peer R, Schuster S, Meiri D, et al. Sumoylation of Arabidopsis heat shock factor A2 (HsfA2) modifies its activity during acquired thermotholerance [J]. Plant Mol Biol, 2010, 74(1/2): 33-45.
35	Garcia-Dominguez M, March-Diaz R, Reyes JC. The PHD domain of plant PIAS proteins mediates sumoylation of bromodomain GTE proteins [J]. J Biol Chem, 2008, 283(31): 21469-21477.
36	Suzuki R, Shindo H, Tase A, et al. Solution structures and DNA binding properties of the N-terminal SAP domains of SUMO E3 ligases from Saccharomyces cerevisiae and Oryza sativa [J]. Proteins, 2009, 75(2): 336-347.
37	Catala R, Ouyang J, Abreu IA, et al. The Arabidopsis E3 SUMO ligase SIZ1 regulates plant growth and drought responses [J]. Plant Cell, 2007, 19(9): 2952-2966.
38	赵媛媛, 刘萍涛, 康建宏. 高温胁迫对马铃薯叶片生理特性及产量影响研究 [J]. 中国农学通报, 2024, 40(21): 35-44.
	Zhao YY, Liu PT, Kang JH. Effects of high temperature stress on physiological characteristics and yield of potato leaves [J]. Chin Agric Sci Bull, 2024, 40(21): 35-44.
39	Wyrzykowska A, Bielewicz D, Plewka P, et al. The MYB33, MYB₆5, and MYB101 transcription factors affect Arabidopsis and potato responses to drought by regulating the ABA signaling pathway [J]. Physiol Plant, 2022, 174(5): e13775.

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