【作者】 迟良；

【导师】 李军；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2013， 博士

【摘要】 消化酶是由消化腺和消化系统分泌的具有消化功能的酶类。消化酶对鱼类的生长发育及新陈代谢过程都起到重要的作用。由于海水鱼类的主要营养来源是蛋白质,因此蛋白酶是鱼类中最主要的消化酶类,其中胃蛋白酶和胰蛋白酶是最重要的两种蛋白消化酶。鱼类早期发育过程中,消化器官的发育及其功能化的过程对于鱼类的生产实践具有重要的指导作用,而蛋白酶的出现也是消化器官功能形成的一个重要的标志,因此,研究蛋白酶的起源发生过程对于了解鱼类的消化过程及消化机制有重要意义。大菱鲆(Scophthalmus maximus)属于鲽形目、鲆科,分布于大西洋北岸,自上世纪90年代引进我国,由于其生长迅速、抗病能力强,营养丰富等特点,目前已是我国重要的经济海水鱼类。本论文研究了大菱鲆消化道的发育及其消化生理的变化,同时结合对蛋白酶基因在发育过程中的起源及定位进行了研究具体,结果如下：1、大菱鲆的消化道在初孵仔鱼为一条未分化的线性消化管。当仔鱼开口之后(3DAH, days after hatching),消化道逐渐发育为口咽腔、食道、胃和肠等部分。食道在2DAH末端开始膨大,上皮细胞逐渐转换成柱状细胞,3DAH时膨大的部分逐渐发育成胃。胃腺在16DAH出现,标志着胃的组织结构发育完善。肠道在2DAH开始分化,3DAH时就分化出前中肠和后肠,至27日龄时基本发育完善。肝胰脏的原基在2DAH出现,并在3DAH出现酶原颗粒,肝胰脏在17日龄时基本发育完善。2、通过蛋白酶活性的检测发现：大菱鲆的胃蛋白酶活性出现的时间在22DAH(43.14±7.57U/prot),晚于胃腺出现的时间,表明胃的功能化与胃的组织学发育并不同步,胃的功能化要晚于组织学的发育。随后胃蛋白酶的活性随着个体的发育逐渐增加。大菱鲆胰蛋白酶在未受精卵及胚胎时期均能检测到其活性,并且在未受精卵的中胰蛋白酶的比活性最高(1320.66±55.63U/prot)在随着胚胎发育逐渐下降,至原肠期时降到最低(77.37±0.58U/prot)。然后又逐渐上升,到18DAH时达(1061.80±102.76U/prot),至22日龄时又迅速下降(213.60±59.65U/prot),此后保持在一个平稳的水平。3、克隆获得了大菱鲆胃蛋白酶原A(pepsinogen A, PGA)、胃蛋白酶原C(pepsinogen C, PGC) cDNA序列全长。并且使用RT-PCR及原位杂交检测了其在大菱鲆早期发育过程中的表达情况。对大菱鲆PGA(tPGA)、PGC(tPGC)蛋白序列分析发现,tPGC较tPGA更加保守,而tPGA在对蛋白底物的结合能力更强。通过RT-PCR及荧光定量PCR检测发现tPGA及tPGC均在22DAH开始表达,在消化道中,tPGA与tPGC均在食道末端与胃的贲门处开始表达。在表达量上,tPGC在发育早期的表达量较高,而tPGA在成鱼中的表达量较高。另外,在不同组织中,tPGA与tPGC在食道和胃的表达量最高,其次在卵巢和肌肉中也有微量的表达。4、克隆获得了大菱鲆胃质子泵基因(H+/K+ATPase)α亚基的cDNA全长序列。并且检测了其在早期发育过程中的表达情况,发现大菱鲆H+/K+ATPase的表达时序跟tPGA、tPGC的表达时序一致,都是在22DAH开始在食道末端和胃的贲门部位表达。5、克隆获得了大菱鲆胰蛋白酶原(Trypsinogen, TG)基因的cDNA序列全长。检测了其在大菱鲆早期发育过程中的表达情况,大菱鲆胰蛋白酶原基因(tTG)的表达贯穿于大菱鲆的整个发育过程。在不同时期发育的卵母细胞中均能检测到tTG的表达,受精后,tTG的表达量随着胚胎发育逐渐降低,至原肠期达到最低,此后逐渐上升,至25日龄左右时,表达量突然降低。tTG基因的表达量与胰蛋白酶活性的变化趋势基本一致。另外,通过对tTG在大菱鲆胚胎中的定位分析发现,在胚胎发育早期,tTG在胚胎中弥散表达,并且在卵黄囊的系膜上也有分布。至孵化期时,tTG的分布逐渐向消化道和头部位置集中。更多还原

