木薯糖转运蛋白基因MeSWEET2b的克隆及其低温胁迫下的功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2025-0857

摘要/Abstract

摘要：

目的研究木薯MeSWEET2b基因在低温胁迫下的功能，为探讨木薯耐寒性的分子机制及选育耐低温新品种提供理论依据。方法利用生物信息学方法分析了MeSWEET2b的序列特性、系统进化关系，在烟草中瞬时表达该蛋白以明确其亚细胞定位。利用RT-qPCR分析不同发育期、不同糖黑暗处理及低温胁迫下MeSWEET2b基因的表达水平。利用病毒诱导的基因沉默技术（VIGS）分析该基因在低温胁迫下的功能。结果 MeSWEET2b蛋白含有7个跨膜结构域，系统进化树分析表明该蛋白属于SWEETs Clade Ⅰ家族，与拟南芥AtSWEET2亲缘关系最近。亚细胞定位显示MeSWEET2b定位于细胞质膜上。RT-qPCR分析表明MeSWEET2b在膨大期（7个月）的叶片中表达水平最高；利用不同糖且在黑暗条件下处理木薯水培苗，发现MeSWEET2b在葡萄糖和果糖中的表达趋势一致；随着胁迫温度降低和时间延长，木薯MeSWEET2b基因的表达水平呈显著上升趋势。利用VIGS技术沉默MeSWEET2b的表达，4 ℃低温胁迫下，与对照相比，MeSWEET2b沉默植株叶片出现明显萎蔫，其葡萄糖和果糖含量显著减少。结论 MeSWEET2b基因沉默导致木薯对低温不耐受，推测该基因在木薯耐低温中发挥重要作用。

关键词: 木薯, 糖转运蛋白, MeSWEET2b, 低温胁迫, VIGS

Abstract:

Objective To study the function of the MeSWEET2b gene in cassava (Manihot esculenta Crantz) under low-temperature stress, and to provide theoretical basis for exploring the molecular mechanism of cassava tolerance to cold and breeding new varieties with low-temperature tolerance. Method Bioinformatics method was used to analyze the sequence characteristics, phylogenetic relationships of the MeSWEET2b gene. Transient expression of the MeSWEET2b protein in tobacco was to determine its subcellular localization. RT-qPCR was adapted to analyze the expressions of the MeSWEET2b gene at different growth stages, under darkness treatments of different sugar and low-temperature stress. Virus induced gene silencing (VIGS) technology was applied to study the function of this gene under low-temperature stress. Result The MeSWEET2b protein had 7 transmembrane domains, and phylogenetic tree analysis indicated that it belonged to the SWEETs Clade I subfamily and had the closest homology with Arabidopsis AtSWEET2. The subcellular localization results showed that MeSWEET2b was located on the cell plasma membrane. The RT-qPCR analysis showed that MeSWEET2b had the highest expression in the leaves during the expansion period (7 months). For the water culture seedlings of cassava treated with different sugars, the expression trends of MeSWEET2b in glucose and fructose were consistent. As the temperature of cold stress decreased and the time extended, the expression of MeSWEET2b in cassava showed a significantly increased. Using VIGS technology to silence MeSWEET2b, under 4 ℃ low-temperature stress, the wilting of the leaves in the MeSWEET2b silenced plants were significantly more severe than that of the control, and the contents of glucose and fructose significantly reduced. Conclusion The silencing of MeSWEET2b gene led to the intolerance of cassava to low temperatures. It is speculated that this gene plays an important role in the cold tolerance of cassava.

Key words: cassava, sugar transporter, MeSWEET2b, low temperature stress, VIGS

薛晶晶, 安飞飞, 罗秀芹, 蔡杰. 木薯糖转运蛋白基因MeSWEET2b的克隆及其低温胁迫下的功能研究[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0857.

XUE Jing-jing, AN Fei-fei, LUO Xiu-qin, CAI Jie. Cloning of Sugar Transporter MeSWEET2b in Cassava and Its Function Analysis Under Low Temperature Stress[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0857.

