热带作物学报

甜橙糖转运蛋白CitSWEET15基因的表达分析与转运活性检测

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郑乾明 ¹ , 王小柯 ¹ , 王红林 ¹ , 解璞 ¹ , 付慧敏 ² , 陈传武 ²^,^*

热带作物学报 | 组学与生物技术 2023,44(10): 1944-1951

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热带作物学报 | 组学与生物技术 2023, 44(10): 1944-1951

甜橙糖转运蛋白CitSWEET15基因的表达分析与转运活性检测

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郑乾明¹, 王小柯¹, 王红林¹, 解璞¹, 付慧敏², 陈传武²^,^*

作者信息

^1.贵州省农业科学院果树科学研究所，贵州贵阳　550006

^2.广西桂北特色经济作物种质创新与利用实验室/广西特色作物研究院，广西桂林　541004

郑乾明（1985—），男，博士，副研究员，研究方向：果实品质形成机理。

通讯作者:

^* 陈传武（CHEN Chuanwu），E-mail: 13707832977@126.com。

Expression Pattern and Transportation Activity Test of Sugar Transporter Gene CitSWEET15 from Sweet Orange

Qianming ZHENG¹, Xiaoke WANG¹, Honglin WANG¹, Pu XIE¹, Huimin FU², Chuanwu CHEN²^,^*

Affiliations

^1.Institute of Pomology Science, Guizhou Academy of Agricultural Science, Guiyang, Guizhou 550006, China

^2.Guangxi Laboratory of Germplasm Innovation and Utilization of Specialty Commercial Crops in North Guangxi/Guangxi Academy of Specialty Crops, Guilin, Guangxi 541004, China

出版时间: 2023-10-25 doi: 10.3969/j.issn.1000-2561.2023.10.002

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摘要

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高等植物糖转运蛋白SWEET（sugars will eventually be exported transporter）家族具有葡萄糖、果糖或蔗糖的转运活性，广泛参与光合产物韧皮部装载和卸载，以及花、果实和种子发育等生理过程。为探讨CitSWEET15基因在甜橙果实发育和成熟期间的生理功能，本研究开展CitSWEET15的基因克隆、表达模式、亚细胞定位和蔗糖转运活性分析。CitSWEET15基因的开放阅读框（open reading frame, ORF）长度为918 bp，编码305个氨基酸，含有7个跨膜结构域。CitSWEET15的理论等电点为6.29，相对分子量为34.15 kDa。系统进化分析表明，CitSWEET15与AtSWEET15、VvSWEET15、LcSWEET15和PuSWEET15为直系同源基因，属于SWEET Ⅲ类。序列比对表明，CitSWEET15与克里曼丁橘、莽山野橘、金柑、柚、宜昌橙和枸橼的SWEET15基因的核苷酸序列一致率为98.69%~99.9%，氨基酸序列一致率为97.38%~100%。CitSWEET15和上述柑橘类SWEET15成员，与AtSWEET13的4个蔗糖识别位点和3个蔗糖转运活性相关位点完全一致，说明CitSWEET15和柑橘类SWEET15成员可能均具有蔗糖转运活性。荧光定量PCR检测表明，CitSWEET15基因在甜橙果实的种子和果肉中表达量较高，果肉中CitSWEET15基因的表达水平在花后80~140 d较低，随着果实发育显著上调，在花后230 d达到最高水平。将CitSWEET15的C端融合绿色荧光蛋白，然后在拟南芥叶肉原生质体中瞬时表达。亚细胞定位分析表明，荧光信号分布在原生质体外缘，CitSWEET15定位于细胞膜。通过在酿酒酵母W303a（W303-1A）菌株中异源表达和蔗糖类似物七叶苷孵育，检测CitSWEET15和AtSWEET10（At5g50790，阳性对照）的蔗糖转运活性。表达CitSWEET15的酵母细胞与AtSWEET10相似，其荧光强度显著高于载体对照，表明CitSWEET15具有蔗糖转运活性。因此，CitSWEET15是定位于细胞膜的蔗糖转运蛋白，在柑橘类物种中序列高度保守，参与甜橙果实成熟期间的质外体蔗糖跨膜转运。本研究结果为了解甜橙果实可溶性糖积累提供基础，为改良果实风味和品质提供候选基因。

