Cloning and Interaction Analysis of StPTST2a Gene in Potato

doi:10.13560/j.cnki.biotech.bull.1985.2025-0043

Abstract

Abstract:

Objective PROTEIN TARGETING TO STARCH (PTST) plays an important role in starch biosynthesis. This study aims to investigate the functional role of StPTST2a in potato (Solanum tuberosum L.). Method RT-PCR was used to clone StPTST2a gene and its amino acid sequence was analyzed by the bioinformatic method. RT-qPCR was performed to examine the expressions of StPTST2a in various organs. Prokaryotic expression system was used to express MBP-tagged StPTST2a in vitro and subsequently the recombinant protein was tested for its binding to starch. Seven starch biosynthesis-related genes were cloned, and the protein-protein interaction analysis was performed with yeast two-hybrid (Y2H) assays and luciferase complementation imaging (LCI) assays. Result StPTST2a encoded a protein of 532 amino acids, and it harboured a CBM48 domain at the C-terminus. StPTST2a was expressed at various tissues, and the transcript level was higher in the leaves than that in other organs. In vitro starch binding assays showed that StPTST2a bound to the starch. Y2H and LCI assays demonstrated that StPTST2a interacted with StSS4, StSS6 and StISA1.1 in yeast cells and in plants. Conclusion StPTST2a form a complex with starch biosynthesis-related genes, which may collaboratively control starch biosynthesis and formation of starch granules.

Key words: Solanum tuberosum L., starch, StPTST2a, protein-protein interaction

HUANG Dan-dan, WU Yun-yi, ZOU Jian-hua, YU Ting, ZHU Yan-hui, YANG Mei-hong, DONG Wen-li, GAO Dong-li. Cloning and Interaction Analysis of StPTST2a Gene in Potato[J]. Biotechnology Bulletin, 2025, 41(7): 172-180.

