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作物学报 ›› 2023, Vol. 49 ›› Issue (1): 129-139.doi: 10.3724/SP.J.1006.2023.24023

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘薯糖转运蛋白IbSWEET15的功能研究

吴旭莉1,2(), 吴正丹1,2,4(), 晚传芳1,2, 杜叶1,2, 高艳1,2, 李賾萱1, 王志前1, 唐道彬1,2,3, 王季春1,2,3, 张凯1,2,3,*()   

  1. 1西南大学农学与生物科技学院, 重庆 400715
    2薯类生物学与遗传育种重庆市重点实验室, 重庆 400715
    3南方山地农业教育部工程研究中心, 重庆 400715
    4广西壮族自治区农业科学院经济作物研究所, 广西南宁 530007
  • 收稿日期:2022-01-18 接受日期:2022-05-05 出版日期:2023-01-12 网络出版日期:2022-05-24
  • 通讯作者: 张凯
  • 作者简介:吴旭莉, E-mail: shirleyswu@163.com;
    吴正丹, E-mail: wudandan0905@163.com第一联系人：**同等贡献
  • 基金资助:
    国家重点研发计划项目(2018YFD1000705);国家重点研发计划项目(2018YFD1000700);中央高校基本科研业务费专项资金(XDJK2020B032);中央高校基本科研业务费专项资金(XDJK2021F001);重庆市技术创新与应用发展专项重点项目(cstc2021jscx-gksbX0022);西南大学种质创制专项研究项目

Functional identification of sucrose transporter protein IbSWEET15 in sweet potato

WU Xu-Li1,2(), WU Zheng-Dan1,2,4(), WAN Chuan-Fang1,2, DU Ye1,2, GAO Yan1,2, LI Ze-Xuan1, WANG Zhi-Qian1, TANG Dao-Bin1,2,3, WANG Ji-Chun1,2,3, ZHANG Kai1,2,3,*()   

  1. 1College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
    2Chongqing Key Laboratory of Biology and Genetic Breeding for Tuber and Root Crops, Chongqing 400715, China
    3Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400715, China
    4Cash Crops Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, Guangxi, China
  • Received:2022-01-18 Accepted:2022-05-05 Published:2023-01-12 Published online:2022-05-24
  • Contact: ZHANG Kai
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    National Key Research and Development Program of China(2018YFD1000705);National Key Research and Development Program of China(2018YFD1000700);Fundamental Research Funds for the Central Universities(XDJK2020B032);Fundamental Research Funds for the Central Universities(XDJK2021F001);Key Project of Chongqing Technology Innovation and Application Development(cstc2021jscx-gksbX0022);Germplasm Creation Research Program of Southwest University

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摘要：

SWEET蛋白在植物生长发育、胁迫响应和糖代谢过程中具有重要的调控作用, 而甘薯SWEET蛋白的研究鲜有报道。开展甘薯SWEET蛋白功能研究, 揭示其在糖转运及淀粉与糖代谢中的功能, 具有重要的理论与实践意义。根据不同淀粉性状甘薯块根中差异表达的SWEET编码基因转录本, 设计引物进行RACE克隆, 获得IbSWEET15全长cDNA序列。利用在线软件对IbSWEET15进行生物信息学分析, 并构建系统发育树明确其分类。通过在本氏烟草中瞬时表达IbSWEET15-GFP融合蛋白, 明确IbSWEET15的亚细胞定位; 以酵母突变体互补试验, 验证IbSWEET15蛋白在酵母中的糖转运功能。通过实时荧光定量PCR (qRT-PCR)分析IbSWEET15在甘薯各器官中的表达特征; 构建IbSWEET15表达载体并通过蘸花法转化拟南芥野生型Col-0, 获得IbSWEET15异源表达拟南芥株系, 比较转基因株系与野生型拟南芥植株的淀粉和糖含量差异, 以明确IbSWEET15在淀粉和糖代谢过程中的功能。IbSWEET15基因开放阅读框为879 bp, 编码292个氨基酸组成的SWEET蛋白, 具有2个MtN3_slv保守结构域及7个跨膜结构域, 属于SWEET蛋白家族第III分支成员。IbSWEET15定位于质膜, 在酵母中无蔗糖和己糖转运功能, 其编码基因在甘薯侧枝中表达量最高, 主茎、叶次之, 块根中最低。IbSWEET15的拟南芥过表达株系的叶片可溶性糖含量显著下降, 而种子可溶性糖和淀粉含量均高于野生型。推测IbSWEET15可能在光合产物源-库运输过程中的韧皮部装载以及植株淀粉和糖积累方面具有重要作用。本研究为揭示IbSWEET15在甘薯淀粉和糖代谢及重要品质性状形成中的功能提供了重要信息。