图/表 10

图1 MeSWEET2b的CDS序列和氨基酸序列*表示终止密码子，下划线表示保守结构域MtN3_slv super family，加粗字体表示跨膜序列

Fig. 1 CDS sequences and amino acid sequences of MeSWEET2b* indicates stop codon, underline indicates conserved domains MtN3_slv super family, bold font indicates transmembrane sequence

图2 MeSWEET2b的系统进化树分析和氨基酸序列比对

Fig. 2 Phylogenetic analysis and acid sequence alignment of MeSWEET2b

图3 MeSWEET2b的亚细胞定位

Fig. 3 Subcellular localization of MeSWEET2b

图4 MeSWEET2b在不同发育期的表达分析*P<0.05，**P<0.01，***P<0.001， the same below

Fig. 4 Expression analysis of MeSWEET2b at different developmental stages

图5 MeSWEET2b在添加不同糖溶液的黑暗处理下的表达分析

Fig. 5 Expression analysis of MeSWEET2b in dark treatment with different sugar solutions

图6 MeSWEET2b在低温胁迫下的表达分析

Fig. 6 Expression analysis of MeSWEET2b under low temperature stress

图7 木薯 MeSWEET2b沉默植株的表达水平分析

Fig. 7 Expression level analysis of cassava MeSWEET2b silenced plants

图8 木薯MeSWEET2b沉默植株低温胁迫下的表型特征

Fig. 8 Phenotypic characteristics of cassava MeSWEET2b silenced plants under low-temperature stress

图9 木薯MeSWEET2b沉默植株低温胁迫下的糖含量

Fig. 9 Sugar content of cassava MeSWEET2b silenced plants under low-temperature stress

图10 木薯MeSWEET2b沉默植株低温胁迫下的生理指标测定

Fig. 10 Analysis of physiological indicators in cassava MeSWEET2b silenced plants under low-temperature stress

