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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (10): 2643-2653.doi: 10.3724/SP.J.1006.2023.34009

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

Identification of potato amylase StBAM9 interacting protein and analysis of the interaction mechanism

DU Juan1(), PENG Xiao-Jun1,2, HOU Juan1,3, LIU Teng-Fei1,4, LIU Zeng1, SONG Bo-Tao1()   

  1. 1National Key Laboratory for Germplasm Innovation and Utilization of Horticultural Crops / Key Laboratory of Potato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs / Huazhong Agricultural University, Wuhan 430070, Hubei, China
    2People's Government of Baren Township, Aktau County, Kizilsu Kirgiz Autonomous Prefecture 845555, Xinjiang, China
    3College of Horticulture, Henan Agricultural University, Zhengzhou 450002, Henan, China
    4College of Food Science and Engineering, Shandong Agricultural University, Tai'an 271018, Shandong, China
  • Received:2023-01-13 Accepted:2023-04-17 Online:2023-10-12 Published:2023-04-26
  • Contact: E-mail: songbotao@mail.hzau.edu.cn
  • Supported by:
    Guizhou Science and Technology Support Plan (the Key Project in Agricultural Field) Biological Breeding Pilot Project (Qiankehe Support [2022] Key 030-3);National Natural Science Foundation of China(31671749)

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Abstract:

Previous research conducted in our laboratory has demonstrated the crucial role of StBAM9 (β-Amylase 9) in the resistance of potatoes to cold-induced sweetening (CIS), although StBAM9 lacks β-Amylase activity. To investigate the mechanism, we generated a yeast two-hybrid library of tuber cDNA following low-temperature storage and screened for potential interacting proteins with StBAM9 as bait. The results revealed that 12 of the identified interacting proteins were common among both full-length and truncated transport peptide StBAM9 bait screens. Among them, four proteins (StDUF842, StTPR01660, StTPR22129, and StTPR45174) had significant interactions with StBAM9 in yeast two-hybrid assay. Subsequently, two of these proteins, StTPR01660 and StTPR4517, were identified as the interactors with StBAM9 through Glutathione-S-transferase (GST) pull-down experiments. Bimolecular fluorescence complementation (BiFC) assays demonstrated that only StTPR01660 was co-localized with StBAM9 on starch granules, while StTPR01660 itself was observed in the cytoplasm. In conclusion, StBAM9 may recruit StTPR01660 from the cytoplasm to starch granules, potentially enabling starch degradation.

Key words: potato, cold-induced sweetening (CIS), starch degradation, β-amylase, protein interaction

