| Objective To gain an insight into the function of FXR1P and guess the relationship between FXR1P and the pathogenesis of fragile X syndrome, yeast two-hybrid system was used to screen proteins interacting with FXR1P.Methods (1) The FXR1 gene was subcloned into the plasmid pGBKT7 in yeast two hybrid system to construct bait plasmid pGBKT7- FXR1. The vector reconstructed was transformed into yeast strain AH109. The BD-Bait protein was examined for the reporter gene LacZ activation and toxicity to yeast strain. (2) The yeast containing bait plasmid pGBKT7- FXR1 was mated with yeast strain Y187 containing human fetal brain cDNA library plasmids. Diploid yeasts were plated on synthetic dropout nutrient medium. And 6-galactosidase filter assay was performed for screening positive clones. (3)The plasmids DNA in positive yeast colonies were isolated, transformed into E.coli Top10 to obtain AD-Library/Top10 transformants. AD-Library plasmids were extracted, digested with two restriction enzymes, and sorted. (4) AD-Library plasmids were transformed into yeast Y187, and then mated with yeast AH109 containing the bait plasmid pGBKT7- FXR1 to retest protein interactions. (5)The AD-Library plasmids in true positive colonies were sequenced and analyzed by bioinformatics techniques.Results (l)The bait plasmid pGBKT7- FXR1 was successfully constructed. And fusion protein had not LacZ activation and toxicity to yeast. (2) 77 positive colonies were obtained by screening the fetal brain cDNA library. (3)After being digested AD-Library plasmids in positive colonies were divided into 15 groups. (4)By means of retesting protein interactions in yeast, 10 colonies were confirmed to be true positive. (5)The sequencing results showed that 3 were right coding sequences, which were scaffold attachment factor B (SAFB), Estradiol 17 beta-dehydrogenase 1(17β-HSD I ) and Bcl-2-associated transcription factor l(BCLAFl). Conclusion (l)The bait plasmid pGBKT7- FXR1 without LacZ activation and...
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