Cloning, Analysis And Rna Interference Of TIP47 Gene In Xinong Sanen Goat

Tail-interacting protein 47, also called mannose 6-phosphate receptors binding protein 1, abbreviated as TIP47, M6PRBP1, PP17, or M6PBP, is a member of the PAT family. It is closely involved in the process of lipid droplet (LD) biogenesis and metabolism. The present study cloned the cDNA of TIP47 from mammary gland of Xinong Saanen Goat using RT-PCR and RACE PCR. Expression of TIP47 mRNA in 12 Xinong Saanen Goat tissues was examined via real-time quantitative PCR. The function of TIP47 gene in dairy goat mammary epithelial cells was studied, using adenovirus vectors constructed for RNA interference. The effect of oleic acid on TIP47 gene expression in mammary epithelial cells was detected. Main results:(1) Complete cDNA of goat TIP47 was 1906 bp in length (GenBank accession No.: HQ846827), including 82 bp of 5'UTR, 1314 bp of CDS, and 510 bp of 3'UTR, coding 437 amino acid residues in total. Similarity of nucleotide sequences between goat TIP47 and cattle (Bos taurus), human (Homo sapiens), mouse (Mus musculus) and porcine (Sus scrofa) TIP47 was 97%, 83%, 74%, and 89%, respectively; homology of amino acid sequences was 98%, 82%, 73%, and 89%, respectively. Molecular weight of goat TIP47 is 47.36 KDa, and isoelectric point is 5.74. An conservative PAT region was detected at the N-terminus of goat TIP47 protein.(2) The mRNA of TIP47 was expressed in dairy goat mammary gland, subcutaneous fat, heart, spleen, lung, pectoral muscle, kidney, rumen, small intestine, liver, hypophysis, ovary tissues examined via RT-qPCR. TIP47 was highly expressed in mammary gland, spleen and lungs, with mammary gland being the highest; and least expressed in muscle.(3) Three pairs of shRNA were constructed onto shuttle vector pENTR/CMV-GFP/U6, and effectiveness was examined by co-transfecting HEK 293 cells with red fluorescence protein vector pDsRed1-C1-TIP47. Two pairs were selected to construct effective adenovirus vectors used for RNA interference. Titer of recombinant adenovirus was 108 U/mL after packaging and amplification in HEK 293 cells; MOI in mammary epithelial cells was 200. Expression of TIP47 in dairy goat mammary epithelial cells was effectively suppressed, expression of FASN and HSL were upregulated, while expression of ATGL was downregulated after RNA interference.(4) Lipid accumulation increased in dairy goat mammary epithelial cells when dealt with oleic acid. Oil red O induction of TIP47 expression showed certain pattern as time increases. Besides, HSL mRNA expression was upregulated by oleic acid treatment, while ADRP mRNA expression was suppressed. It is likely that TIP47 is involved in mammary gland lipid metabolism.In conclusion, the present study cloned complete cDNA of TIP47 from Xinong Saanen goat; the nucleotides and AA sequences are highly homologic with those of cattle, human, mouse and porcine TIP47, and the protein structure contains a conservative functional region. TIP47 is highly expressed in goat mammary gland, and least expressed in muscle. RNA interference with adenovirus vectors could decrease the expression of TIP47 mRNA, increase the expression of FASN and HSL mRNA. Oleic acid treatment revealed certain pattern of TIP47 mRNA expression, and effcet the expression of some genes which involve with lipide storage and metabolism. These results could be the basis of further study of TIP47 gene function.