Table S1

Primers used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 用途
Use
5°-T7 TAATACGACTCACTATAGG 酵母文库构建
Yeast library construction
3°-AD ACTGTGCATCGTGCACCATCTC 酵母文库构建
Yeast library construction
AD-StDUF842-F CCATGGAGGCCAGTGAATTCATGGACGCAATAGCAGCAGC 酵母双杂交Y2H
AD-StDUF842-R AGCTCGAGCTCGATGGATCCTCAATTGTTGAAGCCGAGAG 酵母双杂交Y2H
AD-StTPR01660-F CCATGGAGGCCAGTGAATTCATGTTATTGAGAAGCTCATC 酵母双杂交Y2H
AD-StTPR01660-R AGCTCGAGCTCGATGGATCCCTAAGAAGCAGCAGCCAAGG 酵母双杂交Y2H
AD-StTPR22129-F CCATGGAGGCCAGTGAATTCATGTTGCTAAGGAGTTCTTC 酵母双杂交Y2H
AD-StTPR22129-R AGCTCGAGCTCGATGGATCCTCAAACAGTTATAGAAGCCG 酵母双杂交Y2H
AD-StTPR45174-F CCATGGAGGCCAGTGAATTCATGAGAACTGTCCTTTTCCG 酵母双杂交Y2H
AD-StTPR45174-R AGCTCGAGCTCGATGGATCCCTAGTAAGCTGCAACCGTTG 酵母双杂交Y2H
AD-StSTPK69936-F CCATGGAGGCCAGTGAATTCATGGAGGTAATAGAGAAGGA 酵母双杂交Y2H
AD-StSTPK69936-R AGCTCGAGCTCGATGGATCCTCAAGTTGGTGATTCAGCTA 酵母双杂交Y2H
AD-StSTPK63153-F CCATGGAGGCCAGTGAATTCATGAGTTCCAGAGGTGGTGG 酵母双杂交Y2H
AD-StSTPK63153-R AGCTCGAGCTCGATGGATCCTCATTGTGGTCCCTCTAGCT 酵母双杂交Y2H
AD-StSTPK61170-F CCATGGAGGCCAGTGAATTCATGGAGAAATACGAGCTTGT 酵母双杂交Y2H
AD-StSTPK61170-R AGCTCGAGCTCGATGGATCCTTAGGTGAGACGAACTTCCC 酵母双杂交Y2H
AD-StPP2C-F CCATGGAGGCCAGTGAATTCATGGAAGAAAGTAAAATGAT 酵母双杂交Y2H
AD-StPP2C-R AGCTCGAGCTCGATGGATCCTTAATCTTTCAAGGTATCCA 酵母双杂交Y2H
AD-StGAPDH-F CCATGGAGGCCAGTGAATTCATGGCCAATGGCAAGATCAA 酵母双杂交Y2H
AD-StGAPDH-R AGCTCGAGCTCGATGGATCCTCAAGCCTTGGCCATATGGC 酵母双杂交Y2H
AD-StSHSP12249-F CCATGGAGGCCAGTGAATTCATGGCAACTTCACTTGCTCT 酵母双杂交Y2H
AD-StSHSP12249-R AGCTCGAGCTCGATGGATCCTCACTCAATTTTAACATCAA 酵母双杂交Y2H
AD-StSHSP78006-F CCATGGAGGCCAGTGAATTCATGTCACTGATCCCAAGAAT 酵母双杂交Y2H
AD-StSHSP78006-R AGCTCGAGCTCGATGGATCCTTAACCAGAGATCTCAATGG 酵母双杂交Y2H
AD-StAFTP4-F CCATGGAGGCCAGTGAATTCATGAAGCAAAAGGTTGTTAT 酵母双杂交Y2H
AD-StAFTP4-R AGCTCGAGCTCGATGGATCCTCACATAATGGAGCAATTGG 酵母双杂交Y2H
AD-StSSIV-F CCATGGAGGCCAGTGAATTCATGGAGATGAAGATCTCCAA 酵母双杂交Y2H
AD-StSSIV-R AGCTCGAGCTCGATGGATCCTCAACTACGACTTGCAGCTC 酵母双杂交Y2H
AD-StGWD-F CCATGGAGGCCAGTGAATTCATGAGTAATTCCTTAGGGAA 酵母双杂交Y2H
AD-StGWD-R AGCTCGAGCTCGATGGATCCTCACATCTGAGGTCTTGTCT 酵母双杂交Y2H
StTPR01660-A1-180-F CCATGGAGGCCAGTGAATTCATGTTATTGAGAAGCTCATC 酵母双杂交Y2H
StTPR01660-A1-180-R AGCTCGAGCTCGATGGATCCCTATTTCTGGTAATAGGCAT 酵母双杂交Y2H
StTPR01660-A181-262-F CCATGGAGGCCAGTGAATTCATGATCGAAGCCCACCCAGG 酵母双杂交Y2H
StTPR01660-A181-262-R AGCTCGAGCTCGATGGATCCCTACAGTACGTAGCAGTCAT 酵母双杂交Y2H
StTPR01660-A263-317-F CCATGGAGGCCAGTGAATTCATGGGCTCTTATGCTCGATT 酵母双杂交Y2H
StTPR01660-A263-317-R AGCTCGAGCTCGATGGATCCCTAAGAAGCAGCAGCCAAGG 酵母双杂交Y2H
