Screening Of Dunaliella Salina Mbp Interaceting Protein By Yeast Two-hybrid System

Objective Since the MAR fragment was isolated, MAR and its function have been one of the hotspots in molecular biology. It has been known that MAR could increase transgene expression levels, but so far, only a few MAR-binding protein were isolated. In the previous studies, one MAR-binding protein (MBP) has been isolated from Dunaliella Salina, encoding541amino acids and1623bp in length, prokaryotic expression and functional studies confirmed that the protein could bind to MAR, which localize in the nucleus. The aim of this study was to find the proteins which could interact with MBP through screening of Dunaliella salina cDNA library, using the yeast two-hybrid approach, and explore the features of the MBP gene and its biological significance.Methods Dunaliella Salina total RNA was extracted, primers were designed according to MBP gene sequence of the GenBank database, the MBP gene was amplified by RT-PCR. MBP gene was inserted into the pMD18-T and pGBKT-7vector in turn to construct the yeast two-hybrid "bait" vector. After restriction enzyme digestion, sequencing, the resultant pGBKT-MBP vector was transformed into yeast strain Y187, then the toxicity and self-activation of the "bait" vector was detected. The library (AH109strains, containing library plasmids) was screened wtth Y187strain (containing "bait" plasmid), and then the positive clones were selected by nutrition selection and MEL1gene chromogenic reaction. The positive library plasmid was extracted, the true positive clones were confirmed using small scale yeast two-hybrid and bioinformatic analysis.Results Using cDNA as template,1623bp fragment of MBP gene was successfully amplified by PCR, the fragment was inserted into the pMD18-T and pGBKT-7vector. MBP gene is confirmed by restriction enzyme digestions and sequencing analysis. pGBKT-MBP vector was transformed into yeast strain Y187, the transformed strain could only grow on SD/-Trp plate with white colonies, not on SD/-Trp/-His, the SD/-Trp/-Ade plates, indicating that the "bait" plasmid was no self-activation. In the toxicity test, the OD600of the overnight culture was greater than0.8, indicating that the "bait" plasmid was non-toxic to the yeast strain. After screening the cDNA library of Dunaliella salina, an initial26clones was selected, after restreak the colonies on the X-a-Gal plate several times, final determined seven positive clones. Small scale yeast two-hybrid confirmed that these colonies could interact with MBP.Conclusion The cDNA library of Dunaliella salina was successful screened by Yeast two-hybrid system, some interacting protein such as photosystem I reaction center subunit VI was found, but still need further study...