Studies On Cloning And Analysing Of Lipoprotein Lipase Gene Exon 4, 5 And 6 In Goose

Lipoprotein Lipase(LPL), an acylglycerol hydratase, which is synthesized and secreted by the adipose tissue, cardiac and skeletal muscles, the lactating mammary gland and macrophages, plays a vital role in lipid metabolism and transportation. The paper mainly studied on the cloning and analysing of LPL gene exon 4, 5, 6 and intron 4, 5, 6 sequence and the protein residue it coded in goose, the results showed below:1. Primers P and Q were designed to amplify the LPL gene exon4 to exon 5, exon 5 to intron 6 refer to sequence of the LPL gene in chicken for its high conservation. The LPL gene exon 4 to exon 5 and exon 5 to intron 6 were amplified and sequenced. According the gt-at splicing rule in exons and introns, obtained the LPL gene exon 4, 5, 6 which were 112bp, 234bp, 243bp long and intron 4, 5, 6 in goose which were 1526bp, 187bp, 1102bp long separately. The results were logined in GenBank, ID: DQ105796 and DQ105797.2. Sequence and homologies of LPL gene exon 4, 5, 6 and intron 4, 5, 6 among other 13 species were analyzed by using DNAStar 5.01. The results showed that the homology between goose and chicken was 91.9%. The homologies between goose and mammals (human, sheep, cattle and pig) were 74% to 78%. And the homologies in introns were much smaller, showed there were disparities in introns. The phylogenetic tree of nucleotide from different species of LPL gene exon 4, 5 and 6 was constructed. It showed that the differentiation between goose and mammals started early. And mouse followed. There were evident specificities in differentiation among herbivore pig, primate and sarcophagi.3. The protein coded by LPL gene exon 4, 5 and 6 was deduced and analyzed. The homologies of the protein among different species were calculated. Results showed 196 amino acids were coded by this region. The phylogenetic tree of residue of LPL gene exon 4, 5 and 6 coded protein showed the fish was the earliest differentiate species in 14 species, goose and chicken were the next, mammals were the last. The later differentiations the bigger homologies of amino acids in species.4. The codon usage of LPL gene exon 4, 5 and 6 of goose was calculated. The correlation of codon usage of exon 4, 5 and 6 in LPL gene among 14 species were compared. Different amino acid had different codon usage. For example, Ala prefers to GCU, Arg likes CGC, His uses CAC, Leu uses CUC+CUG, Ser uses AGC, Val uses GUG, Thr uses ACC, which showed the nonrandomness of codon usage. Comparing the results, conspicuous difference was found in the codon usage of different species, in which the similarity coefficient was lower than 0.36 in goose and sarcophagi, 0.4 to 0.5 in goose, human, cattle, sheep and baboon, 0.55 to 0.65 in goose, pig and mouse, and 0.839 in goose and chicken, the highest, which showed the codon usage between goose and chicken was similar.5. Hydrophilicity, average charge, and surface probability of amino acids coded by exon 4, 5 and 6 of LPL gene in goose were analyzed. The results showed the amino acides on the surface of LPL were the ones had hadrohydrophilicity and higher charge density. On the other hand, the area having less surface probability might locate at the furrow of LPL, and these regions had little charge density or negative charge. So the former area might be the binding domain of LPL, and the latter area may be the region which had active or catalytic site.6. PCR-RFLP method was used to analyze the polymorphism of LPL gene intron 4 of goose. Restriction enzymes (PstI, PvuII, HinfI) were used to digest the sequence. There were 2 digest sites in PstI-RFLP, 3 digest sites in PvuII and 3 digest sites in hinfI.But could not find the polymorphism.