Expression And Analysis Of Sequence And Polymorphism Of Porcine Lipoprotein Lipase And Hormone-sensitive Lipase Gene

In the modern animal breeding practice, it is very common that genetic marker and genotype related to the important economic traits on farm animals were identified according to molecular biotechnology, especially the development of DNA molecular marker detection and quantitative trait loci mapping technology in order to select excellent individuals earlier. More suitable marker must be found before using these theories. Lipoprotein lipase and hormone-sensitive lipase are the key enzymes responsible for fat deposition and fat mobilization in white adipose tissue. Mainly in order to find new single nucleotide polymorphisms and useful molecular markers, the molecular genetic base of porcine lipoprotein lipase and hormone-sensitive lipase gene was studied in this paper. In addition, the LPL and HSL gene expression plasmids ware constructed. The expression of recombinant plasmids was also studied. The main results are as follows:1. The extract method of total RNA from pig fat using Trizol reagent was established and improved. The cDNA of HSL and LPL gene in the porcine had been cloned and sequenced. The other related species cDNA are also found from Genbank. So the connection among the different species is revealed in our research.2. The LPL gene intron3, intron5 of the Largewhite and Qingping pig were cloned and sequenced. A lot of mutations were found in these two introns. The mutations numbers include single mutations and missing mutations in introns. Long DNA fragment missing and insertion mutation were not found in these two introns. The cutting sites are GT/AG bases in all these introns. The sequence length of Qingping pig LPL gene intron3, intron5 are 1876bp and 1916bp. The sequence length of Largewhite LPL gene intron3 and intron5 are 1871bp and 1902bp.3. A Nhel polymorphism was detected at the site of pig intron 3 in LPL gene by PCR-RFLP analyzing and sequenceing. There is significant difference on the traits of skin rate and lion eye height in resource family F2 group. Additive effect is dominant in these two traits (p<0.05).4. A EcoT22I loci of pig intron 3 in LPL gene appears in resource family F2 group. The loci exists significant difference in 6-7 rib fat thickness, skin thickness, skin percentage. Additive effect is dominant in these two traits (p<0.05).5 A NcoI loci polymorphism of pig intron 3 in LPL gene is revealed in resource family F2 group. In carcass, lion eye height, lion eye length and lion eye area exist significant difference (p<0.05). On lion eye height, additive effect is dominant, whilein lion eye length and lion eye area, dominant effect is more important. In meat quality, there is significant difference in the traits of pH(LD),pH(BF),meat color (BF) and intramuscular fat. The dominant effect is more important.6. Afal polymorphism is found in intron 5 of LPL gene in porcine. PCR-RFLP analysis in resource family group show that, in the carcass, water loss rate and thorax-waist fat thickness exist significant difference (p<0.05), and additive effect is dominant, but no significant difference, while in meat quality, pH (LD), pH (BF), pH (SC), water holding rate, water loss rate and meat color exist significant difference (p<0.01), additive effect is more important and there is significant different (p<0.01).7. Seal polymorphism is found in intron 5 of LPL gene in porcine, but there are only two genotypes in resource family group.8. The expression of HSL and LPL in E.coli.The complete porcine HSL and LPL gene were amplified by PCR technique and cloned into pMDl8-T vector. They were sequenced by sanger's sequencing technique. Then, they were inserted into downstream of the T7 promoter of an expression vector, pET-28a, to yield the recombinant plasmid pET-28a-HSL/ pET-28a-LPL. After induced by IPTG, a high expression of LPL protein was obtained, but the expression of HSL failed. Enzyme activitie of LPL gene is detected by colorimetric method.9. The expression of HSL and LPL in Pichia pastoris.Coding region of HSL and LPL gene are cloned from porcine, then inserted into pPIC9K expression vector, pPIC9K-#SL/pPIC9K-LPZ,. These plasmids are transmitted into the Pichia pastoris to obtain recombinant protein which have excellent activities.