Construction of Chicken (Gallus gallus) Prox1 Gene Recombinant Eukaryotic Expression Vector and Analysis of Its Expression Characteristics
YUAN Chao1,2, GAO Hong-Bo1,2, ZHOU Lei1,2, HAN Yi-Fan1,2, HU Yan3, DUAN Zhi-Qiang1,2,*
1 Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountains Region of Ministry of Education/Key Laboratory of Animal Genetics, Breeding and Reproduction in Guizhou, Guizhou University, Guiyang 550025, China;
2 College of Animal Sciences, Guizhou University, Guiyang 550025, China;
3 Agricultural and Rural Bureau of Wudang District, Guiyang 550000, China
Abstract：Prox1 (prospero-related homeobox protein 1) protein is a transcription factor that plays a key regulatory role in embryonic development. In order to investigate the subcellular localization of chicken (Gallus gallus) Prox1 and its gene expression pattern in the growth and development of different chicken tissues, the recombinant eukaryotic expression vector pEGFP-C1-Prox1 was constructed based on the pCR2.1-Prox1 cloning vector. The expression and subcellular localization of the recombinant protein were identified by Western blot and fluorescence observation in plasmid-transfected human (Homo sapien) embryonic kidney cells (HEK-293T). In addition, the expression of Prox1 gene in different chicken tissues was detected by qRT-PCR at embryo 14 d (E14 d), 0-day-old (0 d), 7-day-old (7 d), and 14-day-old (14 d). The results showed that pEGFP-C1-Prox1 was successfully constructed and the recombinant protein EGFP-Prox1 was correctly expressed and localized mainly in the nucleus. The qRT-PCR results showed that Prox1 gene expression was highest in the stomach at E14 d and 7 d, but highest in the lung at 0 d and 14 d. The expression pattern showed that Prox1 expression was stable in the eyeball and cerebrum from E14 d to 14 d, but increased in the heart until 7 d. Prox1 expression was on the rise in liver and lung, which showed the lowest expression at E14 d and the highest expression at 14 d. In stomach and leg muscle, Prox1 expression was highest at E14 d, lowest at 0 d, and higher at 7 d, which showed a trend of W pattern. The expression of Prox1 in the chest muscle gradually increased from E14 d to 7 d, and then decreased from 7 d to 14 d. This study provided a reference for further studying the regulation mechanism of Prox1 gene in the growth and development of different chicken tissues.
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