Construction and Expression Analysis of Eukaryotic Fusion Expression Vector Carrying Flag Tag of Ash2l Gene in Chicken (Gallus gallus)
ZHANG Chen, ZUO Qi-Sheng, ZOU Yi-Chen, ZHAO Juan-Juan, ZHANG Ya-Ni, LI Bi-Chun*
Institutes of Agricultural Science and Technology Development/Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China/College of Animal Science and Technology, Yangzhou University Yangzhou, 225009, China
Abstract：Absent, small, or homeotic-like (ASH2L) is an important histone methylation modification enzyme involved in a variety of cell development. However, its function and molecular mechanism in the process of male germ cell development are not clear. The purpose of this study was to study the expression of Ash2l in the formation of chicken (Gallus gallus) male reproductive stem cells and to construct Ash2l eukaryotic fusion expression vector NCBI conserved domain analysis, UniPort, TMHMM Serverv.2.0 and SignalP online websites were used to analyze the conserved domain, hydrophilic and hydrophobic domain, transmembrane domain and signal peptide of ASH2L. qRT-PCR was used to detect the expression of Ash2l in different cells (embryonic stem cells (ESC), primordial germ cell (PGC) and spermatogonial stem cell (SSC)) and tissues of 18.5 d chicken embryo. The recombinant vector pcDNA3.1-Ash2l-Flag was identified by double restriction endonuclease digestion and sequencing. The expression of recombinant protein in PGC was detected by qRT-PCR and Western blot.The binding of recombinant protein and interaction protein was verified by Co-immunoprecipitation (Co-IP). The results showed that ASH2L protein contained 2 conserved domains, SPIa and ryanodine receptor (SPRY) and plant homeodomain (PHD) had histone methylase characteristic domain, and had high hydrophilicity, no transmembrane domain and no signal peptide expression. The results of qRT-PCR showed that the expression of Ash2l in testis was the highest, which was significantly higher than that in other tissues (P<0.01), and that in PGC was significantly higher than that in other cells (P<0.01). The results of double restriction endonuclease digestion showed that Ash2l was correctly inserted into pcDNA3.1 vector without frameshift and mutation. qRT-PCR showed that the recombinant vector in PGC could overexpress Ash2l gene, and Western blot indicated that the recombinant protein was successfully expressed in PGC. Co-IP results showed that the recombinant protein could bind to the interacting proteins Dpy-30 histone methyltransferase complex regulatory subunit (DPY30) and WD repeat containing protein 5 (WDR5) to form a complex. These results provide a reference basis and technical means for the study of the function and mechanism of Ash2l in the formation of male germ cells in chickens.
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