【Abstract】 Proteinases play an important role in digestion and it’s crucial to metabolism because it determines the availability of nutrients for biologic function. Pepsin and trypsin are the two most important proteinase. The study on occurrence of the protease would be significance to production practice and theoretical research on fish development.Turbot (Scophthalmus maximus), an important commercial fish found mainly in the northeastern Atlantic,belongs to the order pleuronectiformes, Bothidae, is characterized by rapid growth and good meat quality. At present, turbot is an important commercial marine species in China. In this study, the ontogenetic development of the digestive system and the enzymatic activity of pepsin and trypsin in turbot were investigated. Then the related genes involved in pepsin and trypsin were isolated, and the expression profile of the homologue genes were characterized during the early stages of turbot development. The main results are as follows:1. At hatching, the digestive tract could not be distinguished, it was a straight, undifferentiated tube composed of columanr epithelium. At3DAH (days after hatching) when larvae started exogenous feeding, the digestive tract was differentiated into buccopharynx, esophagus, stomach and intestine. At2dph, esophagus appeared an expanded lumen, and the anterior zone was lined with stratified squamous epithelium and the dilated posterior zone was lined with a monolayer of columnar epithelium. These layers later develop into the stomach. Gastric glands were formed at16DAH, it’s a sign that stomach were fully developed. Intestine was differentiated at2DAH and was fully developed at27DAH. The liver and pancrease were started to differentiate at2DAH, the zymogen granules were appeared at3DAH. The liver and pancrease were fully developed at17DAH.2. The activity of pesin was firstly detected at22DAH (43.14±7.57U/prot), but was not observed at the time of gastric gland formation(16DAH).We hypothesized that the development of the stomach was not truly complete when gastric gland formation. The activity of trypsin were detected in unfertilized eggs (1320.66±55.63U/prot) and all stages of embryo development. Before gastrula, the activity of trypsin decreased gradually, after gastrula, the activity were ascendant gradully. Then abrupt decrease was observed at22DAH followed by a relatively stable level.3. The full-length cDNA of pepsinogen A (PGA) and pepsinogen C (PGC) homologue were isolated from the turbot. Then the expression patterns of pepsinogen A (tPGA) and pepsinogen C (tPGC) in turbot characterized by RT-PCR and in situ hybridization. The deduced amio acid sequences showed that the tPGC was more strongly conserved than tPGA but tPGA affected a wider range of substrates than tPGC. The initial transcripts of tPGA and tPGC were first detected in the esophagus and cardiac region of the stomach at22DAH. Quantitative expression analysis showed that tPGC was preferentially expressed in early developmental stages, and that the tPGA mRNA expression level was higher in adult fish. In addition, tPGA and tPGC mRNAs were also expressed in muscle and ovary at much lower levels than in stomach and esophagus.4. The full-length cDNA of a subunit of gastric pump gene (H+/K+ATPase) was isolated. Then the expression patterns of H+/K+ATPase were investigated.The initial transcripts H+/K+ATPase were detected at22DAH, were exactly coincide with the expression patterns of pepsinogen genes.This suggested that H+/K+ATPase are very important for supporting the digestive function of stomach.5. The full-length cDNA of trypsinogen gene (TG) was isolated from the turbot. Then the expression patterns of trypsinogen (tTG) in turbot were characterized by RT-PCR and in situ hybridization.The expression of tTG happened throught development, and tTG mRNA were expressed in all stages of oocytes. After the eggs fertilized, The growth rate of tTG genes decreased gradully until gastrula, then it began to increase until about25DAH. Then abrupt decrease was observed at about25DAH followed by a relatively stable level. In addition, the transcripts of tTG were diffuse distribution in the embryo and also detected in the vitelline membrane. As the embryo develops, tTG mRNA were concentrated on the head and digestive tract of larvae at hatching.更多还原