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Cloning, Chromosomal Localization,Prokaryotic Expression and Association Analysis of the Porcine Sar1b Gene

Author: WangXueMin
Tutor: ZhaoShuHong；LiKui
School: Huazhong Agricultural University
Course: Animal Genetic Breeding and Reproduction
Keywords: Pig Sar1b gene Fat metabolism Silico cloning Gene mapping The prokaryotic expression Association analysis
CLC: S828
Type: Master's thesis
Year: 2005
Downloads: 185
Quote: 4
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Abstract


Sar1 (secretion-associated and Ras-related) COP Ⅱ (coat protein complex) type is in the assembly process of the composite body of the envelope protein of the small bubbles plays a role of molecular switch. Study found Sar1b chylomicrons in the regulation of cell from the mitochondria to Golgi transport plays an important role in the human internal base of Sar1b gene mutations related with some fat absorption disorders. In this study, as a candidate gene affect pig fat deposition by its cloning, chromosomal localization, tissue expression analysis of prokaryotic expression, detection and its associated traits analysis of polymorphic loci, for further study of the gene pig fat deposition lay the foundation and provide the molecular genetic basis of their future be applied to molecular marker-assisted selection breeding. The main results are as follows: 1. According to people Sar1b gene cDNA information obtained by bioinformatics and comparative gene group learning strategies pig EST overlapping group designed pig-specific primers object cloning a pig Sar1b gene cDNA encoding District full-length sequence of the sequence the corresponding sequence homology with human and mouse, respectively 93% and 91%, has been submitted to GenBank genes obtained included number AY819557. 2 using somatic cell hybrid panel of the porcine Sar1b gene chromosomal region localization. PCR genotyping data submitted to the online data analysis software for statistical analysis, the results showed that the gene was mapped to pig chromosome 2, the regional positioning results SSC2 (1/2 q24)-q29 (P = 0.8696, correlation coefficient 1 , the risk of error is less than 0.5%). Using radiation hybrid panel on the porcine Sar1b gene fine mapping of chromosome. PCR genotyping data submitted to the online data analysis software for statistical analysis, the results showed that the gene IL4 gene tightly linked (LOD = 8.37), and tightly linked microsatellite markers SW1879 (LOD = 6.68). Western Hubei black pig's liver, small intestine, stomach, heart, lung, spleen, muscle and adipose tissue materials, semi-quantitative RT-PCR analysis of tissue expression pattern of swine Sar1b gene and found that the gene in the eight were expressed in one tissue, but relatively high in fat and liver expression. 5 constructed the porcine Sar1b recombinant prokaryotic expression vector pET28a-Sar1b, detected by SDS-PAGE and Western-blot the prokaryotic expression vector and successfully expressed of porcine Sar1b protein. 6 by PCR-SSCP method of distance in isolated porcine Sar1b gene 3 'untranslated region (UTR), termination codon the last nucleotide 7bp a position to detect a base deletion mutation point. Analysis of the different groups in this base deletion polymorphism of the mutation site to calculate this locus genotype and gene frequency, compared the genotype distribution of differences in different herds. The 7. Tongcheng in the room and Tongcheng County Animal Husbandry Bureau jointly established test groups, large long-pass and grew up through detected Sar1b gene deletion mutation of the 3 'untranslated region polymorphism using SAS8. 2 Generalized linear model analysis of the association of this gene with production traits, the preliminary results of the analysis show that the gene 3 '

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Livestock > Pig
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