Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (15): 3146-3157.doi: 10.3864/j.issn.0578-1752.2020.15.014

• HORTICULTURE • Previous Articles     Next Articles

Screening of Interacting Protein of Tomato SIVQ6 by GST Pull-Down

YUAN GuiBo(),MO ShuangRong,QIAN Ying,ZANG DongNan,YANG Fan,JIANG HongLiang,WU Yuan,DING HaiDong()   

  1. College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, Jiangsu
  • Received:2019-10-17 Accepted:2020-01-29 Online:2020-08-01 Published:2020-08-06
  • Contact: HaiDong DING E-mail:1076416981@qq.com;hdding@yzu.edu.cn

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Abstract:

【Objective】The VQ motif-containing (VQ) proteins are plant-specific proteins with a conserved “FxxhVQxhTG” amino acid sequence, which regulate plant growth and development. SlMPK1 plays an important role in the process of high temperature stress, but its downstream target proteins are poorly understood. Although yeast double hybridization (Y2H) showed that SlVQ6 protein could interact with SlMPK1, there was no further experimental evidence. Therefore, it is especially important to verify the interaction between SlMPK1 and SlVQ6 and to study the interaction network of SIVQ6. 【Method】First, pGEX-4T-1-PC-VQ6 plasmid was constructed. Through design of specific primers based on the SlVQ6 gene sequence, and using the plasmid containing the target gene SIVQ6 as a template to amplify the target gene sequence, the obtained target gene VQ6 was cloned between the restriction sites of BamHⅠ and NotⅠ of the vector pGEX-4T-1, and the pGEX-4T-1-PC-VQ6 was transferred into a TOP10 clone strain. The target protein PC-VQ6 was obtained by IPTG (isopropyl thiogalactoside) inducing expression and the expected GST-SlVQ6 fusion protein was obtained by affinity purification by GST column. Phosphorylation was performed in vitro to further determine whether SlVQ6 is the substrate of SlMPK1. GST-SlVQ6 was used as the bait protein to fix on GST Sepharose Beads and incubated with tomato leaf total protein, and then the incubated fusion material was eluated. The eluate was collected and verified by SDS-PAGE gel electrophoresis. LC-MS/MS was used to detect the candidate proteins that could interact with SlVQ6, and the screened SIVQ6 interacting proteins were used for bioinformatics analysis by GO, KEGG, and protein interaction networks. 【Result】The results showed that we successfully constructed the recombinant gene expression plasmid pGEX4T-1-PC-SlVQ6 and obtained the GST-SlVQ6 fusion protein with a GST tag, of which the molecular weight is about 54 kD. GST-SlVQ6 and His-SlMPK1 were subjected to phosphorylation experiments in vitro. SlVQ6 could be phosphorylated by SlMPK1, however the GST could not be phosphorylated by SlMPK1 as a negative control and there was no autophosphorylation phenomenon in SlVQ6, indicating that SlVQ6 has an interaction with SlMPK1, and SlVQ6 is a downstream substrate of SlMPK1. GST-SlVQ6 fusion protein was used as bait protein (GST as negative control), and the protein bound to SlVQ6 protein in tomato leaf tissue protein was screened by pull-down test. Through SDS-PAGE, liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification, and Mascot protein database search, 37 SlVQ6-bound proteins were identified, including protein kinase Receptor for Activated C Kinase 1B (RACK1B). Bioinformatics analysis of GO, KEGG, and protein interaction networks indicated that these proteins are involved in a variety of signaling pathways, and eight of the ribosomal proteins may be closely related to high temperature stress. 【Conclusion】This research shows that SlVQ6 is a substrate protein of SlMPK1 and 37 proteins may interact with SlVQ6. These proteins are closely related to the stress response and may play an important role in the high temperature tolerance of tomato plants.

