Isolation And Identification Of Interactive Protein Of α Subunit GPA1of G Protein From Arabidopsis

Heterotrimeric G protein (including G protein α, β, γ subunits) which is involved in lotsof signaling pathways can receive extracellular signals via G-protein coupled receptor (GPCR)and transmit it to downstream effectors by three subunits of G protein. So far, little has beenreported about the downstream effectors and signaling pathway related with G-proteincomplexes. Therefore, looking for more novel G protein effectors can contribute to elucidatesignaling pathways related with G protein complex. In this research, we constructed anArabidopsis normalied cDNA library and used G protein α subunit (GPA1) as a bait to screeninteraction proteins in Arabidopsis by the split-ubiquitin system (hunter system), whichprovides a theoretical basis for elucidating signaling pathways of GPA1. In this research, weobtained the following experimental results:1. We constructed a Arabidopsis normalied cDNA library by using switching mechanismat5’-end of RNA transcript (SMART) technique and duplex-specific nuclease (DSN)normalization methods. The storage capacity of the library is1.079×106clones.2.100proteins were acquired by screening the library via split-ubiquitin system.61proteins of them have one transmembrane domains, and26proteins of them have threetransmembrane domains, which proved that split-ubiquitin system can be used to screenmembrane proteins interacting with GPA1.3. We used GPA1as a bait to screen the library by split-ubiquitin system and AtBCB, aGPA1-interacting protein, was identified as a copper ion binding protein. The interactionbetween GPA1and AtBCB occurs in the cell membrane which was verified through BiFC(bimolecular fluorescence complementation).4. The expression pattern analysis showed that GPA1and AtBCB were induced byaluminium stress. Under treated with100μmol/L Al3+, the content of MDA in gpa1-4mutantwas dramatically lower than that in WT (P<0.05), on the contrary, the content of MDA in bcbmutant was higher than WT (P<0.01).5. Furthermore, the expression pattern of genes response to aluminum toxicity, such asALMT1, ALS1and ALS3was analyzed by real-time PCR. The results showed that whetherunder aluminum stress or not, the expression of ALS1and ALS3had no significant differencein mutants and WT. However, under100μmol/L Al3+condition, the expression level of ALMT1in gpa1-4was higher than that in WT, but the expression level of ALMT1in bcb waslower compared with WT.In short, the interaction proteins from the100proteins interacting with GPA1were,GPA1and AtBCB directly interacted with each other in the cell membrane and regulated theexpression of ALMT1together for the response of aluminum stress in plants. In the process ofresistance to aluminum stress in plants, GPA1play negative roles and AtBCB has positiveeffects.