Two-year-old orange-spotted groupers with body weights (bw) of about 420-670 g and gonadosomatic index (the ratio of gonad to body weight without the visceral mass) of 0.2% or less were obtained from Guangdong Daya Bay Fishery Development Center (Huizhou City, Guangdong, PR China) and raised in indoor water tanks at room temperature under a natural photoperiod for approximately 5 days until use. All experiments were performed under license from the Government of the People’s Republic of China and endorsed by the Animal Experimentation Ethics Committee of Sun Yat-sen University.
Total RNA isolation, Gene Cloning, and Construction of the Baculovirus Transfer Vector
Total RNA of grouper pituitary was isolated using TRIzol (Invitrogen) according to the manufacturer’s protocol, and quantified based on the absorbance at 260 nm. The integrity of the RNA was checked with agarose gel electrophoresis. To synthesize cDNA from the total RNA, the mRNA was enriched using oligo(dT) cellulose, and then reverse transcribed for 90 min at 48°C in a reaction of 20 il containing 1 ig RNA, 0.2 mM dNTP, 2 pM T12 adapter, 5U RNAsin, 0.01 M DTT, and 10 U of Superscript reverse transcriptase (Invitrogen). The polymerase chain reaction (PCR) primers used for amplification of grouper lhb and cga subunits were based on the sequences of GenBank (Accession No. AF507939/AY186243 and AY129308). The primer sequences of the oligonucleotides are listed in Table 1. The primers P1 and P3 contained an EcoRI site and an Nhe I site upstream of the ATG start codon, respectively, and the primers P2 and P4 had a HindIII site and a Kpn I site after the stop codon.