2.4| Amplification and isolation of Na+/K+ ATPase α subunit, cytoplasmic carbonic anhydrase, Na+/H+exchanger, and Na+/K+/2Clcotransporter
Amplification and isolation of complementary DNAs (cDNAs) encoding NKA α subunit, CAc, NHE, and NKCC in H. tridens , M. japonicus , and C. dehaani were performed, as previously described (Yamaguchi and Wakahara, 2001). Briefly, the most posterior pair of gills was isolated from crabs, and total RNA was extracted using Isogen reagent (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. cDNAs were synthesized using an oligo (dT) primer and ReverTra Ace (TOYOBO, Osaka, Japan), and resultant cDNAs served as templates for polymerase chain reaction (PCR) amplification. Degenerate primers were designed on the basis of the amino acid sequence of the gene products in Callinectes sapidus, Carcinus maenas ,Portunus trituberculatus , and L. vannamei. The primer sequences used for PCR were as follows: NKA α subunit: forward, 5’-ATGACNGTNGCNCAYATGTGG-3’; reverse, 5’-GGRTGRTCNCCNGTNACCAT-3’; CAc: forward, 5’-TAYGTNTTRGARCARTTYCA-3’ or 5’ -TAYGTNCTNGARCARTTYCA-3’; reverse, 5’-CKRAANGCRTCYAAYTG-3’ or 5’-CKRAANGCRTCNAGYTG-3’; NHE: forward, 5’-AARATHGGNTTYCAYATGAC-3’; reverse, 5’-TGYTCNACRTARTTYTTCAT-3’; NKCC: forward, 5’-AAYATHTGGGGNGTNATG-3’; reverse, 5’-GCRAARCANCCNGCRTADAT-3’.
For amplification of CAc and NKCC cDNA, we performed nested PCR using part of the products from the first amplification as a template. The primer sequences used for nested PCR were as follows: CAc: forward, 5’-CAYTGGGGNAARACNAAYGA-3’; reverse, 5’-CANGGNGGNGTNGTNA-3’; NKCC: forward, 5’-ATHTGGGGNGTNATGYT-3’; reverse, 5’-ARYTCCATNACYTGRAARCT-3’.
In every case, conditions for PCR were as follows: 40 cycles for 30 s at 94°C, 30 s at annealing temperature, and 30 s at 72°C. The annealing temperature for NKA α subunit, CAc, NHE, and NKCC was 50°C, 43°C, 45°C, and 45°C, respectively. Amplified PCR products were electrophoresed and extracted, and then inserted into a pTA2 cloning vector (TOYOBO), and finally, their nucleotide sequences were determined. The sequences of isolated cDNAs were deposited to GenBank (Accession numbers of NKA α subunit, CAc, NHE, and NKCC are LC214855, LC375964, LC572286, and LC572287 for H. tridens , LC214856, LC375965, LC572288, and LC572289 for M, japonicus , and LC572290, LC572291, LC572292, and LC572293 for C. dehaani ). In addition, amino acid sequences were predicted from the cDNA fragment sequences of H. tridens ,M. japonicus , and C. dehaani and compared with those of the shore crab C. maenas (GenBank Accession numbers are AY03550, EU273943, U09274, and AY035548 for NKA α subunit, CAc, NHE, and NKCC, respectively) (Towle et al., 1997; Serrano and Henry, 2008).