Douglas SE, Gallant JW
Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia, Canada B3H 3Z1
J Mar Biotechnol. 1998 Dec;6(4):214-219.
Knowledge of the timing of digestive enzyme expression in developing larvae is essential for evaluating the appropriateness of formulated larval diets. Since little is known at the molecular biological level about the ontogeny of digestive enzyme function in flatfish, we have isolated cDNA clones for key digestive enzymes such as trypsinogen. Portions of trypsinogen genes have been amplified from winter flounder cDNA libraries by PCR using primers based on sequence motifs conserved among trypsinogen genes from other organisms. The PCR products were sequenced, cloned, and used as radioactively labeled probes to screen the libraries. Three distinct trypsinogen cDNAs have been isolated, representing the first cDNAs for winter flounder digestive enzymes. The first type (Flounder 1) is very similar to a trypsinogen sequence reported from a related flounder, Pleuronectes platessa. The second type (Flounder 2) is related to sequences reported from Atlantic salmon, cod, and the Antarctic fish, Paranotothenia magellanica. The third type (Flounder 3) shows limited similarity to the Antarctic fish trypsinogen sequence. All three cDNAs encode a cleavable signal sequence at the amino-terminal end, and Flounder 1 and 2 contain the characteristic acidic sequence for activation of the trypsinogen proenzyme to trypsin. Southern hybridization experiments indicate that Flounder 1 and 3 are single-copy genes, whereas Flounder 2 may be present in more than one copy. In addition, Flounder 2 detects homologs in a wide variety of other fish species. The sequence information will be used to establish RT-PCR assays for larval winter flounder (Pleuronectes americanus) digestive enzyme expression.
了解发育中幼虫消化酶表达的时间对于评估配方幼虫饲料的适宜性至关重要。由于在分子生物学水平上对鲽形目鱼类消化酶功能的个体发育了解甚少，我们已分离出关键消化酶如胰蛋白酶原的cDNA克隆。基于其他生物胰蛋白酶原基因中保守的序列基序设计引物，通过PCR从冬比目鱼cDNA文库中扩增出胰蛋白酶原基因的部分片段。对PCR产物进行测序、克隆，并用作放射性标记探针筛选文库。已分离出三种不同的胰蛋白酶原cDNA，代表冬比目鱼消化酶的首批cDNA。第一种类型（比目鱼1型）与一种相关比目鱼——欧洲比目鱼（Pleuronectes platessa）报道的胰蛋白酶原序列非常相似。第二种类型（比目鱼2型）与大西洋鲑、鳕鱼和南极鱼类——麦哲伦副南极鱼（Paranotothenia magellanica）报道的序列相关。第三种类型（比目鱼3型）与南极鱼类胰蛋白酶原序列的相似性有限。所有三种cDNA在氨基末端均编码一个可切割的信号序列，比目鱼1型和2型含有将胰蛋白酶原激活为胰蛋白酶的特征性酸性序列。Southern杂交实验表明，比目鱼1型和3型是单拷贝基因，而比目鱼2型可能以多个拷贝存在。此外，比目鱼2型在多种其他鱼类物种中检测到同源物。该序列信息将用于建立冬比目鱼（Pleuronectes americanus）幼虫消化酶表达的RT-PCR检测方法。
