| The esterase is a kind of proteolytic enzymes, can be in the water molecules are involved in, through the hydrolysis of triglycerides, phospholipids and cholesterol esters, will cut into acids and alcohols and esters. This enzyme is involved in many biochemical reactions, according to the specific subject matter, protein structure and function, and there are different. A class of enzymes catalyze the hydrolysis of ester bonds. Two kinds of enzymes are usually also includes the catalytic hydrolysis of carboxylates and phosphates. In the esterase catalyzed carboxylic acid ester, the most common such as lipase, can catalyze the hydrolysis of triglycerides and fatty acids. Catalysis of esterase absorbing some of the fat in food, energy and plasma lipoprotein meTableolism plays an important role.Study on lipase has been found, with the function of enzymes including lipoprotein lipase, hepatic lipase, endothelial lipase, pancreaticlipase and so on. In this study, Farmed tilapia as the object of study, for the first time using RT-PCR and RACE method in the whole sequence of HL from liver of Farmed tilapia, LPL gene cloning. The main results are as follows:1. The full-length coding region sequences of HL gene cloning. The sequencing result showed that tilapia containing the full-length coding region of HL gene fragments of 1872bp,5'-UTR is 217bp,3'-UTRis 176bp. ORF is 1479bp, Encoding aprotein of 493 ammo acids,protein molecular weight is 6.3104kD. The polyadenylation signals of polyA is AATAAA.2. Analysis of HL gene of biological information. The sequence homology and phylogenetic analysis showed that Farmed tilapia's HL gene sequence homology is close to tilapia, Micropterus salmoides, Mandarin fish, Red seabream, Epinephelus coioides, Silver carp's HL gene sequence homology, respectively 96.6%?84.68%?80.89%? 79.48%?76.52%?75.89%; is different from Homo sapiens ,Rattus norvegicus, Mus musculus and Acipenser sinensis's HL gene sequence homology, respectively 48.7%?48.99%?49.41%?60.9%. Farmed tilapia have the highest amino acid sequence homology with tilapia, Micropterus salmoides and have the lowest with Homo sapiens?Mus musculus and Acipenser sinensis.The phylogenetic distance relations consistent, showed that Farmed tilapia HL gene is relatively conservative in evolution; Analysis of amino acid composition of HL gene using online software ExPASy,the protein contains 486 amino acids, the leucine content was the highest(10.1%),the second highest is Glycine (7.6%), Tryptophan content was the lowes. Functional analysis found that HL Amino acid is oelectric point is 8.72, The molecular weight is 55303.3 kDa. The total number of negatively charged amino acid residues(AsP+ Glu) is 54, The total number of positively charged amino acid residues (Arg+Lys) is 62. InsTableility index is 39.65, Fat coefficient is 84.24, The overall level of hydrophobicity is-0.328. Application of SignIP to predict the HL protein N-term/nal signal eptide,the result show that Farmed tilapia HL gene containing 30 amino acid signal peptide. Function structure show that there is the catalytic site, lipid binding sites, N-glycosylation sites and a conserved cysteine residues in Farmed tilapia and other fish HL gene sequence. Analysis of the two protein secondary structure of HL showed that, two level of structure with random coil structure of the protein, Alpha helix and extended with scattered throughout the protein.3. The full-length coding region sequences of LPL gene cloning. The sequencing result showed that Farmed tilapia containing the full-length coding region of LPL gene fragments of 2300 bp,5'-UTR is 126 bp, 3'-UTRis 629 bp. ORF is 1545 bp, Encoding aprotein of 515 amino acids,protein molecular weight is 5.813596 kD.4. Analysis of LPL gene of biological information. The sequence homology and phylogenetic analysis of LPL gene showed that, Farmed tilapia LPL amino acid sequence with homology to mammalian is 56.12%-56.54%, to Gallus gallusis 57.35%, to Brachydanio rerio. Mandarin fish, MicroPterus salmoides is 67.51%?92.31%, showed that tilapia HL gene is relatively conservative in evolution; farmed tilapia's HL gene sequence homology is differentr from Mandarin fish's LPL gene sequence homology(67.51%), is close to MicroPterus salmoides's LPL gene sequence homology(92.31%). Application of MEGA3.1 N-J method for constructing Phylogenetic tree,the result show that Red seabream, grouper Epinephelus coioides were clustered into one branch and tilapia were clustered into one clade, Mus musculus and Rattus norvegicus were clustered into one clade and jungle fowl were clustered into one clade, this is the traditional morphological and biochemical characteristics classification and evolutionary status basic consistent. Further prove that cloning of gene is LPL in Farmed tilapia. Analysis of LPL's amino acid composition showed that, the protein contains 513 amino acids, the Threonine content was the highest(9.0%),the second highest is Leucine (7.8%), Cysteine content was the lowes. Functional analysis