Cloning, Analysis And Adenovirus Vector Construction For RNA Interference Of Short-chain Fatty Acid Receptor GPR41 In Dairy Goat

GPR41(G protein-coupled receptor 41) and GPR43(G protein-coupled receptor 43)are the member of G protein-coupled receptor superfamily in vivo, which can be activated by SCFA(short chain fatty acids, SCFA, C≤6),GPR41 and GPR43 can combine with short-chain fatty acids on fatty acid metabolism. These common themes indicated GPR41 and GPR43 have a variety of potential function. The GPR41 and GPR43 entire coding sequence (CDS) was amplified by RT-PCR; real-time fluorescence quantitative-PCR approach was used for detecting the expression levels of GPR41 gene in the tissues of goats; We also did initial research in the regulation of shor-chain fatty acids on GPR41 and other fatty acid related genes in goat mammary epithelial cells; Two recombinant adenovirus vectors (pAd-shRNA-197, pAd-shRNA-951) were generated through LR recombination and were transfected into HEK 293 Cells for packaging and propagation. These work laid a foundation for researching the regulation role on GPR41 and other fatty acid metabolism related genes in goat mammary gland, the main results were as follows:(1) The CDS of goat GPR41 gene was cloned successfully and uploaded to GenBank (GenBank accession HM013824). The CDS of GPR41 was 978bp, coding 326 amino acids. the homology of GPR41 of dairy goat was found to be 96%, 80% and 74% compared with that of bovine,human and mouse, while the amino acid sequence homology to be 97%,76% and 74% respectively. The protein structure analysis showed seven transmembrane helixes were speculated in the GPR41 gene. SignalP prediction analysis indicated that signal peptide sequence of goat GPR41 was located at 1-34 AA, the most likely cleavage site is between the 34 AA and 35 AA.(2) The expression analysis of GPR41 gene revealed that the mRNA of dairy goat GPR41 gene was expressed abundantly in intestine tissue and followed by the mammary gland, but extremely low in the liver tissue during lactation period while during dry period GPR41 gene was highly expressed in the liver tissue ,but lowly in the mammary gland. (3) In mammary gland epithelial cells, Oil red O staining showed adding butyrate and propionate could significantly increased lipid accumulation compared with the control. Quantitative Real-time PCR results showed that propionic acid treatment increased the levels of GPR43,LEPR,TIP47,ATGL and HFABP mRNA but decrease the mRNA expression level of GPR41, FASN and SREBP. The mRNA expression level of all detected genes were upregulated by treatment with butyrate except GPR41(4) The Block-iT^TM shRNA interference system of invitrogen was used in this experiment. According to the goat GPR41 sequence, we designed and synthesized three pairs of complementary single-strand DNA oligonucleotides (shRNA-197,shRNA-442,shRNA-957) which targeting three different sites of GPR41 mRNA. pENTR/CMV-GFP/U6-shRNA and pDsRed1-C1-GPR41 were cotransfected in the HEK293 cell the result shows that entry vector expressing shRNA-197 and shRNA-951 sequences caused an obvious interference effect. Then two recombinant adenovirus vectors (pAd-shRNA-197and pAd-shRNA-951) were generated by LR recombination and adenovirus vector were successfully packed in HEK 293 Cell.In conclusion, we clone GPR41 gene successfully, The homology analysis of dairy goat GPR41 gene indicated dairy goat GPR41 gene share high similarity with that of bovine, human and mouse; The expression of GPR41 mRNA varied greatly in different tissues as well as different period, We speculate that GPR41 may be related to the physiological of dairy goat lactation; In mammary gland epithelial cells adding butyrate and propionate could significantly increased lipid accumulation and result in changes in the related gene expression; we also has constructed adenovirus RNA interference vector of GPR41 Successfully. These results lay a foundation for further study GPR41.