Key words: tomato, SlVQ6, SlMPK1, pull-down, interaction protein

Fig. 1

Construction of fusion expression vector A: Double enzyme digestion of recombinant plasmid pGEX-4T-1-PC-SlVQ6, M: Marker; 1: Pre-digested plasmid; 2: Plasmid after digestion. B: The partial sequence alignment of pGEX-4T-1-PC-SlVQ6"

Fig. 2

Expression and purification of fusion protein A: SDS-PAGE of expression form analysis in Arctic Express, M: Marker; 1: Un-induced; 2: Induced; 3: Supernatant of induction with 0.5 mmol·L-1 IPTG; 4: Precipitate of induction with 0.5 mmol·L-1 IPTG; Arrow: GST-SlVQ6 protein. B: SDS-PAGE of fusion protein purification, M: Marker; 1: Un-purified; 2: Flow through; 3: Elution; Arrow: GST-SlVQ6 protein"

Fig. 3

In-gel analysis of phosphorylation of purified SlVQ6 by SlMPK1 A: His-tagged SlMPK1 and GST-tagged SlVQ6 were used in phosphorylation reactions with 32P labeling, GST was used as a negative control, and the autophosphorylation of GST-SlVQ6 was proved by adding GST-SlVQ6 alone; B: Coomassie blue staining in the below panel"

Fig. 4

GST pull down analysis of SlVQ6-interacting proteins A: The flow chart of GST pull down analysis of SlVQ6-interacting proteins. GST-SlVQ6 (or GST, a negative control) was added to GST sheperose 4B beads and crude protein extract of tomato leaves was added to the beads and incubated for overnight at 4℃. The samples were boiled and then separated by 12% SDS-PAGE gels. The bands were cut out, destained, dried, digested using trypsin, and the LC-MS/MS analysis was performed. SBP: Specific binding proteins; NSBP: Non-specific binding proteins. B: Thirty-seven of SlVQ6-specific binding proteins were identified"

Table 1

Thirty seven of SlVQ6-specific binding proteins identified by GST-pull down"

