Subcellular Localization Of Chicken Prox1 Gene And Its Expression Analysis In Different Tissue Development

Prox1?Prospero-related homeobox protein 1,Prox1?is an important member of the homeo-box transcription factor family,which plays crucial roles in the development of nerves,liver,heart,and eyes in the vertebrate embryonic period.In addition,the expression and function changes of Prox1 gene are also associated with a variety of human cancers.Prox1protein contains the homeobox domain and the homologous domain,which can jointly bind DNA to regulate the transcription of other genes.At present,most of studies focused on the Prox1 gene mainly concentrate in mice and humans,and also in fruit flies,tadpoles,xenopus,goldfish,and zebrafish.Although Prox1 c DNAs of human,mouse,xenopus,goldfish,and zebrafish have been cloned and analyzed,the cloning,subcellular localization,and tissue expression of chicken Prox1 genes during chicken tissue development are unknown.In this study,the complete CDS region of chicken Prox1 gene was cloned and analyzed by bioinformatics analysis.In addition,the subcellular localization of chicken Prox1 protein was studied,and the nuclear localization signal?NLS?determining the nuclear localization of chicken Prox1 protein was characterized.Moreover,q RT-PCR was used to detect the m RNA expression level of Prox1 gene in the periods of chicken E14 d,0 d,7 d and 14 d,and its expression pattern during the development of different chicken embryo tissues was analyzed.1.The cloning and bioinformatics analysis of chicken Prox1 geneAccording to the published chicken Prox1 gene sequence?NM?001005616.1?in Gen Bank,specific primers targeted Prox1 gene CDS region were designed and synthesized.The total RNA reverse transcription product extracted from chicken eyes was used as a template to amplify the CDS region of chicken Prox1 gene by PCR.Physicochemical properties,secondary structure,tertiary structure,subcellular localization,interaction proteins,protein domains and phylogenetic trees were analyzed.The results showed that the chicken Prox1 gene CDS region was successfully amplified from chicken eyes.Sequencing results showed that the CDS region of chicken Prox1 gene was 2211 bp in length,encoding a total of 736 amino acids,with a theoretical p I of 6.62,a relative molecular mass of approximately 82.86 ku,and a molecular formula of C₃₆₀₁H₅₆₇₁N₁₀₄₉O₁₁₃₆S₃₂.Sequence analysis showed that the secondary structure of chicken Prox1 protein was mainly composed of random coils?53.67%?and alpha helix?39.40%?;chicken Prox1 protein was mainly localized in the nucleus,and the cellular proteins interacting with Prox1 protein were LYVE1,PAX6,KDR,VEGFC and FLT4.Nucleotide homology and genetic evolution analysis found that chicken Prox1 gene had the highest nucleotide homology?97.8%?and the closest relationship with quail.The above research provides a basis for the next step in the identification of chicken Prox1 protein nuclear localization signals and tissue expression characteristics.2.The prediction and identification of nuclear localization signal determining the nuclear localization of chicken Prox1 proteinBased on the previously constructed p CR2.1-Prox1 cloning vector,the recombinant eukaryotic expression vector p EGFP-Prox1 was constructed and transfected into HEK-293T cells.The expression of the recombinant protein was detected by Western blotting,and the recombinant protein was observed using an inverted fluorescence microscope.The online software was used to predict putative NLS?putative NLS,p NLS?in chicken Prox1 protein,and the recombinant eukaryotic expression vector of p NLS deletants of chicken Prox1 protein was constructed.After the transfection of plasmids into cells,the NLS was determined by observing the subcellular localization of recombinant protein of the Prox1 protein deletants.At the same time,the identified chicken Prox1 protein NLS was subjected to conservative analysis,and its nuclear import ability of foreign proteins was tested.In addition,the basic amino acid sites in NLS were mutated to identify the key basic amino acid sites in chicken Prox1 protein NLS.The results showed that the recombinant eukaryotic expression vector p EGFP-Prox1 of chicken Prox1 gene was successfully constructed,and the recombinant protein EGFP-Prox1was correctly expressed in plasmid-transfected cells.Fluorescence observation showed that the EGFP-Prox1 recombinant protein was mainly localized in the nucleus.The NLS prediction results showed that there were six p NLS in chicken Prox1 protein.The subcellular localization analysis of the recombinant protein of the p NLS deletants found that p NLS1(¹⁵KRRR¹⁸)and p NLS2(¹⁶³RAKRAR¹⁶⁸)jointly determine the nuclear localization of chicken Prox1 protein.In addition,the chicken Prox1 protein NLS1 and NLS2 motifs were conserved among other avians,humans,and different mammals,and had the similar nuclear import capacity as the SV40 large T antigen NLS.Moreover,the results of basic amino acid point mutation experiments confirmed that K15,R16 and R18,K165,R166 and R168 were the key basic amino acid sites in chicken Prox1 protein NLS1 and NLS2,respectively.The above research results indicate that two highly conserved NLS1(¹⁵KRRR¹⁸)and NLS2(¹⁶³RAKRAR¹⁶⁸)jointly determine the nuclear location of chicken Prox1 protein.3.The expression characteristics analysis of chicken Prox1 gene in chicken tissues at different developmental stagesThe eyes,brains,heart,liver,lungs,stomach,pectoral and leg muscles of chickens were collected at four time points?14 days?E14 d?,0 days?0 d?,7 days?7 d?,and 14 days?14 d??.The total RNA of these tissues was extracted and reversely transcribed into c DNA.The primers for q RT-PCR were designed to detect the expression of Prox1 gene in different chicken tissues.The results showed that the Prox1 gene was highest expressed in the stomach of chickens E14d and 7 d;the highest was expressed in the lungs at 0 d and 14 d.The expression rule shows that Prox1 is stable in the eyes and brain;the expression is stable before 7 d in the heart,and it starts to rise after 7 d;the expression in liver and lung is the lowest at E14 d,the highest expression is at 14 d,and the overall increase The expression was highest in gastric and leg muscles at E14 d,the lowest expression at 0 d,and higher expression at 7 d,showing a"W"expression trend;in the pectoral muscle,the expression from E14 d to 7 d increased gradually.It reached the highest expression at 7 d,then began to decline,and the expression was the lowest at 14 d.The above research lays the foundation for the follow-up research of Prox1gene regulating chicken tissue development.