关键词: 甘薯, IbSWEET15, 可溶性糖, 淀粉

Abstract:

SWEET proteins play important regulatory roles in plant growth and development, stress response and sugar metabolism, but few research has been reported on SWEET proteins in sweet potato. It is of great theoretical and practical significance to carry out functional studies on sweet potato SWEET proteins to reveal their functions in sugar transport and starch and sugar metabolism. Based on the transcripts of SWEET encoding gene differentially expressed in sweet potato storage roots with different starch-related traits, specific primers were designed and the full-length cDNA sequence of IbSWEET15 was cloned using RACE method. IbSWEET15 bioinformatics was performed using online software, and its classification was clarified by phylogenetic tree analysis. The subcellular localization of the protein encoded by IbSWEET15 gene was identified by transient expression of IbSWEET15-GFP fusion protein in Nicotiana benthamiana, while yeast mutant complementation experiments were carried out to identify the sugar transport function of IbSWEET15 in yeast. The relative expression pattern of IbSWEET15 genes in various organs of sweet potato were analyzed by qRT-PCR. IbSWEET15 expression vector was constructed and transformed into Arabidopsis wild-type Col-0 by floral dip method to obtain IbSWEET15 heterologously expressed Arabidopsis lines, and the starch and sugar contents of the transgenic plants were measured and compared with those of wild-type Arabidopsis plants, and the function of IbSWEET15 in starch and sugar metabolism was identified. IbSWEET15, with an open reading frame of 879 bp, encoded a 292 amino acid sucrose transporter protein with two MtN3_slv conserved structural domains and seven transmembrane structural domains, and was a member of clade III of the SWEET protein family. The protein encoded by IbSWEET15 localized at the plasma membrane and did not transport sucrose and hexose in yeast mutant. IbSWEET15 gene had the highest expression in sweet potato branches, followed by stems and leaves, and the lowest expression in storage roots. The leaf soluble sugar contents of the IbSWEET15 heterologously expressed Arabidopsis lines were significantly decreased, while the seed soluble sugar and starch contents were higher than wild type. We proposed that IbSWEET15 played an important role in phloem loading during the source to sink transport of photosynthetic product as well as sugar and starch accumulation. This study provides the critical information for understanding the function of IbSWEET15 in starch and sugar metabolism, and formation of important quality traits in sweet potato.

Key words: sweet potato, IbSWEET15, soluble sugar, starch

表1

本研究所用引物"

