牙鲆发育中ALP基因表达图式及功能分析

The Expression Pattern and Function of Alkaline Phosphatase During the Metamorphosis of Paralichthys olivaceus

为探讨研究碱性磷酸酶(ALP)在牙鲆发育变态中的表达与功能,采用组织学和分子生物学的方法对牙鲆ALP进行了研究。结果如下:1.组织化学染色显示原肠期胚体中即可以检测到ALP的活性。出膜后,在头和背部的活性比较强,而身体的内脏区域,活性较低,卵黄中没有表现出酶活性。仔鱼进食后,ALP开始在消化道中大量的表达,特别是在胃肠道粘膜层。2.牙鲆ALP cDNA全长为1 811 bp,预测能编码476个氨基酸的蛋白质。采用RT-PCR的方法在牙鲆卵到出膜后5天的时候不能检测到ALP基因表达,到变态前的仔鱼体内开始表达。在成鱼胃、肝、鳃、皮肤、皮下肌肉、肾中均能检测到ALP的表达。3.变态前的仔鱼身体总ALP活力约为出膜时的卵中酶活力的4倍,在变态期酶活力变化不大,但是在变态后期上升明显。甲状腺素(T4)在变态中对酶活力增加有明显的促进作用。作为甲状腺素合成抑制剂,硫脲(TU)能降低仔鱼体内酶活性。4.使用ALP的活性抑制剂左旋咪唑(TRM)处理的仔鱼消化功能和头部骨骼的发育受到一定的影响。消化道内食物很少,消化道上皮层中ALP免疫显色反应减弱。头部骨骼中发现较多骨细胞死亡而只留下一些细胞碎片,超过95%的仔鱼不能完成右眼的移位就发生死亡,变态比对照组迟缓。5.定量PCR结果表明,变态中期ALP基因表达量只有初期的1/2,变态后期却明显上升到变态初期的2.5倍,T4和TU处理对ALP基因表达产生影响的在中期和后期表现明显。中期T4组明显高于对照组和TU组,变态后期T4组和TU组均低于对照组。

The expression pattern and function of alkaline phosphatase （ALP） during the metamorphosis of Paralichthys olivaceus were studied by histological and molecular biological approaches. The results are as follows. 1. ALP is present in cells during gastrula stage in the Paralichthys olivaceus embryo. After hatching, ALP is concentrated in the head and notochord. The yolk does not express the enzyme activity all the time. After artificial feeding. The enzyme activity is found to be speeificcally high in the cells of intestinal mucosal epithelium and gastric gland. 2. The full-length cDNA sequence consists of 1811 bp, including an ORF encoding 476 amino acids and a terminal codon. The ALP gene has been examined in liver, gill, skin, kidney, muscle and stomach, it can be detected from 15 d larvae, but can not be detected in the fertilized eggs or 5 d after hatching. 3. Low dose L-tetramisole（TRM） in water can inhibit ALP activity of the larvae, the activity level is only about half of the control group. The function of digestion and assimilation is affected. Immunohistochemical results showed that ALP distributes separately in the mucosal epithelium. Under transmission electrical microscope, a few bone cells decomposed and left many particles of cell organs and cavum. At the end of metamorphosis of the control group, larvae treated with TRM could not finish metamorphosis for failing to translocate the right eyes. At the same time, more than 95 percent of the larvae died： 4. During meta-metamorphosis, ALP gene expression is only half of that in pre-metamorphosis, but in post-metamorphosis, it rises to 2.5 times of pro-metamorphosis. Thyroxine（T4） and thiourea（TU） influence ALP gene expression mainly in cli- max- and post-metamorphosis. In climax-metamorphosis, T4 promotes the expression. However, both T4 and TU restrain ALP gene expression during post-metamorphosis.