StTPR01660-A181-277-F CCATGGAGGCCAGTGAATTCATGATCGAAGCCCACCCAGG 酵母双杂交Y2H
StTPR01660-A181-277-R AGCTCGAGCTCGATGGATCCCTACTCATATTCTTCACCAG 酵母双杂交Y2H
StTPR01660-A278-317-F CCATGGAGGCCAGTGAATTCATGAATGAGGGTGAGGACGA 酵母双杂交Y2H
StTPR01660-A278-317-R AGCTCGAGCTCGATGGATCCCTAAGAAGCAGCAGCCAAGG 酵母双杂交Y2H
StTPR01660-A181-317-F CCATGGAGGCCAGTGAATTCATGATCGAAGCCCACCCAGG 酵母双杂交Y2H
StTPR01660-A181-317-R AGCTCGAGCTCGATGGATCCCTAAGAAGCAGCAGCCAAGG 酵母双杂交Y2H
pET32a-StDUF842-F CTGATATCGGATCCGAATTCATGGACGCAATAGCAGCAGC 原核表达
Prokaryotic expression
pET32a-StDUF842-R TGGTGGTGGTGGTGCTCGAGTCAATTGTTGAAGCCGAGAG 原核表达
Prokaryotic expression
pET32a-StTPR01660-F CTGATATCGGATCCGAATTCATGTTATTGAGAAGCTCATC 原核表达
Prokaryotic expression
pET32a-StTPR01660-R TGGTGGTGGTGGTGCTCGAGCTAAGAAGCAGCAGCCAAGG 原核表达
Prokaryotic expression
pET32a-StTPR22129-F CTGATATCGGATCCGAATTCATGTTGCTAAGGAGTTCTTC 原核表达
Prokaryotic expression
pET32a-StTPR22129-R TGGTGGTGGTGGTGCTCGAGTCAAACAGTTATAGAAGCCG 原核表达
Prokaryotic expression
pET32a-StTPR45174-F CTGATATCGGATCCGAATTCATGAGAACTGTCCTTTTCCG 原核表达
Prokaryotic expression
pET32a-StTPR45174-R TGGTGGTGGTGGTGCTCGAGCTAGTAAGCTGCAACCGTTG 原核表达
Prokaryotic expression
pGEX6p-1-StDUF842-F TCCAGGGGCCCCTGGGATCCATGGACGCAATAGCAGCAGC 原核表达
Prokaryotic expression
pGEX6p-1-StDUF842-R ATGCGGCCGCTCGAGTCGACTCAATTGTTGAAGCCGAGAG 原核表达
Prokaryotic expression
StDUF842-YFPN-F TGTTGATACATATGGGATCCATGGACGCAATAGCAGCAGC 双分子荧光互补BiFC
StDUF842-YFPN-R CCGAATTCACTAGTGTCGACATTGTTGAAGCCGAGAGAAG 双分子荧光互补BiFC
StTPR22129-YFPN-F TGTTGATACATATGGGATCCATGTTGCTAAGGAGTTCTTC 双分子荧光互补BiFC
StTPR22129-YFPN-R CCGAATTCACTAGTGTCGACAACAGTTATAGAAGCCGTGG 双分子荧光互补BiFC
actin-F CAGAAAGGACCTCTACGGTAACATT cDNA检测cDNA detection
actin-R TCTGTGGACGATGGACGGAC cDNA检测cDNA detection
attB1+StDUF842-F GGGGACAAGTTTGTACAAAAAAGCAGGCTCAATGGACGCAATAGCAGCAGC BP反应BP reaction
attB2+StDUF842-R GGGGACCACTTTGTACAAGAAAGCTGGGTATCAATTGTTGAAGCCGAGAG BP反应BP reaction
attB1+StTPR01660-F GGGGACAAGTTTGTACAAAAAAGCAGGCTCAATGTTATTGAGAAGCTCATC BP反应BP reaction
attB2+StTPR01660-R GGGGACCACTTTGTACAAGAAAGCTGGGTACTAAGAAGCAGCAGCCAAGG BP反应BP reaction
attB1+StTPR22129-F GGGGACAAGTTTGTACAAAAAAGCAGGCTCAATGTTGCTAAGGAGTTCTTC BP反应BP reaction
attB2+StTPR22129-R GGGGACCACTTTGTACAAGAAAGCTGGGTATCAAACAGTTATAGAAGCCG BP反应BP reaction
attB1+StTPR45174-F GGGGACAAGTTTGTACAAAAAAGCAGGCTCAATGAGAACTGTCCTTTTCCG BP反应BP reaction
attB2+StTPR45174-R GGGGACCACTTTGTACAAGAAAGCTGGGTACTAGTAAGCTGCAACCGTTG BP反应BP reaction