关键词

蔗糖 / 细胞膜 / 酵母表达 / 果实成熟

Abstract

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The sugar transporter SWEET (sugars will eventually be exported transporter) family has sugar transportation activity for glucose, fructose or sucrose, which is involved in the phloem loading and unloading of photosynthetic product, as well as physiological processes such as flower, fruit and seed development in higher plant. To explore the physiological functions of CitSWEET15 (Cs7g02970) during fruit development and ripening of sweet orange, gene cloning, expression patterns, subcellular localization and sucrose transport activity were performed in the study. The ORF (open reading frame) of CitSWEET15 gene was 918 bp, which encoded 305 amino acids, and possessed seven transmembrane domains. The theoretical isoelectric point of CitSWEET15 was 6.29, and the relative molecular weight was 34.15 kDa. Phylogenetic analysis suggested that CitSWEET15 together with AtSWEET15, VvSWEET15, LcSWEET15 and PuSWEET15 were orthologous genes, and belonged to the SWEET III clade. Sequence alignment showed that nucleotide and amino acid sequence identities of CitSWEET15 with those of Clementine mandarin, Mangshan wild mandarin, Kumquat, Pomelo, Citrus ichangensis Swingle and Citron were 98.69%-99.9% and 97.38%-100%, respectively. CitSWEET15 and the mentioned Citrus SWEET15 members were completely consistent with four sucrose recognition sites and three sucrose transport activity sites of AtSWEET13, which suggested that all CitSWEET15 and Citrus SWEET15 members may have sucrose transport activity. Quantitative real-time PCR analysis demonstrated that the expression of CitSWEET15 was high in the seed and fruit pulp of sweet orange, the expression level of CitSWEET15 in fruit pulp was low at 80-140 days after flowering, then significantly up-regulated as fruit development, and reached the highest at 230 days after flowering. CitSWEET15 was fused with green fluorescent protein at the C-terminus, and then transiently expressed in Arabidopsis mesophyll protoplasts. The subcellular localization analysis suggested the fluorescence signal was distributed around the outer edge of protoplast, indicating that CitSWEET15 was located in the plasma membrane. By heterologous expression in Saccharomyces cerevisiae W303a (W303-1A) strain and incubation in the sucrose analogue Esculin, the fluorescence intensity was detected to test the sucrose transportation activity of CitSWEET15 and AtSWEET10 (At5g50790, positive control). The fluorescent intensity of yeast cells expressing CitSWEET15, as similar as AtSWEET10, was significantly higher than vector control, indicating that CitSWEET15 had sucrose transportation activity. In conclusion, CitSWEET15 is highly conserved in Citrus species, functions as a plasma membrane sucrose transporter, and involves in apoplastic sucrose transport during fruit ripening of sweet orange. The results would provide a basis for understanding the soluble sugar accumulation in sweet orange fruit, and a candidate gene for improving fruit flavor and quality.

Key words

sucrose / plasma membrane / yeast expression / fruit ripening

引用本文

郑乾明, 王小柯, 王红林, 解璞, 付慧敏, 陈传武. 甜橙糖转运蛋白CitSWEET15基因的表达分析与转运活性检测. 热带作物学报, 2023 , 44 (10) : 1944 -1951 . DOI: 10.3969/j.issn.1000-2561.2023.10.002

Qianming ZHENG, Xiaoke WANG, Honglin WANG, Pu XIE, Huimin FU, Chuanwu CHEN. Expression Pattern and Transportation Activity Test of Sugar Transporter Gene CitSWEET15 from Sweet Orange[J]. Chinese Journal of Tropical Crops, 2023 , 44 (10) : 1944 -1951 . DOI: 10.3969/j.issn.1000-2561.2023.10.002

基金

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贵州省科技计划项目(黔科合服企[2019]4002)
广西科技计划项目(桂科 AA22068092)
国家重点研发计划项目(2019YFD1001400)

参考文献

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2023年第44卷第10期

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文章信息

doi: 10.3969/j.issn.1000-2561.2023.10.002

接收时间：2022-11-16
首发时间：2026-03-05
出版时间：2023-10-25

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收稿日期：2022-11-16
修回日期：2022-12-10

基金

贵州省科技计划项目(黔科合服企[2019]4002)

广西科技计划项目(桂科 AA22068092)

国家重点研发计划项目(2019YFD1001400)

作者信息

^1.贵州省农业科学院果树科学研究所，贵州贵阳　550006

^2.广西桂北特色经济作物种质创新与利用实验室/广西特色作物研究院，广西桂林　541004

通讯作者:

^* 陈传武（CHEN Chuanwu），E-mail: 13707832977@126.com。

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https://castjournals.cast.org.cn/joweb/rdzwxb/CN/10.3969/j.issn.1000-2561.2023.10.002

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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