Figures/Tables 6

References 25

[1]	赵宇慈, 许丹, 靳承煜, 等. 马铃薯块茎干物质、淀粉及还原糖含量的检测及相关性分析 [J]. 现代食品科技, 2017, 33(10): 288-293, 280.
	Zhao YC, Xu D, Jin CY, et al. Detection and correlation analysis of dry matter, starch and reducing sugar content in potato tubers [J]. Mod Food Sci Technol, 2017, 33(10): 288-293, 280.
[2]	Zeeman SC, Kossmann J, Smith AM. Starch: its metabolism, evolution, and biotechnological modification in plants [J]. Annu Rev Plant Biol, 2010, 61: 209-234.
[3]	何虎翼, 唐洲萍, 杨鑫, 等. 马铃薯淀粉合成与降解研究进展 [J]. 生物技术通报, 2019, 35(4): 101-107.
	He HY, Tang ZP, Yang X, et al. Research progress on potato starch synthesis and degradation [J]. Biotechnol Bull, 2019, 35(4): 101-107.
[4]	Ohdan T, Francisco PB Jr, Sawada T, et al. Expression profiling of genes involved in starch synthesis in sink and source organs of rice [J]. J Exp Bot, 2005, 56(422): 3229-3244.
[5]	Liu HM, Yu GL, Wei B, et al. Identification and phylogenetic analysis of a novel starch synthase in maize [J]. Front Plant Sci, 2015, 6: 1013.
[6]	He S, Hao X, Wang S, et al. A newly-identified inactive starch synthase simultaneously regulates starch synthesis and carbon allocation in storage roots of cassava [J]. BioRxiv, 2020.
[7]	Cantarel BL, Coutinho PM, Rancurel C, et al. The carbohydrate-active EnZymes database (CAZy): an expert resource for glycogenomics [J]. Nucleic Acids Res, 2009, 37(Database issue): D233-D238.
[8]	Seung D, Soyk S, Coiro M, et al. Protein targeting to starch is required for localising granule-bound starch synthase to starch granules and for normal amylose synthesis in Arabidopsis [J]. PLoS Biol, 2015, 13(2): e1002080.
[9]	Wang W, Wei XJ, Jiao GA, et al. GBSS-BINDING PROTEIN, encoding a CBM48 domain-containing protein, affects rice quality and yield [J]. J Integr Plant Biol, 2020, 62(7): 948-966.
[10]	Seung D, Boudet J, Monroe J, et al. Homologs of protein targeting to starch control starch granule initiation in Arabidopsis leaves [J]. Plant Cell, 2017, 29(7): 1657-1677.
[11]	Seung D, Schreier TB, Bürgy L, et al. Two plastidial coiled-coil proteins are essential for normal starch granule initiation in Arabidopsis [J]. Plant Cell, 2018, 30(7): 1523-1542.
[12]	Peng C, Wang YH, Liu F, et al. FLOURY ENDOSPERM6 encodes a CBM48 domain-containing protein involved in compound granule formation and starch synthesis in rice endosperm [J]. Plant J, 2014, 77(6): 917-930.
[13]	Zhang L, Li N, Zhang J, et al. The CBM48 domain-containing protein FLO6 regulates starch synthesis by interacting with SSIVb and GBSS in rice [J]. Plant Mol Biol, 2022, 108(4/5): 343-361.
[14]	Kerk D, Conley TR, Rodriguez FA, et al. A chloroplast-localized dual-specificity protein phosphatase in Arabidopsis contains a phylogenetically dispersed and ancient carbohydrate-binding domain, which binds the polysaccharide starch [J]. Plant J, 2006, 46(3): 400-413.
[15]	石振明, 杨慧芹, 高冬丽. 马铃薯PTST1基因的克隆、表达及互作分析 [J]. 植物生理学报, 2023, 59(8): 1575-1582.
	Shi ZM, Yang HQ, Gao DL. Cloning of PTST1 and analysis of its expression and interaction in potato [J]. Plant Physiol J, 2023, 59(8): 1575-1582.
[16]	Hochmuth A, Carswell M, Rowland A, et al. Distinct effects of PTST2b and MRC on starch granule morphogenesis in potato tubers [J]. Plant Biotechnol J, 2025, 23(2): 412-429.
[17]	Van Harsselaar JK, Lorenz J, Senning M, et al. Genome-wide analysis of starch metabolism genes in potato (Solanum tuberosum L.) [J]. BMC Genomics, 2017, 18(1): 37.
[18]	Rahman S, Nakamura Y, Li Z, et al. The sugary-type isoamylase gene from rice and Aegilops tauschii: characterization and comparison with maize and Arabidopsis [J]. Genome, 2003, 46(3): 496-506.
[19]	Roldán I, Wattebled F, Mercedes Lucas M, et al. The phenotype of soluble starch synthase IV defective mutants of Arabidopsis thaliana suggests a novel function of elongation enzymes in the control of starch granule formation [J]. Plant J, 2007, 49(3): 492-504.
[20]	Hawkins E, Chen JW, Watson-Lazowski A, et al. STARCH SYNTHASE 4 is required for normal starch granule initiation in amyloplasts of wheat endosperm [J]. New Phytol, 2021, 230(6): 2371-2386.
[21]	Streb S, Delatte T, Umhang M, et al. Starch granule biosynthesis in Arabidopsis is abolished by removal of all debranching enzymes but restored by the subsequent removal of an endoamylase [J]. Plant Cell, 2008, 20(12): 3448-3466.
[22]	Hussain H, Mant A, Seale R, et al. Three isoforms of isoamylase contribute different catalytic properties for the debranching of potato glucans [J]. Plant Cell, 2003, 15(1): 133-149.
[23]	Utsumi Y, Utsumi C, Sawada T, et al. Functional diversity of isoamylase oligomers: the ISA1 Homo-oligomer is essential for amylopectin biosynthesis in rice endosperm [J]. Plant Physiol, 2011, 156(1): 61-77.
[24]	Burton RA, Jenner H, Carrangis L, et al. Starch granule initiation and growth are altered in barley mutants that lack isoamylase activity [J]. Plant J, 2002, 31(1): 97-112.
[25]	Ferreira SJ, Senning M, Fischer-Stettler M, et al. Simultaneous silencing of isoamylases ISA1, ISA2 and ISA3 by multi-target RNAi in potato tubers leads to decreased starch content and an early sprouting phenotype [J]. PLoS One, 2017, 12(7): e0181444.

