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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (1): 129-139.doi: 10.3724/SP.J.1006.2023.24023

• CROP GENETICS & BREEDING ·GERMPLASM RESOURCES ·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2023-01-12 Published:2022-05-24
  • Contact: ZHANG Kai E-mail:shirleyswu@163.com;wudandan0905@163.com;zhangkai2010s@163.com
  • 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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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

Table 1

Primers used in this study"

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

Fig. 1

Characterization of protein encoded by IbSWEET15 A: the analysis of the conserved domain of IbSWEET15 (The red box represents the conserved MtN3_slv structural domain). VvSWEET15 (accession number: P0DKJ5.1): grape (Vitis vinifera) SWEET protein. AtSWEET15 (accession number: Q9FY94.1): Arabidopsis SWEET protein. OsSWEET15 (accession number: Q6K602.1): rice SWEET protein. B: phylogenetic tree of sweet potato IbSWEET15 and IbSWEET10 proteins with Arabidopsis SWEET protein family. C: the prediction of the transmembrane structural domain of IbSWEET15 protein. pCAMBIA1300-GFP (D) and pCAMBIA1300-IbSWEET15-GFP (E) the co-localized with the plasma membrane-located marker protein (red fluorescence). Bars: 20 μm or 10 μm."

Fig. 2

Function identification of Sucrose (A-C) and hexose (D) transport of IbSWEET15 1, 2, and 3 represent the SEY2102 strains that successfully transformed pDR196, pDR196-StSUT1, and pDR196-IbSWEET15 vectors, respectively. Growth of transformed SEY2102 strains in solid SD medium (A) and liquid (B) SD medium containing 2% sucrose and their cell density (OD600 value) (C). Growth of EBY.VW4000 strains transformed with pDR196-ScHXT5 (left), pDR196 (middle), and pDR196-IbSWEET15 (right) vectors on solid SD medium containing 2% maltose or different hexoses (glucose, fructose, mannose, and galactose), respectively. 1×, 20×, 1, 10, 100, and 1000 represent dilution times, respectively. ns: no significant difference. *** means significant difference at the 0.001 probability level. Bar: 0.5 cm."

Fig. 3

Relative expression level of IbSWEET15 genes of various organs in sweet potato XS22: Xushu 22. *** means significant difference at the 0.001 probability level."

Fig. 4

Heterologous expression of IbSWEET15 and morphology of transgenic plants A: the detection of the expression of IbSWEET15 in transgenic Arabidopsis; B: western blot detection of IbSWEET15 protein expression in transgenic plants; C: phenotypic observation of transgenic plants. 219, 246, 266, and 270 represent transgenic lines IbSWEET15-219, IbSWEET15-246, IbSWEET15-266, and IbSWEET15-270, respectively. *** means significant difference at the 0.001 probability level."

Fig. 5

Heterologous expression of IbSWEET15 alters the content of starch and soluble sugars in transgenic Arabidopsis plants * and ** mean significant difference at the 0.05 and 0.01 probability levels, respectively. 219, 246, and 270 represent transgenic lines IbSWEET15-219, IbSWEET15-246, and IbSWEET15-270, respectively."

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