蛋白质ID
UniProt ID		 拟南芥ID
At ID		 基因IDa
Gene ID		 描述b
Description
R4KMW8 ATCG00490 二磷酸核酮糖羧化酶大链Ribulose bisphosphate carboxylase large chain
K4DC90 AT2G24200 Solyc12g010020 胞质氨肽酶Cytosol aminopeptidase
K4BQD5 AT5G58420 Solyc04g016350 40S核糖体蛋白S4 (RPS4D) 40S ribosomal protein S4 (RPS4D)
K4CAN7 AT2G20450 Solyc06g083820 60S核糖体蛋白l14（RPL14A） 60S ribosomal protein L14 (RPL14A)
K4ASC2 AT1G65930 Solyc01g005560 异柠檬酸脱氢酶/ NADP+异柠檬酸脱氢酶,推定 Isocitrate dehydrogenase / NADP+ isocitrate dehydrogenase, putative
K4CPX9 AT1G32990 Solyc08g083350 PRPL11;核糖体的结构组成 PRPL11; Structural constituent of ribosome
K4BWB5 AT5G14320 Solyc05g005880 核糖体蛋白S13,叶绿体（CS13） 30S ribosomal protein S13, chloroplast (CS13)
H1ZXA8 AT3G12580 热休克蛋白70亚型2 Heat shock protein 70 isoform 2
K4BEV0 AT3G04770 Solyc03g019780 RPSAb;核糖体的结构组成 RPSAb; Structural constituent of ribosome
K4CLA3 AT4G33010 Solyc08g065220 AtGLDP1（拟南芥黄曲霉毒素P-蛋白1）AtGLDP1 (Arabidopsis thaliana glycine decarboxylase P-protein 1)
K4BMD5 AT1G74070 Solyc03g119860 肽基脯氨酸顺反异构酶亲环素型家族蛋白 Eptidyl-prolyl cis-trans isomerase cyclophilin-type family protein
K4B1M5 AT5G49840 Solyc01g102990 ATP结合/ATP酶/核苷三磷酸酶/核苷酸结合
ATP binding/ATPase/nucleoside-triphosphatase/ nucleotide binding
K4DEU5 AT1G48630 Solyc12g040510 RACK1B_AT;核苷酸结合 RACK1B_AT; Nucleotide binding
P07369 AT1G29930 Solyc03g005780 CAB1,AB140,CAB140,LHCB1.3
K4D831 AT2G37270 Solyc11g042610 ATRPS5B;核糖体的结构组成ATRPS5B; Structural constituent of ribosome
K4CP59 AT4G11650 Solyc08g080590 ATOSM34|ATOSM34（渗调蛋白34）ATOSM34|ATOSM34 (osmotin 34)
K4CAM4 AT4G28730 Solyc06g083690 谷氨酸家族蛋白 Glutaredoxin family protein
K4C3Z3 AT3G62410 Solyc06g009630 CP12-2,CP12|CP12-2;蛋白结合 CP12-2, CP12|CP12-2; protein binding
K4BQ07 AT2G43560 Solyc04g015040 免疫亲和蛋白Immunophilin
K4ASQ6 AT5G06480 Solyc01g006900 MD-2相关脂质识别含域蛋白MD-2-related lipid recognition domain-containing protein
Q9XGI9 AT2G27450 Solyc11g068540 NLP1,ATNLP1,CPA;N-氨基甲酰腐胺酰胺酶NLP1, ATNLP1, CPA; N-carbamoylputrescine amidase
K4C3Z6 AT4G02080 Solyc06g009660 ASAR1,ATSARA1C,ATSAR2|ATSAR2;GTP结合 ASAR1, ATSARA1C, ATSAR2|ATSAR2; GTP binding
Q8LPU1 AT4G11650 Solyc08g080620 ATOSM34|ATOSM34（渗调蛋白34）ATOSM34|ATOSM34 (osmotin 34)
K4CMN4 AT2G35040 Solyc08g075160 AICARFT/IMPCHase双酶家族蛋白AICARFT/IMPCHase bienzyme family protein
K4D621 AT1G12050 Solyc11g012160 延胡索酰乙酰乙酸酶,推定 Fumarylacetoacetase, putative
K4B1B8 AT2G47170 Solyc01g100870 ARF1A1C;GTP结合/磷脂酶激活剂/蛋白结合ARF1A1C; GTP binding / phospholipase activator/ protein binding
K4BB24 AT5G66530 Solyc02g085100 Aldose 1-变旋酶家族蛋白 Aldose 1-epimerase family protein
K4CNE8 AT4G17040 Solyc08g077890 ATP依赖的Clp蛋白酶水解亚基,推定 ATP-dependent Clp protease proteolytic subunit, putative
Q9XG54 AT1G76690 Solyc10g086220 OPR2,ATOPR2|OPR2
K4BG20 AT3G49910 Solyc03g044000 60S核糖体蛋白L26（RPL26A）60S ribosomal protein L26 (RPL26A)
K4DA30 AT5G22300 Solyc11g068730 NIT4;3-氰基丙氨酸水合酶/氰基丙氨酸腈水解酶NIT4; 3-cyanoalanine hydratase/cyanoalanine nitrilase
K4C952 AT3G11710 Solyc06g073330 ATP结合/氨酰基-tRNA连接酶/赖氨酸-tRNA连接酶ATP binding/aminoacyl-tRNA ligase/lysine-tRNA ligase
K4D3M1 AT3G09630 Solyc10g084350 60S核糖体蛋白L4/L1（RPL4A）60S ribosomal protein L4/L1 (RPL4A)
K4CJ96 AT2G44050 Solyc08g015660 COS1;6,7-二甲基-8-核苷鲁嗪合酶COS1; 6,7-dimethyl-8-ribityl lumazine synthase
K4CSE1 AT3G25530 Solyc09g018790 GHBDH,ATGHBDH;3-羟丁酸脱氢酶GHBDH, ATGHBDH; 3-hydroxybutyrate dehydrogenase
K4C8R4 AT3G04120 Solyc06g071920 GAPC,GAPC-1,GAPC1|GAPC1
K4D3F2 AT3G52960 Solyc10g083650 2类过氧化物酶,推定Peroxiredoxin type 2, putative

Fig. 5

GO analysis of SlVQ6 interacting protein A, B: GO functional enrichment analysis of SlVQ6 interacting proteins (Biological process, Cellular component, Molecular function)"

Fig. 6

KEGG analysis of SlVQ6 interacting proteins A: KEGG signaling pathway function classification of SlVQ6 interacting proteins; B: Statistics of pathway enrichment of SlVQ6 interacting proteins by KEEG. Differentially expressed genes are displayed by scatter plots. The degree of KEEG enrichment is measured by the enrichment factor, q value, and the number of genes enriched in this pathway"

Fig. 7

Putative interaction network of SlVQ6 proteins in tomato Functional interacting network models were integrated using the STRING tool, and the confidence parameters were set at a 0.40 threshold. Blue node: Ribosomal protein; Green node: Nucleotide binding; Red node: Oxidoreductase activity"

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