| Oriental river prawn, Macrobrachium nipponense, subordinated to Decapoda, Palaemonidae, extensively distributed in China. It is an important economic spieces for freshwater aquaculture in China. In recent years, its economic characters seriously degenerated, and the benefit of oriental river prawn farms declined remarkably due to small size and precocious puberty. Crustacean hyperglycemic hormone (CHH) family, synthe-sized and secreted by the eyestalk of crustaceans, play a central role in the regulation of numerous physiological functions such as energetic metabolism, growth, reproduction. In the present study, we characterized full-length cDNAs encoding two important eyestalk hormones of the CHH family, GIH (gonad-inhibting hormone) and CHH, from the eyestalk of oriental river prawn.The isolation of high quality total RNA from oriental river prawn eyestalk is a precondition of cDNA cloning of these neurohormones. In this paper, eyestalks of Macrobrachium nipponense were studied as the material for seeking the best extraction method of total RNA by using RNAiso Plus, RNeasy Mini Kit, Unizol with some improvements in the predecesors. The quality of the total RNA was detected and analyzed by ordinary agarose gel electrophoresis, UV spectrophotometer and RT-PCR. The results showed that the total RNA isolated by RNAiso method had mass pigments. The total RNA obtained by improved RNAiso method had been partially dissolved and got less pigments. Although less pigment inhibit the activity of RTase to some extent, it still could be used for RT-PCR. The RNeasy Mini Kit method could get rid of pigments more efficiently, which inhibit the activity of RTase, and obtain integrate total RNA but with serious DNA contamination. The method of RNAiso or Unizol combing with RNeasy Mini Kit could get high purity total RNA without pigments and DNA. However, the total RNA extracted by the method of Unizol combing with RNeasy Mini Kit had much better integrity than by the method of RNAiso combing with RNeasy Mini Kit. Therefore, Unizol combing with RNeasy Mini Kit should be the best method for total RNA extraction in Macrobrachium nipponense eyestalk, and the total RNA obtained by this method could be used in the subsequent molecular biological experiments.Two isoforms of oriental river prawn GIH cDNA encoding GIH-A and GIH-B were cloned by means of RT-PCR and RACE (rapid amplification of cDNA ends) methords. The full-length sequence of GIH-A cDNA is918bp in size with a455bp5’-untranslated ragion, a333bp open reading frame and a118bp3’-untranslated region [excluding poly(A)]. The deduced preproGIH-A consists of a32-aa signal peptide and a78-aa mature peptide. The full-length sequence of GIH-B cDNA is1350bp in size with a205bp5’-untranslated region, a339bp open reading frame and a794bp3’-untranslated ragion [excluding poly(A)]. The deduced preproGIH-B consists of a34-aa signal peptide, a78-aa mature peptide. The deduced mature peptides of GIH-A and GIH-B, both possessing the six conserved cysteine residues in the same relative position, have a glycine residue at position12of the mature peptides, which is characterized by the type-II neuropepdides of CHH family. According to the alignment of mature peptides amino acid sequence deduced from GIH-A and GIH-B cDNA with CHH family mature peptides amino acid sequence of other crustaceans, GIH-A and GIH-B share respectively94%and67%identity with GIH of Rimicaris Kairei, and71%and59%identity with GIH of Homarus americanus, and exhibit39-60%,35-51%,28-34%identity with MIH (molt-inhibiting hormone)/MOIH (mandibular organ-inhibiting hormone)/CHH of other crustaceans, respectively. Phylogenetic analysis of the CHH family indicates that GIH-A and GIH-B are clustered at first with GIH of Rimicaris Kairei which belonging to Caridea, followed with GIH of Nephropidae, then with type-Ⅱ neuro-pepdides of CHH family from Decapoda.The full-length cDNA sequence of Macrobrachium nipponense CHH was cloned by means of RT-PCR and RACE methods. The full-length sequence of CHH cDNA is1017bp in size with a241bp5’-untranslated region, a408bp open reading frame and a355bp3’-untranslated region [excluding poly(A)]. The deduced preproCHH consists of a26-aa signal peptide,33-aa CPRP (CHH-precursor related peptide) and a78-aa mature peptide. The deduced mature peptides of CHH, possessing the six conserved cysteine residues in the same relative position, lack the glycine residue at position12of the mature peptides. According to the alignment of mature peptide amino acid sequence deduced from CHH cDNA with mature peptides amino acid sequence of CHHs of other crustaceans, Macrobrachium nipponense CHH mature hormone exhibits the highest identity(99%) with CHH from Macrobrachium rosenbergii and Macrobrachium lanchesteri, and shares52-69%identity with CHH from other crustaceans which belonging to Pleocyemata. Phylogenetic analysis of the CHHs indicates that all crustaceans are clustered into two main branches:Pleocyemata and Dendrobranchiata, Macrura CHHs cluster with Caridea CHHs at first, then cluster with Branchyra CHHs, finally cluster with Penaeidea CHHs.In general, this is the first time to clone the GIH and CHH for oriental river prawn, and the results accumulated the data of background for further research of gene expression, function and regulation. It is also helpful for the resolution of precocious puberty in oriental river prawn. |
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