AQPs Genes Cloning And Tissues Expression In Rana Nigromaculate And Bufo Gargarizans

Aquaporins(AQPs) are a kind of transmembrane protein family which are integral membrane proteins. They are associated with the rapid transport of water. AQPs are water-selective channels that function to increase plasma membrane water permeability in response to osmotic pressure, and they play an critical role in the maintenance of fluid homeostasis in all the organisms. Therefore, AQPs were cloned and their expressions in different tissues were analyzed to speculate their physiological functions,and to predict their roles in vertebrate osmoregulation and thermoregulation throughe water vaporation of body surface. They may establish academic groundwork for elucidating their novel biological functions.A phylogenetic analysis of amphibian and mammalian AQPs suggests that anuran AQPs can be assigned to six clusters: types 1, 2, 3, and 5, and two anuran-specific types, designated as a1 and a2 (The letter"a"represents anuran)In this study, we first cloned part fragments of AQP1,AQP3,AQP5 and keepinghouse genesβ-actin and Glyceraldehyde phosphate dehydro-genase (GAPDH) in different tissues of R.nigromaculate and B. gargarizans, as well as R.nigromaculate AQPa2 genes cDNAs by RT-PCR.Then We examined gene expression of AQP1 mRNA in different tissues of R.nigromaculate and B.gargarizans by semi-quantitative RT-PCR.The specific contents were included:Firstly, searching for theAQP1,AQP3,AQP5,AQPa2 and keepinghouse genes such asβ-actin /GAPDH genes sequences of X.tropicalis,H.chrysoscelis,H.japonica and so on in Genbank, we designed the specific primers.Extracted total RNA from the different tissues in R. nigromaculate and B.gargarizans, and amplified part fragments of AQP1,AQP3,AQP5,AQPa2,β-actin,GAPDH gene cDNAs by RT-PCR,connected these fragments into PMD18-T vetors, and then transformed them into competent cells.Blue-white spots selection was carried out . After positive cloning plasmids were identified ,and they were sent to sequencing.Secondly, According toβ-actin as control gene,the expression of AQP1gene was examined in the different tissues of R.nigromaculate and B.gargarizans by semi-quantitative RT-PCR.The results of researches showed that: firstly,Searching for similarities of being sequenced results in Genebank ,we discovered that cloned 11 gene sequences of two amphibians in experiments were respectively correspondence with AQP1, AQP3, AQP5, AQPa2,GAPDH andβ-actin gene fragments of different species. So it was confirmed that cloned sequences were respectively part sequences of AQPA,AQP3,AQP5,AQPa2,β-actin and GAPDH genes cDNAs of R.nigromaculate, and part sequences of AQP1,AQP3,AQP5,β-actin and GAPDH genes cDNAs of B.gargarizans.Amino acid sequences of being cloned gene fragments were deduced by DNAMAN software, and their homologies with corresponding amino acid sequences of other species were analyzed.The results demonstrated that AQPA amino acid sequence of R.nigromaculate was the most similarity with R.esculenta chip, that is 97.6%, and genetic distance was the smallest; but AQP1 Amino acid sequence of B.gargarizans had lower similarity with other species. AQP3 amino acid sequence of R.nigromaculate had the most similarity with H.chrysoscelis and H. japonica, 96.9%, and genetic distance was the smallest; AQP3 amino acid sequence of B.gargarizans had the most similarity with H.chrysoscelis and H.japonica, that were both 91.9%,and genetic distance was the smallest, moreover its similarity with R.nigromaculate was 90.7%. AQP5 amino acid sequence of R.nigromaculate and B.gargarizans had higher similarity with B.marinus, 95.9%,and genetic distance was the smaller. AQPa2 amino acid sequence of R.nigromaculate had higher similarity with B.marinus AQP-t3,B.marinus AQP-t2,H.japonica AQP-h3 and H.japonicaAQP-h2, respectively83.7%,66.3%,83.2% and 66.3%. Phylogenetic analysis of the AQPs indicated that amphibian AQPs can be classified into six clusters, and clustering results were basally coincident with morphological characteristics and traditional classified evolution status. AQP1,AQP3 and AQP5 gene R.nigromaculate and B.gargarizans with corresponding genes of mammalian were the same subtribe. The RT-PCR results showed that AQPAgene mRNA had all been expressed in urinary bladder, dorsad skin, ventral pelvis skin, ventral skin, brain and kidney of R.nigromaculate; and expressions of AQP1gene mRNA had been examined in urinary bladder, dorsad skin, ventral pelvis skin, ventral skin, lung and kidney of B.gargarizans. Analytical results of gel electrophoresis bands indicated that the expression level of AQPA gene in urinary bladder of R.nigromaculate was highest, and higher in brain, but that was the weakest in ventral pelvis skin and dorsad skin; AQP1gene had the highest expression in dorsad skin of B.gargarizans, and higher in ventral pelvis skin. The expression quantitative differences of AQP1mRNA in different tissues may be related to the different capacity of their water transport; and the high expression of urinary bladder is likely to be relevant to the main ways of uptaking water by urinary bladder in amphibians.