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Screening Of Novel Proteins That Interact With G Protein Or CaM Using Yeast Two-hybrid System

Posted on:2004-05-09Degree:DoctorType:Dissertation
Country:ChinaCandidate:L X SongFull Text:PDF
GTID:1100360095957690Subject:Cell biology
Abstract/Summary:PDF Full Text Request
The yeast two-hybrid system is an effective method for the study of protein-protein interaction in recent years. It is widely used in the study of life science because it provides a genetic means of identifying proteins that physically interact in vivo. In plant, many important signal molecules were isolated by this system. We tried to establish the yeast two-hybrid technique due to its importance in the field of signal transduction.G protein and calmodulin are two important signal molecules, they implicated in many cellular processes and have essential physiological functions in plant. In order to study the upstream and downstream components in G protein and calmodulin signal pathways, a yeast two-hybrid system was used to screen interaction partners of these two proteins. This work provides a good foundation for the establishment of yeast two-hybrid system in our lab.We firstly used G protein a subunit as bait. The GPA1 gene was obtained via PCR amplification and was cloned in the two-hybrid DNA binding domain vector pGBKT7 The combinant plasmid was designated as pGBKT7-Ga. β-galactosidase assay indicatedthat Got did not have the property of self-activation. After pGBKT7-Ga was transformed to yeast pJ69-4A, we transformed Arabidopsis vegetative tissue two-hybrid cDNA library plasmids to yeast pJ69-4A containing pGBKT7-Ga. Four positive clones were obtained by the selection of autotrophic phenotype and β-galactosidase assay. Sequencing and Genbank blasting demonstrated that these four had the same sequences and encoded part of Arabidopsis choloroplast carbonic anhydrase (CA). The coding region contained 251 amino acid, from N terminal amino acid 86 to C terminus. We cloned full-length cDNA of CA coding region via PCR amplification, but a negative result was obtained when the interaction of full-length CA and Ga was tested in yeast two-hybrid system. Then we cloned the mature CA fragment and the same region of the selected CA fragment from library plasmid to vector pGADT7, but neither did they interact with Ga in the assay of two-hybrid system.Then we used Arabidopsis CaM2 isoform as bait to screen the two-hybrid library. The combinant bait plasmid pGBKT7-ACaM2 was constructed. We obtained two positive clones after screening. Sequencing and blasting results showed that the two clones had the same sequences and encoded part of KED like protein in Arabidopsis. The coding region was from N terminal amino acid 240 to C terminus. We tested the interaction of these two proteins using in vitro transcription and translation system and coimmunoprecipitation. It was shown that the two proteins could interact in vitro and the interaction was Ca2+ dependent. Further we cloned full-length cDNA of the KED like protein coding region via PCR amplification and confirmed its interaction with ACaM2 in yeast two-hybrid system. We also predicted the CaM binding domain of KED like protein and showed that the CaMBD lies in its C terminus.
Keywords/Search Tags:Arabidopsis, yeast two-hybrid system, G protein, CaM, coimmunoprecipitation
PDF Full Text Request
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