【作者】 郑凯迪；

【导师】 李英文；

【作者基本信息】 西南大学 ， 水产养殖， 2007， 硕士

【摘要】 胰蛋白酶（EC3.4.21.4）属于丝氨酸家族，是鱼类消化道内的一种主要的碱性蛋白质水解酶，其前体形式为胰蛋白酶原前体，在脊椎动物中，由胰脏的腺泡细胞粗面内质网（RER）上的核糖体产生，然后运输到高尔基体，再进入胰腺运输管，最后释放到肠腔，在那里被肠黏膜细胞分泌的肠激酶活化成有活性的胰蛋白酶，从而行使其消化功能。影响胰蛋白酶活性的因素很多，如消化道内的不同部位、温度、pH、饲料组成、食性等，这些因素对胰蛋白酶的分泌分别起着正负调节的作用，而且即使是同一因素，由于其数量或者程度不同对胰蛋白酶活性所产生的影响也不同。胭脂鱼（Myxocyprinus asiaticus），主要分布于长江中上游的干支流水域中，是我国名贵淡水野生鱼类，由于其不但肉味鲜美、营养丰富，而且幼鱼具有比较高的观赏价值，所以具有巨大的养殖前景。胰蛋白酶是一种十分重要的蛋白质消化酶类，但目前还没有研究从分子水平具体阐明胰蛋白酶对胭脂鱼消化的作用机制，研究胭脂鱼胰蛋白酶原前体基因可以填补这一研究的欠缺，为进一步探讨胰蛋白酶在胭脂鱼消化上发挥的作用建立一定的分子生物学基础。同时由于很多因素都会对胰蛋白酶的活性产生影响，在不同程度上调控胰蛋白酶的活性，所以本文研究不同蛋白含量饲料对胰蛋白酶活性产生的影响，以期为胭脂鱼等名优鱼类养殖过程中的生物饲料技术提供理论或现实基础，同时该研究对于阐明脊椎动物胰蛋白酶原前体基因的分子进化也有一定的学术意义。本研究运用RACE技术克隆了胭脂鱼胰蛋白酶原前体基因，并对其核苷酸序列及推导的氨基酸序列以及预测的蛋白质高级结构进行了分析。同时，本研究还设计了不同蛋白含量的饲料，饲喂胭脂鱼30d，每5d取样一次检测了在此过程中胰蛋白酶活性的变化过程。主要研究结果如下：1．胭脂鱼胰蛋白酶原前体基因全长cDNA序列在对多种鲤科鱼类的胰蛋白酶原前体基因进行了多重比对后设计了4条特异性中间片段引物，以来自胭脂鱼肝胰脏组织的mRNA为模板扩增得到了胭脂鱼胰蛋白酶原前体中间片段的序列。根据得到的中间片段的序列设计了2条特异性3’RACE引物、1条反转录引物和3条5’RACE特异性引物，从而得到了胭脂鱼胰蛋白酶原前体的3’端序列和5’端序列。分别从得到的3’端序列和5’端序列设计特异性引物扩增胭脂鱼胰蛋白酶原前体全长cDNA。从胭脂鱼肝胰脏总RNA中扩增得到的胰蛋白酶原前体全序列为921bp，包括5’端非编码区（untranslation region，UTR）47bp，蛋白质编码区741bp，3’端非编码区133bp。2．胭脂鱼胰蛋白酶原前体基因推导氨基酸序列的分析胭脂鱼胰蛋白酶原前体基因开放阅读框编码247个氨基酸，包括15个氨基酸的信号肽和8个氨基酸的激活肽。所得序列具有丝氨酸蛋白酶的保守序列：（1）含有保守的His（H63）、Asp（D107）和Ser（S200）活性三联体，并且在催化位点附近的Ala、Gly、Asp、Ser、Gly等也都很保守。（2）具有典型的保守的底物结合位点Asp（D194），该残基位于底物结合沟的底部，并通过电荷作用而稳定底物裂解位点的Lys或Arg残基，因而也决定着胰蛋白酶的专一性。（3）胰蛋白酶N末端的氨基酸序列为Ile-Val-Gly-Gly。（4）具有6个（30／160、48／64、132／233、139／206、171／185、196／220）二硫键。其基因基本结构和从其它脊椎动物中克隆的胰蛋白酶原前体的基因结构一致。3．胭脂鱼胰蛋白酶原前体基因的同源性分析胭脂鱼胰蛋白酶原前体的氨基酸序列与许多动物的胰蛋白酶原前体基因具有较强的相似性。尤其是底物作用位点和活性三联体是完全保守的。除此之外在底物作用位点附近的一些氨基酸残基也具有比较高的保守性，还有丝氨酸家族特有的组成3个二硫键的6个半胱氨酸残基在所有真核动物中都是保守的，脊椎动物中一般都有6个二硫键，这些位点的氨基酸残基也非常保守。