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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front Agric Chin    2011, Vol. 5 Issue (1) : 64-71    https://doi.org/10.1007/s11703-011-1063-9
RESEARCH ARTICLE
The physical interaction between LdPLCs and Arabidopsis G beta in a yeast two-hybrid system
Jinglei SUN1, Xiuhua LIU2, Yanyun PAN1()
1. College of Life Science, Agricultural University of Hebei, Baoding 071001, China; 2. College of Basic Medical, Hebei University, Baoding 071002, China
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Abstract

Phosphoinositide-specific phospholipase C plays pivotal roles in a host of physiologic processes in both animals and plants. Animal PI-PLC is regulated by heterotrimeric G-protein. Plant PI-PLCs are structurally close to the mammalian PI-PLC-ζ isoform, and it is not testified what regulated this isoform enzyme. In this paper, two isoform genes of LdPLC (Pan, 2005) and three subunits of heterotrimeric G-protein in BoldItalic were amplified and recombinated with plasmids of a yeast two-hybrid system. Using this system, we provided the evidence that LdPLC1 and Gβ subunit could be able to interact with each other. This result indicated that LdPLC1 might be regulated by G-protein.

Keywords phosphoinositide-specific phospholipase C (PI-PLC)      heterotrimeric G-protein      yeast two-hybrid     
Corresponding Author(s): PAN Yanyun,Email:pyycell@163.com   
Issue Date: 05 March 2011
 Cite this article:   
Jinglei SUN,Xiuhua LIU,Yanyun PAN. The physical interaction between LdPLCs and Arabidopsis G beta in a yeast two-hybrid system[J]. Front Agric Chin, 2011, 5(1): 64-71.
 URL:  
https://academic.hep.com.cn/fag/EN/10.1007/s11703-011-1063-9
https://academic.hep.com.cn/fag/EN/Y2011/V5/I1/64
name of ORFsgeneprimerprimersreferences
GPA1forward primer5′ ggaattccatatgggcttactctgcagtag 3′Ma et al., 1990
reverse primer5′ cggaattctcataaaaggccagcctccag 3′
AGB1forward primer5′ ggaattccatatgtctgtctccgagctc 3′Weiss et al., 1993
reverse primer5′ cgggatcctcaaatcactctcctgtgtc 3′
AGG1forward primer5′ggaattccatatggaagcgggtagctcc 3′Mason and Botela, 2001
reverse primer5′ cgggatcctcaaagaatggagcagcc 3′
LdPLC1LdPLC1forward primer5′ ggaattccatatgaatgccccgctgtcacac 3′Pan et al., 2005
reverse primer5′ cgggatccctatatatatacatagggattacaaactatc 3′
LdPLC2LdPLC2forward primer5′ ggaattccatatgctgagcggattgtcgatcag 3′
reverse primer5′ cgggatcctcactcaatttctacagaactaaagc 3′
Tab.1  PCR primer sets for the amplification of each ORF
name of the co–transformed yeast strainrecombined plasmid of pGBKT7recombined plasmid of pGADT7
αβpGBKT7-GαpGADT7-Gβ
αγpGBKT7-GαpGADT7-Gγ
βγpGBKT7-GβpGADT7-Gγ
α0pGBKT7-GαpGADT7
β0pGBKT7-GβpGADT7
γ0pGBKT7-GγpGADT7
αL1pGBKT7-GαpGADT7-LdPLC1
βL1pGBKT7-GβpGADT7-LdPLC1
γL1pGBKT7-GγpGADT7-LdPLC1
αL2pGBKT7-GαpGADT7-LdPLC2
βL2pGBKT7-GβpGADT7-LdPLC2
γL2pGBKT7-GγpGADT7-LdPLC2
Tab.2  Name of the co–transformed yeast strain and recombine plasmid
Fig.1  PCR amplification of G-protein α/β/γ subunits and restriction analysis of the recombined plasmid
Note: M1, M3 and M4 represent PCR markers. M2 represents λDNA/dIII markers. 1-10 represent Gα PCR products, pGBKT7/I+ , pGBKT7- Gα/I+ RI, GβPCR products, Gγ PCR products, pGADT7-Gβ/ I+ H, pGBKT7-Gβ/ I+ HI, pGADT7-Gγ/ I+ H, pGBKT7-Gγ/ I+ H and pGBKT7-Gα/ I+ RI, respectively.
Fig.2  Restriction analysis of plasmids recombined with LdPLCs and pGADT7
Note: M1 and M2 are PCR markers. M3 is λ DNA/dIII marker.1-4 are LdPLC1 PCR products, LdPLC2 PCR products, pGADT7-LdPLC1 / I+ H and pGADT7-LdPLC2/ I+ HI, respectively.
name of the co–transformed yeast strainplasmid combinationgrowth on SD/-Trp-Leu mediumgrowth on SD/-Trp-Leu-His-Ade mediumx-gal filter analysis
α0pGBKT7-Gα + pGADT7+--
β0pGBKT7-Gβ + pGADT7+--
γ0pGBKT7-Gγ + pGADT7+--
Tab.3  Autonomous activation analysis of three G-protein subunits
Fig.3  Autonomous activation assay of G-protein α, β and γ subunits
Note: x-gal filter results of α0, β0 and γ0 from SD/-Trp-Leu medium are negative and pcl1 is positive control.
name of the co–transformed yeast strainplasmid combinationgrowth on SD/-Trp-Leu mediumgrowth on SD/-Trp-Leu-His-Ade mediumx-gal filter analysis
αβpGBKT7-Gα + pGADT7-Gβ++-
αγpGBKT7-Gα + Pgadt7-Gγ+--
βγpGBKT7-Gβ + pGADT7-Gγ+++
γβpGBKT7-Gγ + pGADT7-Gβ+++
Tab.4  The interaction analysis between the Gα/β/γ subunits
Fig.4  The interaction analysis of G-protein α/β/γ subunits
Note: A represents growth on SD medium (SD/-Trp-Leu-His-Ade) and B represents x-gal filter analysis.
name of yeast stainplasmid recombinationgrowth on SD/-Trp-Leu mediumgrowth on SD/-Trp-Leu-His-Ade mediumx-gal filter analysis
ααL2pGADT7-Gα+pGBKT7-LdPLC2+--
ααL1pGADT7-Gα +pGBKT7-LdPLC1+--
ββL2pGADT7-Gβ+ pGBKT7-LdPLC2++-
ββL1pGADT7-G+pGBKT7-LdPLC1+++
γγL2pGADT7-G+pGBKT7-LdPLC2+--
γγL1pGADT7-G+pGBKT7-LdPLC1+--
Tab.5  The interaction analysis of Gα/Gβ/Gγ subunits with LdPLCs
Fig.5  The growth of the co-transformated yeast clones on SD/-Trp-Leu-His-Ade medium (a) and their β-gal assay (b)
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