2.4| Amplification and isolation of
Na+/K+ ATPase α subunit, cytoplasmic
carbonic anhydrase, Na+/H+exchanger, and
Na+/K+/2Cl–cotransporter
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).