拟南芥IQM4互作蛋白的筛选和鉴定

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

摘要/Abstract

摘要：

拟南芥IQM4由AT2G26190编码,是1个含有IQ基序的Ca²⁺不依赖型钙调素结合蛋白,筛选和鉴定与IQM4互作的蛋白为进一步阐明IQM4所涉及的信号途径奠定基础。通过酵母双杂交筛选拟南芥幼苗cDNA文库,进而通过一对一的酵母双杂交试验和双分子荧光互补试验验证蛋白之间的互作关系。结果表明,获得15个与IQM4互作的候选蛋白;确定了CAT2和FBA1与IQM4蛋白存在互作关系,表明IQM4与CAT2及FBA1在植物细胞中存在相互作用。

关键词: IQM4互作蛋白, 拟南芥, 酵母双杂交, 双分子荧光互补

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

杨华杰, 周玉萍, 范甜, 吕天晓, 谢楚萍, 田长恩. 拟南芥IQM4互作蛋白的筛选和鉴定[J]. 生物技术通报, 2021, 37(11): 190-196.

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.

图/表 7

表1 本试验所用的引物序列

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

图1 IQM4编码序列的PCR扩增 M:5000 Marker;1和2:IQM4扩增条带

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

图2 IQM4蛋白自激活及毒性检测 A:pGBKT7-IQM4所表达的融合蛋白毒性的检测;B:IQM4自激活效应的检测

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

图3 CAT2及FBA1编码序列的PCR扩增

Fig. 3 PCR amplification of CAT2 and FBA1 coding sequences

图4 IQM4互作候选蛋白的酵母双验证 SD-TL:缺Trp和Leu培养基;SD-TLHA/X-α-gal:缺Trp、Leu、His和Ade/+X-α-Gal培养基。1:pGADT7-T/pGBKT7-53,阳性对照;2:pGADT7/pGBKT7,阴性对照;3-17分别为BD-IQM4/AD-CAT2、FBA1、GAPA-2、ATDI19、FBA2、FBA5、LHCA2、LHCB1.1、LHB1B2、LHCB3、RBCS3B、CRB、CPN60B、RBCS1A和GAPC

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

图5 BiFC载体构建验证图

Fig. 5 Verification of BiFC vector construction

图6 IQM4互作蛋白的双分子荧光互补试验

Fig. 6 Bimolecular fluorescence complementation assay of IQM4 interacting proteins

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