引物名称Primer name	引物序列 Primer sequence （5′-3′）	引物用途 Primer usage
pMAL-C2X-StPTST2a-F	GGATCGAGGGAAGGATTTCAATGGTTTTGACCCAGTTCGC	体外淀粉结合实验
pMAL-C2X-StPTST2a-R	CAGGTCGACTCTAGAGGATCCTTATGTGACAATGAGAAGATT	In vitro starch-binding assays
pGBKT7-StPTST2a-F	CATATGGCCATGGAGGCCATGGTTTTGACCCAGTTCG	酵母双杂交实验
pGBKT7-StPTST2a-R	CTAGTTATGCGGCCGCTGCAGTTATGTGACAATGAGAAG	Yeast two-hybrid assays
pGADT7-StSS1-F	CATATGGCCATGGAGGCCAGTATGGGGTCTCTGCAAACAC	酵母双杂交实验
pGADT7-StSS1-R	CGATGCCCACCCGGGTGTCATCTGACATATGGAGGATC	Yeast two-hybrid assays
pGADT7-StSS2-F	CATATGGCCATGGAGGCCAGTATGGAGAATTCCATTCTTC	酵母双杂交实验
pGADT7-StSS2-R	CGATGCCCACCCGGGTGTCACCACTGATACTTAGCAG	Yeast two-hybrid assays
pGADT7-StSS3-F	CATATGGCCATGGAGGCCAGTATGGACTCACTTACAGTTCT	酵母双杂交实验
pGADT7-StSS3-R	CGATGCCCACCCGGGTGCTATTCTAACTTTCTAGCAGC	Yeast two-hybrid assays
pGADT7-StSS4-F	CATATGGCCATGGAGGCCAGTATGGAGATGAAGATCTCCAA	酵母双杂交实验
pGADT7-StSS4-R	CGATGCCCACCCGGGTGTCAACTACGACTTGCAGCTCTT	Yeast two-hybrid assays
pGADT7-StSS5-F	ATGGCCATGGAGGCCAGTATGATCACGGAGATGCTTTC	酵母双杂交实验
pGADT7-StSS5-R	CGATGCCCACCCGGGTGTTACAAGTTTTTTATTGCTGTA	Yeast two-hybrid assays
pGADT7-StSS6-F	CATGGAGGCCAGTATGGATTTCACATCCGGCCTGTC	酵母双杂交实验
pGADT7-StSS6-R	CGATGCCCACCCGGGTGTCAAAGCAAGTGACGTCTAAG	Yeast two-hybrid assays
pGADT7-StISA1.1-F	ATGGCCATGGAGGCCAGTATGGTACGGCAGTTCATCAA	酵母双杂交实验
pGADT7-StISA1.1-R	CGATGCCCACCCGGGTGCTATGCATCATCAGCAGATG	Yeast two-hybrid assays
pCAMBIA-nLUC-StPTST2a-F	GAACAGGGGGACGAGCTCATGGTTTTGACCCAGTTCG	荧光素酶互补实验
pCAMBIA-nLUC-StPTST2a-R	CCCGGGACGCGTACGAGATCTGTGTGACAATGAGAAGATTA	Luciferase complementation assays
pCAMBIA-cLUC-StSS4-F	CTCGTACGCGTCCCGGGGCATGGAGATGAAGATCTCC	荧光素酶互补实验
pCAMBIA-cLUC-StSS4-R	CGTCCTTGTAGTCCATTTGTTTCAACTACGACTTGCAGCTCTTGC	Luciferase complementation assays
pCAMBIA-cLUC-StSS6-F	CTCGTACGCGTCCCGGGGCATGGATTTCACATCCGG	荧光素酶互补实验
pCAMBIA-cLUC-StSS6-R	GTCCTTGTAGTCCATTTGTTTCAAAGCAAGTGACGTCTAAGTAC	Luciferase complementation assays
pCAMBIA-cLUC-StISA1.1-F	GTACGCGTCCCGGGGCATGGTACGGCAGTTCAT	荧光素酶互补实验
pCAMBIA-cLUC-StISA1.1-R	CGTCCTTGTAGTCCATTTGTTCTATGCATCATCAGCAGATGATAG	Luciferase complementation assays
qPCR-StPTST2a-F	CCAGCTTCCTCATCTGGTAGATC	荧光定量PCR
qPCR-StPTST2a-R	CATGTCGTGAGGCTTCCATTTTT	Quantitative real-time PCR

