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作物学报 ›› 2020, Vol. 46 ›› Issue (7): 1120-1127.doi: 10.3724/SP.J.1006.2020.94173

• 研究简报 • 上一篇    下一篇

甘薯蔗糖转运蛋白基因IbSUT3的克隆及功能分析

王丹丹1,**,柳洪鹃1,**,王红霞2,张鹏2,*(),史春余1,*()   

  1. 1 山东农业大学农学院, 山东泰安 271018
    2 中国科学院分子植物科学卓越创新中心 / 植物生理生态研究所植物分子遗传国家重点实验室, 上海 200032
  • 收稿日期:2019-11-11 接受日期:2020-03-24 出版日期:2020-07-12 网络出版日期:2020-04-03
  • 通讯作者: 张鹏,史春余
  • 作者简介:王丹丹, E-mail: wdd201712@126.com|柳洪鹃, E-mail: liumei0535@126.com
    ** 同等贡献
  • 基金资助:
    国家自然科学基金青年基金项目(31701357);山东省薯类产业创新团队首席专家项目(SDAIT-16-01)

Cloning and functional analysis of the sweet potato sucrose transporter IbSUT3

WANG Dan-Dan1,**,LIU Hong-Juan1,**,WANG Hong-Xia2,ZHANG Peng2,*(),SHI Chun-Yu1,*()   

  1. 1 College of Agronomy, Shandong Agricultural University, Tai’an 271018, Shandong, China
    2 National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China
  • Received:2019-11-11 Accepted:2020-03-24 Published:2020-07-12 Published online:2020-04-03
  • Contact: Peng ZHANG,Chun-Yu SHI
  • About author:** Contributed equally to this work
  • Supported by:
    National Natural Science Foundation of China Youth Fund(31701357);Shandong Agriculture Innovation Team(SDAIT-16-01)

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摘要：

蔗糖作为光合产物的主要运输形式, 其跨膜运输主要由蔗糖转运蛋白介导。本研究采用RACE技术, 从甘薯[Ipomoea batatas (L.) Lam.]中克隆得到甘薯蔗糖转运蛋白基因IbSUT3 (GenBank登录号为MN233361)。IbSUT3基因全长1607 bp, 开放阅读框为1518 bp, 编码505个氨基酸。IbSUT3蛋白预测分子量为53.82 kD, 等电点(pI)为9.19, 含有12个跨膜结构域。序列比对表明, IbSUT3属于Group I亚族, 与其他物种的SUTs蛋白高度相似, 但与Group IV亚族的蔗糖转运蛋白在进化上具有明显差别。利用酵母表达体系SUSY7/ura3证明IbSUT3编码有功能的蔗糖转运蛋白。亚细胞定位发现, IbSUT3蛋白定位在烟草原生质体膜上。实时荧光定量PCR结果表明, IbSUT3基因在甘薯不同组织中均有表达, 但在叶片中表达最高; 非生物胁迫(低温、高盐、干旱)和外源脱落酸均可诱导IbSUT3基因在叶片中的表达, 表明该基因响应多种非生物胁迫, 参与植物对脱落酸的响应。

关键词: 蔗糖转运蛋白, IbSUT3基因, 亚细胞定位, 功能分析, 逆境胁迫

Abstract:

Sucrose, as the main transporter of photosynthate, is transported by sucrose transporters. In this study, we cloned IbSUT3 gene from sweet potato (Ipomoea batatas Lam. GenBank accession number MN233361), by RACE technique. The full-length sequence of the gene is 1607 bp and the complete open reading frame is 1518 bp, encoding 505 amino acids. The predicted protein has the molecular weight of 53.82 kD and isoelectric point of 9.19, containing 12 transmembrane domains. Multiple sequence alignment analysis indicated the protein, belonging to Group I, was significantly different from Group IV in evolution and shared high similarities with SUTs from other plant species. IbSUT3 had the function of transporting sucrose verified by SUSY7/ura3 system. Subcellular localization showed that IbSUT3 protein was located in the tobacco protoplasts plasma membrane. The qRT-PCR results showed that IbSUT3 highly expressed in leaves and induced by drought, high salt, low temperature and exogenous abscisic acid, suggesting that IbSUT3 is involved in response to various abiotic stresses, including abscisic acid.

Key words: sucrose transporters, IbSUT3 gene, subcellular localization, function analysis, adversity stresses

表1

引物信息"

引物Primer 序列 Sequence (5'-3') 目的 Purpose
E3-F AGATAGTAATGGTGGCCTCCATTGC EST序列扩增
E3-R GCGAAGCCAATGAGGAAGACAGC EST sequence amplification
Sp-F1 AGATAGTAATGGTGGCGTCCATTGC 3'端和5'端RACE扩增
Sp-F2 GCGTCCTTTATGTGGCTTTGTGG 3' and 5'-RACE amplification
Sp-R1 TGAGATTGGCGCAGTAAACG
Sp-R2 GCGGCGAAGCCAATGAGGAA
IbSUT3-F CCATTCAGGCTGCGCAACT IbSUT3全长扩增
IbSUT3-R TTAAGAAATTAATTCCAAGGTCCAT IbSUT3 full-length amplification
SUT3Q-F CGGAAAATCCTCCTTGCAAGTG 荧光定量PCR
SUT3Q-R GACGCCACCATTACTATCTTCC Real-time PCR
Actin-F CTGGTGTTATGGTTGGGATGG
Actin-R GGGGTGCCTCGGTAAGAAG
SUT3m-F CATCGATATGGAGAGAGACTCCGTTAACG 酵母功能验证
SUT3m-R CCCCGGGTCAGTGGAAACCACCACCAGAT Yeast function verification
SUT3g-F ACGCGTCGACATGGAGAGAGACTCCGTTAACGGAA 亚细胞定位
SUT3g-R GGACTAGTACGTGGAAACCACCACCAGATAGCTCA Subcellular localization

图1

IbSUT3基因扩增结果 A: IbSUT3基因EST序列扩增结果; B: IbSUT3 3'-RACE, 5'-RACE扩增结果; C: IbSUT3全长扩增结果。"

图2

IbSUT3蛋白跨膜区预测"

图3

IbSUT3编码的氨基酸序列与其他物种SUTs的多重比对"

图4

IbSUT3与其他植物SUTs蛋白的系统发生分析"

图5

IbSUT3功能分析 A: 转化的酵母菌株在含2%蔗糖培养基上的生长情况; B: 转化的酵母菌株在含2%葡萄糖培养基上生长情况。"

图6

IbSUT3亚细胞定位分析"

图7

IbSUT3基因在不同组织中的表达 L: 叶片; B: 柄; J: 茎; WR: 白须根; DR: 发育根; MR: 成熟根。图柱上不同小写字母表示在0.05水平上显著差异。"

图8

IbSUT3在胁迫处理下的表达模式 图柱上不同小写字母表示在0.05水平上显著差异。"

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