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Quantitative real-time polymerase chain reaction (RT-qPCR) is a preferred method for rapid and reliable quantification of gene expression study. However the transcription levels of target genes may be misinterpreted due to differential expression of the reference gene which was thought to express stably. Thus, a systematic evaluation on the expression stability of nine frequently used reference genes across various tissues (including leaf, root, stem, sepal, peal, lip and gynostemium) and treatments (including phytohormone and stress treatments) in Cymbidium kanran was performed in this study. The results demonstrated that expression stability of the candidate genes varied greatly across different sample pools. Based on geNorm and NormFinder analysis, Tubulin (TUB), Actin (ACT) and Ribosomal protein S3 (RPS3) were considered to be the most suitable reference genes when all 25 samples were taken together. In addition, ACT and TUB were also the suitable internal controls in C. kanran with ABA, NaCl, cold and CuSO4 treatments. RPS3 and TUB exhibited the most stable expression pattern in NAA, 6-BA and GA treatments, while ACT and RNA polymerase beta (RpoB) could be used as the reliable internal control genes under PbAC stress. In different tissues, RPS3 and RpoB could be served as the reliable internal controls, while RpoB and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were ranked top in heat treatment. On the other hand, 18S ribosomal RNA (18S), ribosomal protein S19 (RPS19) and photosystem I P700 apoprotein B (PsaB) performed poorly in expression stability analysis across all sample pools, and they could not be used as internal controls for RT-qPCR studies in C. kanran.

Myostatin (MSTN), belonging to transforming growth factor (TGF) beta superfamily, is a negative regulator of muscular development in mammals and fish. The expression of MSTN can be regulated by hormone and environmental factors. Sex steroids, androgen and estrogen can affect sex differentiation and also can affect muscle development. However, few documents are concerned about the effects of sex steroids on MSTN expression in fish. Here, we report the cloning and expressional analyses of MSTN in rare minnow (Gobiocypris rarus), an emerging model fish in China. The MSTN cDNA of rare minnow that has an open reading frame of 1128 base pairs (bp) encodes a protein of 375 amino acids belonging to fish MSTN1 with typical structures like a prodomain, a mature MSTN domain, a conservative hydrolytic site (RIRR) and 9 conservative cysteine residues in mature MSTN. MSTN was detectable in all the examined tissues by RT-PCR that indicated the ubiquitous expression of MSTN in adult tissues. MSTN was detectable from zygote till hatching showed the maternal deposition of MSTN mRNA from oocytes and continuous expression during embryogenesis. Treatment of rare minnow with 1 mu g/L 17 alpha-methyl testosterone (MT) or 17 alpha-ethynyl estradiol (EE2) significantly impaired the body growth and increased the expression of MSTN mRNA detected by quantitative real time PCR. However, low dosage of MT (10 ng/L) had no significant effects on body growth and MSTN expression. The results suggest that the effects of exogenous sex steroids on growth can be mediated through MSTN. It is also suggested that low concentration of sex steroid (less than 10 ng/L) may have little effect on MSTN expression. (C) 2012 Elsevier B.V. All rights reserved.

Article history:Received 5 November 2011Received in revised form 21 December 2011Accepted 22 December 2011Available online 4 January 2012Keywords:Freshwater musselHyriopsis schlegeliiDefensinmRNA expressionImmune response Defensin is one of the most important antimicrobial peptides(AMPs) in bivalves and is involved in the im-mune responses. Herein, we reported the isolation and initial characterization of a defensin from the fresh-water pearl mussel, Hyriopsis schlegelii (designated as Hs-defn) and its immune response for the first time.The full length Hs-defn cDNA was 410 bp long and consisted of a 51 bp 5'UTR, a 161 bp 3'UTR with amRNA instability motif (AATAAA) and an open reading frame(ORF) of 198 bp encoding a polypeptide of 65amino acids. The deduced amino acid sequence of Hs-defn shared certain common features of AMPs, suchas α-helical structure, net positive charge, lower molecular mass and high hydrophobic residue ratio. Homo-logue searching against GenBank database suggested that Hs-Defn was also phylogenetically close to thedefensin isoforms identified from other mollusks. Furthermore, predicted with homology-modeling method,the three-dimensional structureofHs-Defndemonstrated a significantsimilaritywith otherknown defensinssuch as oyster C. gigas and insect P. terraenovae defensins. Phylogenetic analysis results further revealed thatHs-defn is a member of invertebrate defensin family.Results of tissue specific analysis showed that the expression level of Hs-defn mRNA was the highest in hae-mocytes, moderate in gill, hepatopancrease, mantle and intestine, the lowest in foot, adductor muscle, gonadand kidney. After pathogenic Aeromonas hydrophila stimulation, the Hs-defn mRNA expression was signifi-cantly up-regulated at2 hand12 hin haemocytes, gill, mantle andhepatopancrease. These resultsimplicatedthat Hs-Defn was a constitutive and inducible acute-phase protein which was perhaps involved in the im-mune defense of pearl mussel.© 2011 Elsevier B.V. All rights reserved.

Neuronal apoptosis sculpts the developing brain, and nearly all identified classes of neurons seem to be produced "in excess" during development. FoxO transcription factors regulate apoptosis in vitro in deprived of neurotrophins. It is unknown if FoxO3a is involved in the development of neurons. Here, we report a role of FoxO3a during neuronal development in zebrafish. By using in situ hybridization, we revealed that FoxO3a transcripts in zebrafish were gradually confined to regions of the central nervous system during embryonic development, including the forebrain, midbrain, midbrain-hindbrain boundary and hindbrain. By using FoxO3a morpholino antisense oligonucleotides, we observed that FoxO3a loss-of-function led to neural developmental defects, including increased neural apoptosis as detected by acridine orange and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling. These defects could be partially rescued by the injection of FoxO3a mRNA. In this study, we found that FoxO3a loss-of-function resulted in the decreased expression of neuronal markers as determined by in situ hybridization and relative quantitative real-time PCR. Furthermore, the activation of FoxO3a was required for the maintenance of neuron survival but not necessary for the induction of neurogenesis. Our results indicated that FoxO3a might be essential for the maintenance of neural development in zebrafish. Therefore, this work provides novel evidence of FoxO3a in the embryonic neurodevelopment from zebrafish to other mammals.