胭脂鱼胰蛋白酶cDNA 克隆以及不同蛋白含量日粮和饥饿对mRNA 表达和酶活力的影响
MOLECULAR CLONING OF TRYPSIN AND THE EFFECT OF DIETARY PROTEIN LEVELS AND STARVATION ON TRYPSIN mRNA EXPRESSION AND ENZYME ACTIVITY IN JUVENILE CHINESE SUCKER (MYXOCYPRINUS ASIATICUS BLEEKER)
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摘要: 为检测不同蛋白含量的日粮和饥饿对胭脂鱼(Myxocyprinus asiaticus)幼鱼胰蛋白酶活性和mRNA表达的影响, 首先用RACE 和PCR 的方法从胭脂鱼幼鱼的肝胰脏组织中克隆得到胰蛋白酶cDNA 全长, 然后用半定量RT-PCR 和酶活性检测方法分别检测了经不同蛋白含量日粮(酪蛋白含量分别为35%、45% 和 55%)投喂和饥饿处理后的胭脂鱼幼鱼的胰蛋白酶mRNA 表达水平和胰蛋白酶活力的变化。结果显示, 胭脂鱼胰蛋白酶cDNA 全长为912 bp。投喂蛋白质含量适中(45%酪蛋白)日粮组的试验鱼胰蛋白酶活性和mRNA 水平高于投喂高蛋白水平日粮组(55%酪蛋白)和低蛋白水平日粮组(35% 酪蛋白); 饥饿明显降低mRNA水平和胰蛋白酶活性; 胰蛋白酶活性的变化滞后于mRNA 水平的变化。胰蛋白酶活力的变化与mRNA 水平的变化之间未呈现直接相关性。因此, 胭脂鱼胰蛋白酶合成可能是一个由多种因素共同调控的复杂过程。Abstract: The effect of dietary protein and starvation on the expression of trypsin was evaluated in the Chinese sucker (Myxocyprinus asiaticus Bleeker). The complete trypsin cDNA was cloned from juvenile Chinese sucker pancreatic tissue by using RACE and PCR methods. We used semi-quantitative RT-PCR and enzymatic activity measurements to quantify mRNA expression and trypsin activity in fish that were either starved or fed differing levels of dietary casein (35%, 45% and 55%). The results showed that the Chinese sucker trypsin cDNA sequence was 912 bp in length. Trypsin activity and mRNA levels were higher in fish that were fed moderate (45% casein) levels of protein than those that were fed high or low levels. Starvation significantly decreased mRNA expression level and trypsin activity. The changes in trypsin activity tended to lag behind the changes in mRNA levels. There was no direct relationship between the trypsin activity and mRNA level. Given this, the trypsin synthesis is a complex process regulated by a balance of several factors in the Chinese sucker.
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