Screening and Identification of IQM4-Interacting Proteins in Arabidopsis thaliana

doi:10.13560/j.cnki.biotech.bull.1985.2021-0235

Abstract

Abstract:

The IQM4 protein in Arabidopsis thaliana encoded by AT2G26190 is a Ca²⁺-independent calmodulin-binding protein with an IQ motif. Screening and identifying the IQM4-interacting protein would lay a foundation for further understanding the signal pathways involved by IQM4. Yeast two-hybrid experiment was used to screen A. thaliana seedling cDNA library,one-to-one yeast two-hybrid system and bimolecular fluorescence complementary experiment were adapted to validate the interactions among proteins. The results showed that the 15 candidates of IQM4-interacting proteins were obtained,and the interaction of IQM4 with CAT2 or FBA1 was confirmed,indicating that IQM4 interacted with CAT2 or FBA1 in plant cells.

Key words: IQM4-interacting protein, Arabidopsis thaliana, yeast two-hybrid, bimolecular fluorescence complementation

YANG Hua-jie, ZHOU Yu-ping, FAN Tian, LV Tian-xiao, XIE Chu-ping, TIAN Chang-en. Screening and Identification of IQM4-Interacting Proteins in Arabidopsis thaliana[J]. Biotechnology Bulletin, 2021, 37(11): 190-196.

Figures/Tables 7

Table 1 Primer sequences used in this experiment

基因Gene	引物名称Primer name	引物序列Primer sequence（5'-3'）
IQM4	IQM4-F	TCAGAGGAGGACCTGCATATGATGGCTTCGGCTACTTTCTCTG
	IQM4-R	CTAGTTATGCGGCCGCTGCAGCTAACAGTTTACTTGAACTCTAGGGCTT
	BF-IQM4—F	CGAGCTCAAGCTTCGAATTCGATGGGTCTTTCTCTTTCTTTGCTC
	BF-IQM4-75-R	GTACCGTCGACTGCAGAATTCCTAACAGTTTACTTGAACTCTAGGGCTT
CAT2	CAT2-F	GCCATGGAGGCCAGTGAATTCATGGATCCTTACAAGTATCGTCCAG
	CAT2-R	ATGCCCACCCGGGTGGAATTCTTAGATGCTTGGTCTCACGTTCA
	BF-CAT2-F	TCGAGCTCAAGCTTCGAATTCGATGGATCCTTACAAGTATCGTCCAG
	BF-CAT2-R	GTACCGTCGACTGCAGAATTCTTAGATGCTTGGTCTCACGTTCA
FBA1	FBA1-F	GCCATGGAGGCCAGTGAATTCATGGCGTCAAGCACTGCG
	FBA1-R	ATGCCCACCCGGGTGGAATTCTTAGTAGGTGTAGCCTTTTACAAACATAC
	BF-FBA1-F	TCGAGCTCAAGCTTCGAATTCGATGGCGTCAAGCACTGCG
	BF-FBA1-R	GTACCGTCGACTGCAGAATTCTTAGTAGGTGTAGCCTTTTACAAACATA

Fig. 1 PCR amplification of IQM4 coding sequence M:5000 Marker. 1 and 2:Amplified bands of IQM4

Fig. 2 Self-activation and toxicity detection of IQM4 protein A:Toxicity detection of the fusion protein expressed by pGBKT7-IQM4.B:Detec-tion of self-activation effect of IQM4

Fig. 3 PCR amplification of CAT2 and FBA1 coding sequences

Fig. 4 Validation of IQM4 interacting candidate proteins by yeast two hybrid system SD-TL:-Trp-Leu medium. SD-TLHA/X-α-Gal:-Trp-Leu-His-Ade /+X-α-Gal medium. 1:pGADT7-T/pGBKT7-53,positive control. 2:pGADT7/pGBKT7,negative control. 3-17 is BD-IQM4/AD-CAT2,FBA1,GAPA-2,ATDI19,FBA2,FBA5,LHCA2,LHCB1.1,LHB1B2,LHCB3,RBCS3B,CRB,CPN60B,RBCS1A,and GAPC,respectively

Fig. 5 Verification of BiFC vector construction

Fig. 6 Bimolecular fluorescence complementation assay of IQM4 interacting proteins

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