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Cloning, Analysis and Adenovirus Vector Construction for RNA Interference of Short-chain Fatty Acid Receptor GPR41 in Dairy Goat

Author: SunYuZuo
Tutor: LuoJun
School: Northwest University of Science and Technology
Course: Genetics
Keywords: Dairy goat G protein-coupled receptor 41 Clone Tissue expression The adenovirus RNA interference carrier
CLC: S852.65
Type: Master's thesis
Year: 2011
Downloads: 58
Quote: 0
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Abstract


GPR41 (G protein-coupled receptor 41) and GPR43 (G protein-coupled receptor 43) is a living body, the G protein coupled receptor superfamily members, which may be a short-chain fatty acids (the SHORT Chain fatty acids, of SCFA, C ≤ 6 ) activation, studies have shown that GPR41 and GPR43 short chain fatty acid binding has a potential role in the regulation of lipid metabolism. This experiment using the RT-PCR method to clone the CDS region of the short-chain fatty acid receptor GPR41 in dairy goats, dairy goats of different organizations GPR41 expression using real-time quantitative PCR, short-chain fatty acid salt processing goat mammary epithelial cells, analysis of short-chain salt of a fatty acid GPR41 and lipid metabolism related genes. At the same time build a dairy goat GPR41 gene recombinant adenovirus RNA interference vector to provide experimental evidence, analysis of for GPR41 gene function studies of the GPR43 gene and other lipid metabolism gene expression and regulation. In this study, the following results: (1) the cloned milk goat GPR41 gene CDS region, GenBank accession the included number: HM013824. The the goat mammary GPR41 gene CDS region of full-length 978bp, encoding 326 amino acids; dairy goats GPR41 gene the CDS nucleotide sequence and cattle after Phylogenetic analysis showed that, similar to human and mouse were 96%, 78% and 74% amino acid similarity of 97%, 76% and 74%; transmembrane structure analysis revealed that GPR41 has seven transmembrane region; SignalP predictive analysis show that the the GPR41 signal peptide sequence is located 1-34AA, possible cutting bit The point is in between 34Aa and 35AA. (2) The GPR41 gene's expression in the the dairy goat small intestine, breast, spleen, rumen, subcutaneous fat and liver 6 organization analysis found that dairy goat lactation the second highest breast tissue gene expression in the small intestine, liver tissue minimum dry period highest expression in the liver, small intestine, followed by breast expression lowest. Mammary epithelial cells after treatment in (3) propionate and butyrate, can obviously observed lipid accumulation. Real-time quantitative PCR results caused GPR43 propionate processing dairy goat mammary epithelial cells the LEPR, TIP47, ATGL and HFABP genes were up-regulated, the GPR41 and SREBP genes downregulated; butyrate treatment of mammary epithelial cells results in addition to GPR41 all other detected genes upregulated genes. (4) Invitrogen's BLOCK-iT TM shRNA adenovirus interference system, according to the dairy goat GPR41 gene sequence, designed and synthesized three pairs of shRNA interference sequences (shRNA-197, shRNA-442 shRNA-951) to form double-stranded template annealing to build three shRNA the shuttle vector pENTR/CMV-GFP/U6-shRNA, these three shuttle vector expressing the gene of the red fluorescent protein vectors pDsRed1-C1-GPR41 co-transfected HEK-293 cells were screened by two valid interference sequences (shRNA-197 and shRNA-951), recombinant vector pAd-shRNA-197 and pAd-shRNA-951 by homologous recombination in HEK 293 cells packaged adenovirus particles. In summary, the first cloned GPR41 gene with human, bovine, mouse GPR41 gene similarities were higher, and the nucleotide and amino acid analysis found; different organizations and different periods of the dairy goat GPR41 gene The expression shows that GPR41 may be associated dairy goat lactation physiology; propionate and butyrate treatment dairy goat mammary epithelial cells may promote breast cell lipid accumulation and cause-related changes in the expression level of the gene. Build the GPR41 gene recombinant adenovirus vector of interference, laid the foundation for the study of gene function.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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