Fig. 1

Titer and inserted fragment detection of yeast cDNA Library A: titer and inserted fragment detection of the entry cDNA library by colony PCR; B: titer and inserted fragment detection of the expression cDNA library by colony PCR."

Table 1

Yeast library screening results by using StBAM9 or StBAM9-P as a bait"

饵蛋白
Bait protein		 测序转化子数
Number of sequencing
transformers		 潜在互作蛋白个数
Number of potential interaction proteins		 相同潜在互作蛋白个数
Number of proteins with the same potential interaction
StBAM9 68 29 12
StBAM9-P 118 75

Fig. 2

WEGO analysis of potential interacting proteins of StBAM9 or StBAM9-P Red and gray columns show the analysis of StBAM9 and StBAM9-P, respectively."

Table 2

Basic information of 12 candidate interacting proteins of StBAM9"

序号
Serial number		 基因名称
Gene name		 PGSC编号
PGSC number		 注释
Annotation		 CDS
(bp)
1 StDUF842 PGSC0003DMT400042117 含DUF842结构域的蛋白质
DUF842 domain containing protein		 450
2 StTPR01660 PGSC0003DMT400001660 含四肽重复结构域的蛋白
Tetratricopeptide repeat domain-containing protein		 951
3 StTPR22129 PGSC0003DMT400022129 含四肽重复结构域的蛋白
Tetratricopeptide repeat domain-containing protein		 1083
4 StTPR45174 PGSC0003DMT400045174 含四肽重复结构域的蛋白
Tetratricopeptide repeat domain-containing protein		 909
5 StSHSP12249 PGSC0003DMT400012249 线粒体小分子热休克蛋白
Mitochondrial small heat shock protein		 636
6 StSHSP78006 PGSC0003DMT400078006 17.6 kD I类热休克蛋白
17.6 kD class I heat shock protein		 465
7 StGAPDH PGSC0003DMT400044944 胞质甘油醛-3-磷酸脱氢酶
Glyceraldehyde-3-phosphate dehydrogenase, cytosolic		 1017
8 StAFTP4 PGSC0003DMT400044590 金属离子结合蛋白
Metal ion binding protein		 396
9 StSTPK61170 PGSC0003DMT400061170 渗透应激激活蛋白激酶
Osmotic stress-activated protein kinase		 1083
10 StSTPK63153 PGSC0003DMT400063153 丝氨酸/苏氨酸蛋白激酶
Ser/Thr protein kinase		 1515
11 StSTPK69936 PGSC0003DMT400069936 丝氨酸/苏氨酸蛋白激酶
NEK8Serine/threonine-protein kinase Nek8		 1425
12 StPP2C PGSC0003DMT400067098 蛋白磷酸酶
2CProtein phosphatase 2C		 1128

Fig. 3

Interaction validation of StBAM9 and 12 candidate interaction proteins by yeast two hybrid"

Fig. 4

In vitro interaction validation of StBAM9 with StTPR01660 and StTPR45174"

Fig. 5

StBAM9 interacts with StTPR01660 on starch granules Bar: 25 μm."

Fig. 6

StTPR01660 is located in the cytoplasm Bar: 25 μm."

Fig. 7

Validation of yeast two hybrid interaction between the TPR domain of StTPR01660 and StBAM9"

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