参考文献 41

[1]	Williams LE, Lemoine R, Sauer N. Sugar transporters in higher plants—a diversity of roles and complex regulation [J]. Trends Plant Sci, 2000, 5(7): 283-290.
[2]	Wormit A, Trentmann O, Feifer I, et al. Molecular identification and physiological characterization of a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transport [J]. Plant Cell, 2006, 18(12): 3476-3490.
[3]	Rosa M, Prado C, Podazza G, et al. Soluble sugars: Metabolism, sensing and abiotic stress: a complex network in the life of plants [J]. Plant Signal Behav, 2009, 4(5): 388-393.
[4]	Ruan YL, Jin Y, Yang YJ, et al. Sugar input, metabolism, and signaling mediated by invertase: roles in development, yield potential, and response to drought and heat [J]. Mol Plant, 2010, 3(6): 942-955.
[5]	Sami F, Yusuf M, Faizan M, et al. Role of sugars under abiotic stress [J]. Plant Physiol Biochem, 2016, 109: 54-61.
[6]	Zhu JK. Abiotic stress signaling and responses in plants [J]. Cell, 2016, 167(2): 313-324.
[7]	Jeena GS, Kumar S, Shukla RK. Structure, evolution and diverse physiological roles of SWEET sugar transporters in plants [J]. Plant Mol Biol, 2019, 100(4/5): 351-365.
[8]	Pil Joon Seo JP. An Arabidopsis senescence-associated protein SAG29 regulates cell viability under high salinity [J]. Planta, 2011, 233(1): 189-200.
[9]	Klemens PAW, Patzke K, Deitmer J, et al. Overexpression of the vacuolar sugar carrier AtSWEET16 modifies germination, growth, and stress tolerance in Arabidopsis [J]. Plant Physiol, 2013, 163(3): 1338-1352.
[10]	Wang L, Yao LN, Hao XY, et al. Tea plant SWEET transporters: expression profiling, sugar transport, and the involvement of CsSWEET16 in modifying cold tolerance in Arabidopsis [J]. Plant Mol Biol, 2018, 96(6): 577-592.
[11]	Zhou AM, Ma HP, Feng S, et al. A novel sugar transporter from Dianthus spiculifolius, DsSWEET12, affects sugar metabolism and confers osmotic and oxidative stress tolerance in Arabidopsis [J]. Int J Mol Sci, 2018, 19(2): 497.
[12]	Zhou AM, Ma HP, Feng S, et al. DsSWEET17, a tonoplast-localized sugar transporter from Dianthus spiculifolius, affects sugar metabolism and confers multiple stress tolerance in Arabidopsis [J]. Int J Mol Sci, 2018, 19(6): 1564.
[13]	Hu LP, Zhang F, Song SH, et al. CsSWEET2, a hexose transporter from cucumber (Cucumis sativus L.), affects sugar metabolism and improves cold tolerance in Arabidopsis [J]. Int J Mol Sci, 2022, 23(7): 3886.
[14]	Zeng CY, Chen Z, Xia J, et al. Chilling acclimation provides immunity to stress by altering regulatory networks and inducing genes with protective functions in cassava [J]. BMC Plant Biol, 2014, 14(1): 207.
[15]	An D, Yang J, Zhang P. Transcriptome profiling of low temperature-treated cassava apical shoots showed dynamic responses of tropical plant to cold stress [J]. BMC Genom, 2012, 13(1): 64.
[16]	Li SX, Yu X, Cheng ZH, et al. Global gene expression analysis reveals crosstalk between response mechanisms to cold and drought stresses in cassava seedlings [J]. Front Plant Sci, 2017, 8: 1259.
[17]	胡梅珍. 木薯叶片蔗糖质外体装载模式 [D]. 海口: 海南大学, 2016.
	Hu MZ. Sucrose apoplastic loading pattern in cassava leaf [D]. Haikou: Hainan University, 2016.
[18]	薛蓓蓓, 覃丽芳, 董明右, 等. 木薯SWEETs基因家族生物信息学及表达特性研究 [J]. 基因组学与应用生物学, 2019, 38(1): 260-268.
	Xue BB, Qin LF, Dong MY, et al. Bioinformatics analysis and expressional characteristics of cassava SWEETs gene family [J]. Genom Appl Biol, 2019, 38(1): 260-268.
[19]	Cohn M, Bart RS, Shybut M, et al. Xanthomonas axonopodis virulence is promoted by a transcription activator-like effector-mediated induction of a SWEET sugar transporter in cassava [J]. Mol Plant Microbe Interact, 2014, 27(11): 1186-1198.
[20]	刘秦, 马畅, 冯世鹏, 等. 木薯SWEET1基因的分子克隆、亚细胞定位与功能分析 [J]. 分子植物育种, 2017, 15(7): 2502-2509.
	Liu Q, Ma C, Feng SP, et al. Molecular cloning, subcellular localization and function analysis of a MeSWEET1 gene from Manihot esculenta [J]. Mol Plant Breed, 2017, 15(7): 2502-2509.
[21]	朱柏光, 李闯, 张雪娇, 等. 木薯MeSWEET3b的基因克隆及功能分析 [J]. 分子植物育种, 2022, 20(3): 733-741.