用DNAstar软件的MgeAlign对几种脊椎动物胰蛋白酶进行相似性和趋异性比较发现，除了那些特殊位点之外，组成脊椎动物的胰蛋白酶原前体的氨基酸并不是特别保守，胭脂鱼的胰蛋白酶原前体的氨基酸序列与斑马鱼（zebrafish）的相似性最高为84.0％，而与其他脊椎动物比较相似性要低一些，大都在60-70％之间。如与人（Human）Ⅰ、鸡（Chicken）P1、大西洋鲑（Atlantic salmon）Ⅰ、Ⅱ、Ⅲ、挪威大鼠（Norway rat）Ⅰ、家鼠（house mouse）Ⅱ、河豚（Fugu rubripes）、非洲爪蛙（African clawed frog）、日本凤尾鱼（Japanese anchovy）Ⅰ、Ⅱ、大西洋鳕（Atlantic cod）Ⅰ、白斑角鲨（spiny dogfish）、七鳃鳗（sea lamprey）A1、B1、美洲拟鲽（winter flounder）Ⅰ、Ⅱ、猪（pig）、狗（dog）Ⅰ、牛（cattle）Ⅱ的相似性分别是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％。用鼠的酪氨酸酶（Tyrosinase）作为外类群，采用Clustal X的N-Jmethod对序列排序并构建系统进化树，系统进化树分析总体表现为同属内种间亲缘关系最近，同目内科间亲缘关系次之，不同目间的亲缘关系最远，聚类的先后顺序同样揭示出目科属种不同分类阶元的亲缘关系的远近。这种分析的结果与利用胰蛋白酶基因保守片段进行系统进化分析所得的结果是一致的。在胰蛋白酶上体现的进化趋势符合现有的理论，即：首先是依次分化出了软骨鱼，硬骨鱼，两栖类和鸟类，最终分化出了哺乳动物。4．胭脂鱼胰蛋白酶原前体基因推导蛋白的分析及其结构预测对预测得到的胰蛋白酶原前体基因氨基酸序列进行分析，得到蛋白的基本参数，其原子组成为C₁₁₅₈H₁₈₀₀N₃₂₄O₃₅₇S₁₇。分子量Mw=26518Da，等电点pI=7.95。在氨基酸组成上，丝氨酸（Ser）是含量最高的氨基酸（11.0％）：其次是天冬酰氨（Asn）和亮氨酸（Leu）都占8.9％；再次是丙氨酸（Ala）占7.3％；含量最低的氨基酸是组氨酸（His）仅为1.6％。从整体上来看，碱性氨基酸（组氨酸His、赖氨酸Lys、精氨酸Arg）和酸性氨基酸（天冬氨酸Asp、谷氨酸Glu）分别占8.68％和6.1％，这与胰蛋白酶呈碱性相吻合。对胰蛋白酶原前体蛋白的二级结构预测（SOPMA）表明，该蛋白质的二级结构主要由四种形式组成，即α-螺旋（Alpha helix），占17.07％；随机卷曲（random coil），占45.93％；β-转角（Beta turn），占9.35％；延伸链（extended strand），占27.64％。在胰蛋白酶原前体的二级结构中延伸链和随机卷曲所占比例相当高，延伸链主要位于α-螺旋和随机卷曲之间。N-末端以α-螺旋的形式存在。对胭脂鱼胰蛋白酶原前体基因的三级结构进行同源建模预测，得到其同源三维模型。5．不同蛋白含量的饲料对胭脂鱼胰蛋白酶活性的影响试验中设置了6个试验组及1个对照组，试验组中投喂水蚯蚓及蛋白含量分别为55％、45％、35％、25％的饲料，对照组投喂蛋白含量36％的全价配合饲料，还有1个饥饿组。结果发现：投喂水蚯蚓组的胭脂鱼生长速度明显快于其他组，并且其胰蛋白酶活性明显呈现持续上升趋势；而蛋白含量在55％和25％的试验组，胰蛋白酶活性在试验期间呈现出逐渐下降的趋势；而蛋白含量为35％的试验组胰蛋白酶活性呈现出微小的下降趋势；45％的组，呈现出明显的上升趋势；饥饿组胰蛋白酶活性出现了先下降后上升的现象，但总体表现为下降趋势；并且这几个试验组，除了35％的组试验结果与对照组相比差异不显著之外，其余几组与对照组相比差异都极显著。更多还原

【Abstract】 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.更多还原