Screening of candidate molecules interacting with protein kinase Pkmyt1 using yeast two hybrid technique

LIU Chao MENG Zhi-chao LUAN Zhi-dong REN Li-li LIU Yi-meng XIAO Jian-ying*

doi:10.16098/j.issn.0529-1356.2017.01.009

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Acta Anatomica Sinica ›› 2017, Vol. 48 ›› Issue (1) : 48-53. DOI: 10.16098/j.issn.0529-1356.2017.01.009

Cell and Molecules Biology

Screening of candidate molecules interacting with protein kinase Pkmyt1 using yeast two hybrid technique

LIU Chao¹MENG Zhi-chao² LUAN Zhi-dong¹REN Li-li³ LIU Yi-meng¹XIAO Jian-ying^4*

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Abstract

Objective Protein kinase Pkmyt1 negatively regulates the mitosis of mouse 1-cell stage fertilized eggs, but the specific mechanism is not clear. Therefore, the yeast two hybrid system was used to screen the candidate proteins interacting with Pkmyt1 from human ovary cDNA library in order to provide a new clue for the study of Pkmyt1 regulating the early development of mouse fertilized eggs. Methods Mice ovarian tissue cDNAs was used as a template to build pGBKT7-Pkmyt1 bait plasmid, and pGBKT7-Pkmyt1 was transformed into yeast competent cells at SD/Trp(SDO), SD/Trp/X-α-Gal(SDO/X) and SD/Trp/X-α-Gal/AbA plates(SDO/X/A) medium to detect the toxicity and self-activation ability of yeast and detect its expression in yeast using Western blotting method. The pGBKT7-Pkmyt1 containing yeast cells was fused with human ovary cDNA library, followed by screening positive clones by PCR, plasmid extraction, DNA sequencing and detection the yeast self-activated ability again, the use of bioinformatics analysis of selected proteins and embryo development. Results The enzyme digestion analysis and Blast analysis showed that the pGBKT7-Pkmyt1 plasmid was successfully constructed. When the plasmid was transferred into golden Y2H, the SDO medium was evenly grown, and no clones were grown on the SDO/X/A plate, and the protein was detected by Western blotting to detect the expression of Pkmyt1 in the yeast cells. After PCR validation, the positive clones were inserted into fragments. 182 proteins which interact with Pkmyt1 were obtained by preliminary screening, and 46 proteins were further verified by the recovery experiment with Pkmyt1. Conclusion Totally 46 proteins were found through the selection of the interaction between Pkmyt1 and they regulated in mouse oocyte maturation and early development of fertilized eggs.

Key words

Pkmyt1 / Two hybrid system / Protein-protein interaction / Western blotting / Mouse

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LIU Chao MENG Zhi-chao LUAN Zhi-dong REN Li-li LIU Yi-meng XIAO Jian-ying. Screening of candidate molecules interacting with protein kinase Pkmyt1 using yeast two hybrid technique[J]. Acta Anatomica Sinica. 2017, 48(1): 48-53 https://doi.org/10.16098/j.issn.0529-1356.2017.01.009

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