found that HL Amino acid isoelectric point is 8.54, The molecular weight is 57.8538 kDa. The total number of negatively charged amino acid residues(Asp+Glu) is 53, The total number of positively charged amino acid residues(Arg+Lys) is 58. InsTableility index is 30.98, Fat coefficient is 79.81, The overall level of hydrophobicity is-0.323. Application of SignIP to Predict the HL protein N-terminal signal peptide,the result show that Farmed tilapia HL gene containing 24. amnio acid signal peptide. Function structure show that there is the catalytic site, lipid binding sites, N- glycosylation sites and a conserved cysteine residues in Farmed tilapiaand other fish HL gene sequence. Analysis of the two protein secondary structure of HL showed that, two level of structure with random coil structure of the protein, Alpha helix and extended with scattered throughout the protein.5. The activity level of HL expression in the liver for the different levels of choline, lipid level and feeding frequency and feeding. Appying the method of qRT-PCR to detect liver of Farmed tilapia in different levels of choline, lipid level and feeding frequency, feeding on hepatic lipase activity levels in the liver expression. The results showed that choline levels were 750mg/kg and 1000mg/kg, with the level of fat Farmed tilapia liver gene expression of HL (4%,8% and 12%) increases, the difference between the groups was significant (p< 0.01).When fat levels for the same amount of choline levels in 4%, with Farmed tilapia liver HL gene expression (500mg/kg,750mg/kg and 1000mg/kg) increased first and then decreased and increased. And the fat level was 8% and 12%, the HL gene expression in liver volume with choline level has been in a rising state; Different feeding level and the effects of feeding frequency on Farmed tilapia liver HL mRNA expression of the experiment, when the feeding frequency was 2 times/d, Farmed tilapia liver HL mRNA expression with the level of fat (2%-10%) increased firstly and then decreased and increased latter, except the fat level is 4%, the expression of other fat the level of HL mRNA in the liver was higher than that of the control group (2/d,2% higher level of fat).Between 10% and 2%, fat 8%, between 4% and 6% of fat level were significantly different (P<0.05), there was significant difference between the 4% and 10%(p<0.01); Feeding frequency was 3 /d, each level of fat liver HL mRNA expression was higher than that of the control group (2/d,2% fat), and with the increase of fat content, liver HL mRNA expression also showed a rising trend. The fat level of 2%and other groups, between 4% and 8%, fat 10%, between 6% and 10% level of fat liver HL mRNA expression was significantly different (p<0.01), between 8% and 10% level of fat liver HL mRNA expression was significantly different (P< 0.05), the other group difference is not significant (P> 0.05).6. The activity level of LPL expression in the liver for the different levels of choline, lipid level and feeding frequency and feeding. Applying the method of qRT-PCR to detect the different levels of choline, lipid level and feeding frequency, the expression of hepatic lipase activity feed investment level in the liver of Farmed tilapia liver. The results showed that Choline levels for 500mg/kg, with the level of fat (4%,8% and 12%) increased the Farmed tilapia liver gene expression of LPL decreased first and then increased. Choline levels for 750mg/kg, with the level of fat (4%,8% and 12%) increased the Farmed tilapia liver gene expression of LPL the rise and then decline, significant differences between the groups (p<0.01). Choline levels for 1000mg/kg, with the LPL gene expression in liver fat levels (4%,8% and 12%) increased and showed a rising trend, significant differences between the groups (p< 0.01);Analysis of fatty liver in constant, LPL gene expression changes in the amount of fat, at 4% level, the liver LPL gene expression with increased choline levels and increased first and then decreased, but the levels of fat in the liver was 12%, the expression of LPL gene decreased firstly and then increased.The fat level of 8%, with the rising levels of choline, the liver LPL gene expression has been showing an upward trend; feeding frequency 2/d, with the increase of dietary lipid levels, tilapia liver LPL mRNA expression decreased firstly and then increased and then decreased and increased trend, significant differences in each group (p<0.01). When the feeding frequency 3/d, fat level was 2% and 4%, Farmed tilapia liver LPL mRNA expression was lower than that of the control group (2 /d,2% levels of fat), and with the rising level of fat decreased with no significant difference between the groups (P> 0.05). But when the fat level of 6% to 10%, Farmed tilapia liver LPL mRNA expression increased with the level of fat and showed a rising trend, and the expression was much higher than that of the control group, significant differences between the groups (p<0.01). |
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