Cloning,Expression Of EIF3L?EIF3H?EEF1D Gene And Their Effects On Wool Growth-related Factors In Sheep

The growth and development of wool are affected by many factors.The process of henogenesis and periodic physiological regulation both have a positive effect on the determination of wool trait related genes and the exploration of regulatory network.Eukaryotic translation initiation factor 3L?EIF3L?,eukaryotic translation initiation factor 3H?EIF3H?,eukaryotic translation elongation factor?EEF1D?are cell factors,which involved in the regulation of protein translation and are closely related to cell activity.The study uses Merino Sheep Fibroblast total RNA as template,to get CDS encoding sequence of EIF3 L,EIF3H and EEF1 D gene of mRNA by RT-PCR.After enzyme digestion and purification,it respectively connected to the prokaryotic expression vector pet28?alpha?,eukaryotic expression vector pcDNA3.1?-?,then builded the prokaryotic expression vector and the eukaryotic expression vector about EIF3 L,EIF3H,EEF1 D gene.The results showed that the similarity of the obtained gene sequence was 100%,99.91% and 100%,respectively,and the nucleotide base at position 1055 bp of EIF3 H sequence was changed from A to G.The amino acids encoding asparagine?Asn / N?were mutated into AGC serine?Ser / S?.Sequence analysis showed that EIF3 L,EIF3H,EEF1 D nucleotide sequences and amino acids encoded with amino acids,Rabbit and other animals,the nucleotide sequence and the encoded amino acid sequence are homogenous.The recombinant prokaryotic expression plasmid was transformed into E.coli host strain and induced by IPTG at a final concentration of 0.75 mmol·L^-1 for 3.5 h at 37 ?,respectively.The protein expressed in inclusion body was produced,including the molecular weight of EIF3 L protein.The size of EIF3 H protein is about 40 kDa,the molecular weight of EEF1 D is about 40 kDa,and the protein molecule has good reactivity by Western blot.The recombinant eukaryotic expression vectors were transiently transfected into the primary cultured Merino fine wool sheep,and then the total RNA and total protein were extracted from the transfected cells.After RT-q PCR and Western blot were detected,the results showed that the mRNA expression and expression of EIF3 L and EIF3 H were up-regulated in normal fibroblasts,and the expression of EEF1 D mRNA was not up-regulated and the protein expression was increased.In the detection of wool-related gene expression,it was found that the mRNA expression of fibroblasts transfected with recombinant eukaryotic expression vector was up-regulated and down-regulated in different degrees.The results showed that the mRNA and protein of EIF3 L,EIF3H and EEF1 D were in the heart,liver,and liver of Merino fine wool sheep and Kazak coarse wool sheep,and the expression of EIF3 L,EIF3H and EEF1 D mRNA and protein were analyzed by RT-qPCR and Western blot.Spleen,lung,kidney,skin,muscle tissues are different degrees of expression and have a certain difference.