| Prolactin (PRL) is a polypeptide hormone produced mainly in the anterior pituitary, having a wide range of biological functions in vertebrates. PRL exerts its actions by binding to a single-pass transmembrane receptor (PRLR) belonging to the class-1cytokine receptor superfamily. In mammary gland, PRL initiates and maintains lactation and it is the hormone primarily responsible for the synthesis of milk proteins, lactose, lipids and all major components of milk. In this experiment, RT-PCR methods were used for the isolation and cloning of the whole cDNA of PRLR in Xinong Saanen dairy goats, real-time fluorescence quantitative-PCR (RT-qPCR) approach was used for detecting the expression levels of PRLR gene in10tissues of goats. In addition, three pairs of short hairpin RNA oligo nucleotides (shRNAs) based on the obtained sequence of goat PRLR gene were designed, before being connected into adenovirus vectors to construct recombinant adenovirus vector used for RNA interference. Recombinant adenovirus vectors were transfected into HEK293cells for its packaging and amplification, before further used in RNA interference experiments. The major results are as follows:We report here two types of PRLR CDs, including the long form PRLR (1PRLR)and short form PRLR(sPRLR), owing to the alternative splicing of the same PRLR mRNA. The1746bp nucleotide sequence of1PRLR CDs region, coding577amino acid residues. Homology of nucleotide sequences between goat1PRLR, sheep, cow, pig, human and mouse was99%,94%,88%,80%and80%. The homology encoding amino acid was94%,91%,78%,67%and62%, respectively. The894bp nucleotide sequence of sPRLR CDs region, coding227amino acid residues. Homology of nucleotide sequences between goat1Pr1R, sheep, cow, pig(PRLR-SFla), human(PRLR-SFla)and human(PRLR-SFlb) was99%,96%,88%,85%and85%. The homology encoding amino acid was98%,95%,79%,72%and73%, respectively.Mature1PRLR protein is instability. The protein structure analysis showed that the estimated protein chemical formula is C2826H4343N709O852S24and its molecular weight is62.6522ku; the mature short PRLR protein stability, and chemical formula is C1442H2189N347O394S13with31.1071ku molecular weight. Both1PRLR and sPRLR have one transmembrane domain; signal peptides of them have the same sequence "MKENAASRVLFILLLLYASLLNG". Both the ECD (extracellular domain) of1PRLR and sPRLR protein fold in a β-sandwich formed by two antiparallel (3-shects.(1) The higher PRLR expression levels are in the lung, liver, small intestine, followed by muscle, rumen, subcutaneous fat, breast, and the PRLR expression in the kidney, heart and spleen are lower.(2) In mammary gland epithelial cells, adding PRL could significantly increased the mRNA levels of CSN2, LALBA, FASN, ACC, PPARγ, SLC2A1, LEPR(P<0.01), and the expression of SCD,SREBP, Akt1, the difference was significant (P<0.05);(3) Recombinant adenovirus carrying shRNA which targets goat PRLR was successfully constructed, packaged and amplified. We transfected recombinant adenovirus into mammary epithelial cells of Xinong Saanen dairy goat for48h,60h and72h. The results show that487recombinant adenovirus reduced the PRLR gene expression in mammary epithelial cells by83%.(4) In that case, milk protein genes CSN2and LALBA expression reduced significantly (P<0.05); the SLC2A1expression in experimental group was significantly decreased (P<0.01); ACC and FASN expression were decreased (P>0.05); while SCD expression was increased significantly (P<0.05); triglyceride hydrolyses ATGL mRNA expression levels decreased, the difference was extremely significant (P<0.01). Lipid droplets increased in mammary epithelial cells of experiment group. The total triglyceride levels increased and total cholesterol increased (P>0.05) in experiment group. The expression of fatty acid metabolism regulatory factor Aktl decreased by52%(P<0.01), SREBP1decreased by12%(P>0.05), PPARy decreased by76%(P<0.01). |
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