引物名称
Primer name		 引物序列
Primer sequences (5°-3°)		 引物用途
Primer usage
15-5-1 TGCTCCCGAGAAAGGAAACCATGAGACC 5°-RACE特异引物5°-RACE specific primer
15-5-2 GAGTGGCGTTCTTCTTGATGAAAGC 5°-RACE巢扩引物5°-RACE nested amplification primer
15-3-1 TAGCCTTGTTGGTTGGATTTGCGTGGCT 3°-RACE特异引物3°-RACE specific primer
15-3-2 AATTGGTGCACCCTGTGGATTCA 3°-RACE巢扩引物3°-RACE nested amplification primer
C15-Fwd ATGAAAACTGAGTGTGCC 基因克隆
C15-Rev GGCATTAATAGAATTCATATATAACATGC Gene cloning
SC15-Fwd GAGCTCATGGCAATTCTAGATCTTCATCACC 亚细胞定位载体构建
SC15-Rev GGATCCGTTCTCCATCGCCGCCGG Subcellular localization vector construction
SI15-Fwd TCGACTAGTGGATCCATGGCAATTCTAGATCTTCA 酵母表达载体构建
SI15-Rev TCCAAAGCTGGATCCGTTCTCCATCGCCGCCGGCG Yeast expression vector construction
T15-Fwd CACCATGGCAATTCTAGATCTTCATCAC 植物表达载体构建
T15-Rev GTTCTCCATCGCCGCCGGCGGAG Plant expression vector construction
IbH2B-QF GTGCCGGAGACAAGAAGAAG 内参引物
IbH2B-QR CTTGCTGGAGATTCCGATGT Control primer
IbUBI-QF CTTGCTGGAGATTCCGATGT 内参引物
IbUBI-QR CTTGATCTTCTTCGGCTTGG Control primer
AtACTIN-QF ACACTGTGCCAATCTACGAGGGTT 内参引物
AtACTIN-QR ACAATTTCCCGCTCTGCTGTTGTG Control primer
IbSWEET15-QF TGGTGCACCCTGTGGATTCAGGGA qRT-PCR检测
IbSWEET15-QR TCACTTGATGGACTCAGCCGGCCA qRT-PCR detection
Fbar CGACATCCGCCGTGCCACCGA 转基因植株鉴定
Rbar GTACCGGCAGGCTGAAGTCCAGC Transgenic plant identification

图1

IbSWEET15编码蛋白分析 A: IbSWEET15蛋白保守结构域分析(红色框内代表MtN3_slv保守结构域)。VvSWEET15 (登录号为P0DKJ5.1): 葡萄(Vitis vinifera) SWEET蛋白; AtSWEET15 (登录号为Q9FY94.1): 拟南芥SWEET蛋白; OsSWEET15 (登录号为Q6K602.1): 水稻SWEET蛋白。B: 甘薯IbSWEET15、IbSWEET10蛋白和拟南芥SWEET蛋白家族的系统发育树。C: IbSWEET15蛋白跨膜结构域预测。pCAMBIA1300-GFP (D)和pCAMBIA1300-IbSWEET15-GFP (E)分别与细胞膜标记蛋白(红色荧光)共定位于质膜。标尺为20 μm或10 μm。"

图2

IbSWEET15的蔗糖(A~C)和己糖(D)转运功能鉴定 1、2、3分别表示成功转化pDR196、pDR196-StSUT1和pDR196-IbSWEET15载体的SEY2102菌株。转化的SEY2102菌株在含2%蔗糖的固体SD培养基上(A)和在液体SD培养基中的生长情况(B)及其菌体密度(OD600值) (C)。分别转化了pDR196-ScHXT5 (左), pDR196 (中), pDR196-IbSWEET15 (右)载体的EBY.VW4000菌株在含2%麦芽糖或不同己糖(葡萄糖、果糖、甘露糖、半乳糖)的固体SD培养基中的生长情况(D)。1×、20×、1、10、100、1000分别表示稀释倍数。ns表示无显著差异。***表示在0.001水平差异显著。标尺为0.5 cm。"

图3

IbSWEET15在甘薯各器官中的表达量 XS22: 徐薯22。***表示在0.001水平差异显著。"

图4

IbSWEET15在拟南芥中的异源表达及转基因植株的形态 A: 转基因拟南芥中IbSWEET15的表达量检测; B: 免疫印迹检测转基因植株中IbSWEET15蛋白表达; C: 转基因植株的表型观察。219、246、266、270分别代表转基因株系IbSWEET15-219、IbSWEET15-246、IbSWEET15-266和IbSWEET15-270。***表示在0.001水平差异显著。"

图5

IbSWEET15的异源表达改变了拟南芥淀粉和可溶性糖含量 *、**分别表示在0.05、0.01水平差异显著。219、246、270分别代表转基因株系IbSWEET15-219、IbSWEET15-246和IbSWEET15-270。"

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