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作物学报 ›› 2023, Vol. 49 ›› Issue (10): 2643-2653.doi: 10.3724/SP.J.1006.2023.34009

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

马铃薯淀粉酶StBAM9互作蛋白的鉴定及其互作机制分析

杜鹃1(), 彭晓君1,2, 侯娟1,3, 刘腾飞1,4, 刘增1, 宋波涛1()   

  1. 1果蔬园艺作物种质创新与利用全国重点实验室 / 农业农村部马铃薯生物学与生物技术重点实验室 / 华中农业大学, 湖北武汉 430070
    2阿克陶县巴仁乡人民政府, 新疆克孜勒苏柯尔克孜自治州 845555
    3河南农业大学园艺学院, 河南郑州 450002
    4山东农业大学食品科学与工程学院, 山东泰安 271018
  • 收稿日期:2023-01-13 接受日期:2023-04-17 出版日期:2023-10-12 网络出版日期:2023-04-26
  • 通讯作者: 宋波涛, E-mail: songbotao@mail.hzau.edu.cn
  • 作者简介:E-mail: juandu@mail.hzau.edu.cn
  • 基金资助:
    贵州省科技支撑计划(农业领域重点项目)生物育种先导性项目(黔科合支撑[2022]重点030-3);国家自然科学基金项目(31671749)

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 Published:2023-10-12 Published online: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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摘要：

本实验室前期研究表明β-淀粉酶9 (StBAM9)在马铃薯抗低温糖化中具有重要作用, 但其并无β-淀粉酶活性。为了研究StBAM9在马铃薯抗低温糖化中的功能机制, 我们构建了低温贮藏后块茎cDNA酵母双杂交文库, 并以StBAM9蛋白为诱饵, 对其进行了互作蛋白的筛选和分析, 结果显示分别以全长StBAM9和截去转运肽的StBAM9为诱饵筛选到的候选互作蛋白中有12个是共有的。酵母双杂交结果显示有4个蛋白(StDUF842、StTPR01660、StTPR22129和StTPR45174)与StBAM9互作。进一步通过谷胱甘肽-S-转移酶融合蛋白沉降技术验证表明, 其中的2个蛋白StTPR01660和StTPR4517与StBAM9互作。双分子荧光互补结果显示只有StTPR01660与StBAM9互作于淀粉粒上, 而StTPR01660自身定位于细胞质。因此, 我们推测StBAM9可能通过从细胞质中招募StTPR01660到淀粉粒上发挥淀粉降解的功能。

关键词: 马铃薯, 低温糖化, 淀粉降解, β-淀粉酶, 蛋白互作

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

附表1

本研究所用的引物"

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

图1

酵母cDNA文库滴度检测及其插入片段分析 A: 次级cDNA文库滴度及插入片段菌落PCR检测; B: 表达cDNA文库滴度及插入片段菌落PCR检测。"

表1

StBAM9和StBAM9-P的酵母文库筛选结果"

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

图2

StBAM9和StBAM9-P潜在互作蛋白的WEGO分析 红色和灰色柱子分别表示StBAM9和StBAM9-P的分析结果。"

表2

StBAM9的12个候选互作蛋白的基本信息"

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

图3

StBAM9和12个候选互作蛋白的酵母双杂交互作验证结果"

图4

StBAM9分别与StTPR01660和StTPR45174的体外互作验证结果"

图5

StBAM9与StTPR01660互作于淀粉粒 标尺: 25 μm。"

图6

StTPR01660定位于细胞质 Autofluorescence, 叶绿体自发荧光; 标尺: 25 μm。"

图7

StTPR01660的TPR结构域与StBAM9的酵母双杂互作验证结果"

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