Aquaporin 4 is a ubiquitously expressed isoform in the dogfish (Squalus acanthias) shark
Aquaporin 4 is a ubiquitously expressed isoform in the dogfish (Squalus acanthias) shark
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DOI：
10.3389/fphys.2011.00107
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发表时间：
2012-01-01
影响因子：
4
通讯作者：
Zeidel, Mark
中科院分区：
文献类型：
--
作者：
Cutler, Christopher P.;MacIver, Bryce;Zeidel, Mark
利用退化PCR技术从水通道蛋白4 (AQP4) cDNA中扩增出角鲨同源基因，并进行克隆和测序。然后使用5‘和3’ RACE技术获得完整的编码区。与其他物种AQP4氨基酸序列比对表明，角鲨AQP4与高等脊椎动物序列同源性较高（高达65.3%），而与Agnathan（38.2%）或硬骨鱼（57.5%）的同源性较低。Northern blotting表明，角鲨mRNA约为3.2 kb，在直肠腺（鲨鱼的分泌液器官）中高度表达。半定量PCR进一步表明，AQP4是普遍存在的，在所有被测组织中均有表达，但在某些组织中表达水平较低，其中在肝脏>鳃>肠中表达。对角鲨组外部环境盐度的处理表明，当角鲨分阶段适应120%或75%海水盐度时，直肠腺、肾脏、食道/心胃中AQP4 mRNA的表达均无变化。而使用同一实验的RNA样本进行的定量PCR实验显示，在120%的sw驯化角鲨中，鳃AQP4 mRNA的表达丰度显著降低了63.1%。通过测定非洲爪蟾卵母细胞中AQP4表达的影响来确定角鲨AQP4的功能。与对照组相比，表达AQP4的角鲨卵母细胞的渗透性（P-f）显著增加，这与ph值不变。与其他物种的AQP4一样，氯汞处理卵母细胞的渗透性没有显著降低。同样，表达AQP4的卵母细胞也没有表现出增强的尿素或甘油渗透性，这也与其他物种中AQP4的水选择性一致。
The dogfish ortholog of aquaporin 4 (AQP4) was amplified from cDNA using degenerate PCR followed by cloning and sequencing. The complete coding region was then obtained using 5' and 3' RACE techniques. Alignment of the sequence with AQP4 amino acid sequences from other species showed that dogfish AQP4 has high levels (up to 65.3%) of homology with higher vertebrate sequences but lower levels of homology to Agnathan (38.2%) or teleost (57.5%) fish sequences. Northern blotting indicated that the dogfish mRNA was approximately 3.2 kb and was highly expressed in the rectal gland (a shark fluid secretory organ). Semi-quantitative PCR further indicates that AQP4 is ubiquitous, being expressed in all tissues measured but at low levels in certain tissues, where the level in liver > gill > intestine. Manipulation of the external environmental salinity of groups of dogfish showed that when fish were acclimated in stages to 120% seawater (SW) or 75% SW, there was no change in AQP4 mRNA expression in either rectal gland, kidney, or esophagus/cardiac stomach. Whereas quantitative PCR experiments using the RNA samples from the same experiment, showed a significant 63.1% lower abundance of gill AQP4 mRNA expression in 120% SW-acclimated dogfish. The function of dogfish AQP4 was also determined by measuring the effect of the AQP4 expression in Xenopus laevis oocytes. Dogfish AQP4 expressing-oocytes, exhibited significantly increased osmotic water permeability (P-f) compared to controls, and this was invariant with pH. Permeability was not significantly reduced by treatment of oocytes with mercury chloride, as is also the case with AQP4 in other species. Similarly AQP4 expressing-oocytes did not exhibit enhanced urea or glycerol permeability, which is also consistent with the water-selective property of AQP4 in other species.