	Zhu BG, Li C, Zhang XJ, et al. Molecular cloning and function analysis of a MeSWEET3b gene from Manihot esculenta [J]. Mol Plant Breed, 2022, 20(3): 733-741.
[22]	Fan XW, Sun JL, Cai Z, et al. MeSWEET15a/b genes play a role in the resistance of cassava (Manihot esculenta Crantz) to water and salt stress by modulating sugar distribution [J]. Plant Physiol Biochem, 2023, 194: 394-405.
[23]	薛晶晶, 安飞飞, 罗秀芹, 等. VIGS技术鉴定木薯糖转运蛋白Mesweet18的功能研究 [J]. 生物技术进展, 2022, 12(6): 888-893.
	Xue JJ, An FF, Luo XQ, et al. Study on the function of cassava sugar transporter Mesweet18 by VIGS [J]. Curr Biotechnol, 2022, 12(6): 888-893.
[24]	薛晶晶, 安飞飞, 朱文丽, 等. 木薯糖转运蛋白MeSWEET18的克隆与功能分析 [J]. 热带作物学报, 2023, 44(6): 1083-1090.
	Xue JJ, An FF, Zhu WL, et al. Cloning and functional analysis of sugar transporter MeSWEET18 in cassava [J]. Chin J Trop Crops, 2023, 44(6): 1083-1090.
[25]	薛晶晶, 韦卓文, 罗秀芹, 等. 木薯糖转运蛋白基因MeSWEET17b的克隆及表达分析 [J]. 广西植物, 2024, 44(12): 2212-2221.
	Xue JJ, Wei ZW, Luo XQ, et al. Cloning and expression analysis of sugar transporter protein gene MeSWEET17b in cassava [J]. Guihaia, 2024, 44(12): 2212-2221.
[26]	Rabbi IY, Kayondo SI, Bauchet G, et al. Genome-wide association analysis reveals new insights into the genetic architecture of defensive, agro-morphological and quality-related traits in cassava [J]. Plant Mol Biol, 2022, 109(3): 195-213.
[27]	Fernandez-Pozo N, Rosli HG, Martin GB, et al. The SGN VIGS tool: user-friendly software to design virus-induced gene silencing (VIGS) constructs for functional genomics [J]. Mol Plant, 2015, 8(3): 486-488.
[28]	Tuo DC, Zhou P, Yan P, et al. A cassava common mosaic virus vector for virus-induced gene silencing in cassava [J]. Plant Meth, 2021, 17(1): 74.
[29]	Lin IW, Sosso D, Chen LQ, et al. Nectar secretion requires sucrose phosphate synthases and the sugar transporter SWEET9 [J]. Nature, 2014, 508(7497): 546-549.
[30]	Durand M, Mainson D, Porcheron B, et al. Carbon source-sink relationship in Arabidopsis thaliana: the role of sucrose transporters [J]. Planta, 2018, 247(3): 587-611.
[31]	Gwon S, Park J, Huque AM, et al. The Arabidopsis SWEET1 and SWEET2 uniporters recognize similar substrates while differing in subcellular localization [J]. J Biol Chem, 2023, 299(12): 105389.
[32]	Ho LH, Klemens PAW, Neuhaus HE, et al. SlSWEET1a is involved in glucose import to young leaves in tomato plants [J]. J Exp Bot, 2019, 70(12): 3241-3254.
[33]	Wilson MC, Mutka AM, Hummel AW, et al. Gene expression atlas for the food security crop cassava [J]. New Phytol, 2017, 213(4): 1632-1641.
[34]	Eom JS, Chen LQ, Sosso D, et al. SWEETs, transporters for intracellular and intercellular sugar translocation [J]. Curr Opin Plant Biol, 2015, 25: 53-62.
[35]	Morii M, Sugihara A, Takehara S, et al. The dual function of OsSWEET3a as a gibberellin and glucose transporter is important for young shoot development in rice [J]. Plant Cell Physiol, 2020, 61(11): 1935-1945.
[36]	Zhou YN, Cui XY, Hu AN, et al. Characterization and functional analysis of pollen-specific PwSWEET1 in Picea wilsonii [J]. J For Res, 2020, 31(5): 1913-1922.
[37]	Geng YQ, Wu MJ, Zhang CM. Sugar transporter ZjSWEET2.2 mediates sugar loading in leaves of Ziziphus jujuba mill [J]. Front Plant Sci, 2020, 11: 1081.
[38]	Yao LN, Ding CQ, Hao XY, et al. CsSWEET1a and CsSWEET17 mediate growth and freezing tolerance by promoting sugar transport across the plasma membrane [J]. Plant Cell Physiol, 2020, 61(9): 1669-1682.
[39]	Fang T, Rao Y, Wang MZ, et al. Characterization of the SWEET gene family in Longan (Dimocarpus longan) and the role of DlSWEET1 in cold tolerance [J]. Int J Mol Sci, 2022, 23(16): 8914.
[40]	Liu XZ, Zhang Y, Yang C, et al. AtSWEET4, a hexose facilitator, mediates sugar transport to axial sinks and affects plant development [J]. Sci Rep, 2016, 6: 24563.
[41]	Yang GX, Xu HF, Zou Q, et al. The vacuolar membrane sucrose transporter MdSWEET16 plays essential roles in the cold tolerance of apple [J]. Plant Cell Tissue Organ Cult, 2020, 140(1): 129-142.