引物名称Primer name	引物序列 Primer sequence （5′-3′）	引物用途 Primer usage
pMAL-C2X-StPTST2a-F	GGATCGAGGGAAGGATTTCAATGGTTTTGACCCAGTTCGC	体外淀粉结合实验
pMAL-C2X-StPTST2a-R	CAGGTCGACTCTAGAGGATCCTTATGTGACAATGAGAAGATT	In vitro starch-binding assays
pGBKT7-StPTST2a-F	CATATGGCCATGGAGGCCATGGTTTTGACCCAGTTCG	酵母双杂交实验
pGBKT7-StPTST2a-R	CTAGTTATGCGGCCGCTGCAGTTATGTGACAATGAGAAG	Yeast two-hybrid assays
pGADT7-StSS1-F	CATATGGCCATGGAGGCCAGTATGGGGTCTCTGCAAACAC	酵母双杂交实验
pGADT7-StSS1-R	CGATGCCCACCCGGGTGTCATCTGACATATGGAGGATC	Yeast two-hybrid assays
pGADT7-StSS2-F	CATATGGCCATGGAGGCCAGTATGGAGAATTCCATTCTTC	酵母双杂交实验
pGADT7-StSS2-R	CGATGCCCACCCGGGTGTCACCACTGATACTTAGCAG	Yeast two-hybrid assays
pGADT7-StSS3-F	CATATGGCCATGGAGGCCAGTATGGACTCACTTACAGTTCT	酵母双杂交实验
pGADT7-StSS3-R	CGATGCCCACCCGGGTGCTATTCTAACTTTCTAGCAGC	Yeast two-hybrid assays
pGADT7-StSS4-F	CATATGGCCATGGAGGCCAGTATGGAGATGAAGATCTCCAA	酵母双杂交实验
pGADT7-StSS4-R	CGATGCCCACCCGGGTGTCAACTACGACTTGCAGCTCTT	Yeast two-hybrid assays
pGADT7-StSS5-F	ATGGCCATGGAGGCCAGTATGATCACGGAGATGCTTTC	酵母双杂交实验
pGADT7-StSS5-R	CGATGCCCACCCGGGTGTTACAAGTTTTTTATTGCTGTA	Yeast two-hybrid assays
pGADT7-StSS6-F	CATGGAGGCCAGTATGGATTTCACATCCGGCCTGTC	酵母双杂交实验
pGADT7-StSS6-R	CGATGCCCACCCGGGTGTCAAAGCAAGTGACGTCTAAG	Yeast two-hybrid assays
pGADT7-StISA1.1-F	ATGGCCATGGAGGCCAGTATGGTACGGCAGTTCATCAA	酵母双杂交实验
pGADT7-StISA1.1-R	CGATGCCCACCCGGGTGCTATGCATCATCAGCAGATG	Yeast two-hybrid assays
pCAMBIA-nLUC-StPTST2a-F	GAACAGGGGGACGAGCTCATGGTTTTGACCCAGTTCG	荧光素酶互补实验
pCAMBIA-nLUC-StPTST2a-R	CCCGGGACGCGTACGAGATCTGTGTGACAATGAGAAGATTA	Luciferase complementation assays
pCAMBIA-cLUC-StSS4-F	CTCGTACGCGTCCCGGGGCATGGAGATGAAGATCTCC	荧光素酶互补实验
pCAMBIA-cLUC-StSS4-R	CGTCCTTGTAGTCCATTTGTTTCAACTACGACTTGCAGCTCTTGC	Luciferase complementation assays
pCAMBIA-cLUC-StSS6-F	CTCGTACGCGTCCCGGGGCATGGATTTCACATCCGG	荧光素酶互补实验
pCAMBIA-cLUC-StSS6-R	GTCCTTGTAGTCCATTTGTTTCAAAGCAAGTGACGTCTAAGTAC	Luciferase complementation assays
pCAMBIA-cLUC-StISA1.1-F	GTACGCGTCCCGGGGCATGGTACGGCAGTTCAT	荧光素酶互补实验
pCAMBIA-cLUC-StISA1.1-R	CGTCCTTGTAGTCCATTTGTTCTATGCATCATCAGCAGATGATAG	Luciferase complementation assays
qPCR-StPTST2a-F	CCAGCTTCCTCATCTGGTAGATC	荧光定量PCR
qPCR-StPTST2a-R	CATGTCGTGAGGCTTCCATTTTT	Quantitative real-time PCR

基因 Gene	基因号 Gene ID	花 Flower	叶 Leaf	叶柄 Petiole	茎 Stem	匍匐茎Stolon	发育块茎Young tuber	成熟块茎 Mature tuber
StSS1	PGSC0003DMG402018552	25.61	63.81	33.58	22.92	14.46	19.21	14.80
StSS2	PGSC0003DMG400001328	21.37	59.03	37.83	50.03	45.99	124.37	207.38
StSS3	PGSC0003DMG400016481	9.03	18.75	15.66	10.55	10.48	35.72	40.04
StSS4	PGSC0003DMG400008322	10.81	6.04	7.51	6.26	14.39	8.27	4.13
StSS5	PGSC0003DMG400030619	3.71	1.25	1.42	3.84	14.25	42.0	57.44
StSS6	PGSC0003DMG402013540	2.28	2.00	7.30	4.10	8.72	13.75	13.21
StISA1.1	PGSC0003DMG400020699	5.29	19.7	11.29	7.52	16.16	38.48	42.26

基因 Gene	基因号 Gene ID	花 Flower	叶 Leaf	叶柄 Petiole	茎 Stem	匍匐茎Stolon	发育块茎Young tuber	成熟块茎 Mature tuber
StSS1	PGSC0003DMG402018552	25.61	63.81	33.58	22.92	14.46	19.21	14.80
StSS2	PGSC0003DMG400001328	21.37	59.03	37.83	50.03	45.99	124.37	207.38
StSS3	PGSC0003DMG400016481	9.03	18.75	15.66	10.55	10.48	35.72	40.04
StSS4	PGSC0003DMG400008322	10.81	6.04	7.51	6.26	14.39	8.27	4.13
StSS5	PGSC0003DMG400030619	3.71	1.25	1.42	3.84	14.25	42.0	57.44
StSS6	PGSC0003DMG402013540	2.28	2.00	7.30	4.10	8.72	13.75	13.21
StISA1.1	PGSC0003DMG400020699	5.29	19.7	11.29	7.52	16.16	38.48	42.26