Cloning And Sequence Analysis Of CDNA Encoding Pretrypsinogen And Effect Of Feed On Trypsin Enzyme Activities In Myxocyprinus Asiaticus

Pancreatic trypsin(EC3.4.21.4) is a member of the large and diverse serine peptidase. It is one of the most important alkaline proteolytic enzymes in alimentary canal of fish. Trypsinogen is it's non-activity precursor.In vertebrates, trypsinogen is synthesized on ribosomes attached to the pancreatic acinar cell's rough endoplasmic reticulum (RER), then transported to the Golgi complex, concentrated and stored within secretory granules, secreted into the pancreatic duct, and ultimately discharged into intestines, where it is activated by enteropeptidase.and play a role of digestion of food.There are mang factores affect the activity of pancreatic trysin,for example,the different part of alimentary canal,temperature,pH,the composition of feed,feeding habits and so on.Those factors have a positive or negative regulation.Even if the same factor also can bring different effects on fish because of different quantites or degrees.Chinese sucker is a rare and economical fresh water species in China, it is mainly distributed in the upper and medium reaches of Changjiang River and its branches. Chinese sucker is very delicious and nutritious, and because of it's beautiful color it also be fed as pet fish.Feeding Chinese sucker have a bright future. Pancreatic trysin is an important digestive enzyme, but the molecular mechanisms of it in controlling Chinese sucker food digestion have not been reported.In the research the cDNA encoding pancreatic trypsin from Chinese sucker was cloned and sequenced, at the same time, its potential amino acid sequence was deduced and the corresponding protein structure was predicted and analysed.Meanwhile it is also been studied that the relationship of dietary protein rations and activities of enzyme.The main results are as follows:1. Full-length cDNA sequence of pretrypsinogen of Chinese suckerBased on multiple alignment of a number of other fish pretrypsinogen genes, four special primers of fragment sequences were designed for amplification of the center fragmengt of pretrypsinogen genes and the sequence was acquired from the cDNA templet of the pancreas of Chinese sucker. Based on acquired sequence two special primers of 3'cDNA ends were designed for amplification of the 3'cDNA ends, a special reverse transcription primer and two pairs of special primers on 5'cDNA ends were designed, so the sequence of the 3'cDNA ends and 5'cDNA ends were acquired from the cDNA templet of the pancreas of Chinese sucker. Then two special primers were designed for amplification of the full-length cDNAs sequences according to 5' and 3'cDNA ends of Chinese sucker pretrypsinogen gene. The full-length cDNA of pretrypsinogen of Chinese sucker is 774bp with 47bp 5'untranslated region and 133bp 3'untraslated region. The cDNA includes an open reading frame of 741bp encoding the 247 amino acid residues of pretrypsinogen.2. Deduced amino acid sequence analysis from pretrypsinogen cDNA of Chinese suckerThe open reading frame of Chinese sucker pretrypsinogen gene was 741bp in length, encoded 247amino acids residues ,it was composed of an signal peptide(15 amino acids residues), an activation peptide(8 amino acids residues), It contains a typical character shared by serine peptidase family : the catalytic triad His63, Asp107 and Ser200 and the obligatoryAsp194. In addition, 12 cysteine residues are found, forming six conserved cystine bridge.3. Homology analysis of Chinese sucker pretrypsinogen geneThe cDNA sequence of Chinese sucker pretrypsinogen gene don't demonstrated very high similarity with other vertebrates, but in the special loci, especially in the signal region, activation region, catalytic triad region and cystine bridge are very conservation. Similarity of the amino acid sequence of Chinese sucker pretrypsinogen compared with zebrafish, HumanⅠ, ChickenPl,Atlantic salmonⅠ,Ⅱ,Ⅲ,Norway ratⅠ,house mouseⅡ,Fugu rubripes,African clawed frog,Japanese anchovyⅠ,Ⅱ,Atlantic codⅠ,spiny dogfish, sea lampreyA1.B1,winter flounderⅠ,Ⅱ, pig, dogⅠ, cattleⅡrespectively are 84.0%,67.2%,65.6%,64.5%,65.8%,77.3%,67.1%,67.1%,51.0%,67.5%,65.0%,66.4%,60.8%,64.6%,64.0%,63.9%,49.4%64.9%,75.3%,71.5%,73.5%.Taking mouse tyrosinase as outgroup , phylogenetic analysis of known pretrypsinogen sequences with various vertebrates revealed the relationship within a genus or species is most closely ,next is within an order.4.Pretrypsinogen structure prediction of Chinese suckerThe predicted pretrypsinogen protein of Chinese sucker is 247 aa long and has a Mw of 26518Da and a pI of7.95. In composition of amino acid, proportions of serine is relatively large, accounting for 11.0%; secondly, asparagine and leucine are all 8.9%; and then, alanine is 7.3%, histidine is relatively lowest, all accounting for 1.6%. The proportion of acidic amino acid and basic amino acid is 8.63% and 6.1% respectively. Chinese sucker pretrypsinogen protein contains four main secondary structures:α-helix, random coil,β-turn and extended strand. Extended strand is the dominant secondary structure and its proportions is 27.64%.5. The effects of different dietary protein ratios on activities of trypsin enzyme in Chinese suckerThe effect s of dietary protein levels (protein from 35% to 55%) on the pancreas trypsin enzyme activities in Chinese sucker were studied. Four experimental feeds with dietary protein content of 25%,35%,45%,55% were formulated to rear juvenile Chinese sucker and also have two additional groups and feed Limnodrilus hoffmeisteri and fasted respectively.The 30-day feeding experiment showed that pancreas trypsin activities in the Chinese sucker increased with the increase in dietary protein levels when dietary protein levels increased from 25% to 45% , and decreased as the dietary protein level was reach to 55%. There was a significant difference in the activities among the treatments except for the term of treated by 35% protein level. The Chinese sucker fed the Limnodrilus hoffmeisteri had the highest growth rate and activitiesi.