Xiangshan Ji

Cloning, Characterization, and Expression Analysis of Hepcidin Gene from Red Sea Bream (Chrysophrys major)

ABSTRACT A cDNA encoding hepcidin was isolated from a library of cDNA from spleen of red sea bream (Chrysophrys major) by expressed sequence tag analysis. The expression of the hepcidin mRNA in various tissues was examined. Challenge of red sea bream with Escherichia coli DH5α elevated hepcidin mRNA levels in spleen, gill, liver, and intestine.

MHC polymorphism and disease resistance to Vibrio anguillarum in 12 selective Japanese flounder (Paralichthys olivaceus) families.

Genetic variation in the major histocompatibility complex (MHC) class IIB was tested in Japanese flounder (Paralichthys olivaceus) for survival after challenge with bacterial infection. The material consisted of 6000 Japanese flounder from 60 families challenged with Vibrio anguillarum, which causes significantly different mortality in flounder families. Five individuals from each of six high-resistance (HR) and six low-resistance (LR) families were screened for their MHC class IIB genotypes using sequence analysis. High polymorphism of MHC IIB gene and at least three loci were discovered in Japanese flounder and the rate of d(N) occurred at a significantly higher frequency than that of d(S) in PBR. Among 60 individuals, 76 alleles were discovered and 15 alleles were used to study associations between alleles and resistance to disease. We found highly significant associations between resistance towards infectious disease caused by V. anguillarum and MHC class IIB polymorphism in Japanese flounder. Some alleles appeared in both HR and LR families, while some alleles were only discovered in HR or LR families. One allele, Paol-DAB*4301, was significantly more prevalent in HR families than in LR families (P=0.023). Paol-DAB*0601, Paol-DAB*0801, Paol-DAB*2001, Paol-DAB*3803 were discovered in two HR families with high frequency. One allele, Paol-DAB*1601, was discovered in three LR families. The steady heredity of MHC class IIB alleles was observed, and the family having Paol-DAB*4301 alleles was confirmed with higher resistance to V. anguillarum. This study confirmed the association between alleles of MHC class IIB gene and disease resistance, and also detected some alleles which might be correlated with high bacterial infection resistance. The disease resistance-related MHC markers could be used for molecular marker-assisted selective breeding in the flounder.

Molecular cloning, genomic structure, polymorphism and expression analysis of major histocompatibility complex class IIA and IIB genes of half-smooth tongue sole (Cynoglossus semilaevis).

Major histocompatibility complex (MHC) genes play an important role in the immune response of vertebrates. Its function is to present foreign peptide to the T-cell. In order to study the function and molecular polymorphism of class II genes in teleost, the full lengths of MHC class IIA and IIB cDNA were cloned from half-smooth tongue sole by homology cloning and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). Genomic organizations, molecular polymorphism, and expression profiles of class IIA and IIB were examined to study the function in fish. As in other teleost, four exons and three introns were identified in half-smooth tongue sole class IIA gene, five exons and four introns were identified in class IIB gene. The deduced amino acid sequence of class IIA had 27.3-69.8% identity with those of mammal and teleost. Nine class IIA alleles were identified from four individuals. Four different alleles observed in a single individual may infer the existence of two loci at least. The deduced amino acid sequence of class IIB had 7.9-71.9% identity with those of other species. Fifteen class IIB alleles were identified. Six different alleles observed in a single individual may suggest that there are at least three loci in class IIB genes. Real-time quantitative RT-PCR demonstrated that the MHC class IIA and IIB were ubiquitously expressed in twelve normal tissues. Challenge of half-smooth tongue sole with the pathogenic bacteria, Vibrio anguillarum, resulted in significant changes in the expression of MHC IIA and IIB mRNA in three tissues.

Growth differences and differential expression analysis of pituitary adenylate cyclase activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH) between the sexes in half-smooth tongue sole Cynoglossus semilaevis

Pituitary adenylate cyclase activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH) are regulators of growth hormone secretion. In this article, we examined the difference in growth and mRNA expression of PACAP and GHRH between the sexes in half-smooth tongue sole, an important cultured fish species indicating sexually growth dimorphism in China. Firstly, a significant body weight difference between females and males was first observed at 7 months (P<0.05) and at 18 onths the mean body weight of the females (771.0±44.3 g) was as much as 4.9 times higher than that of males (130.6±6.0 g). As a result, half-smooth tongue sole, Cynoglossus semilaevis, is a good model to investigate the effects of growth-related genes expression on sexual growth dimorphism. Secondly, the cDNAs encoding PRP/PACAP and GHRH were isolated. Two differently processed mRNA transcripts of PRP/PACAP (PRP-encoding and PRP splice variant) were found. PACAP and GHRH mRNA was highly abundant in brain and less abundant in other tissues. However, PACAP mRNA was expressed in most brain regions, and was lower in the cerebellum. GHRH mRNA was predominantly expressed in the hypothalamus and weakly expressed in all areas of the brain examined. Ontogenetic expression analysis indicated that PACAP and GHRH mRNA was detected in the early stages of embryogenesis. Finally, differential expression showed that there was no significant difference of the expression level of PACAP or GHRH between the sexes before 8 months of age. However, between 9 and 12 months of age, the GHRH mRNA expression level in males was significantly higher than in females (P<0.05), which might be associated with GH deficiency in males. In contrast, the male PACAP mRNA expression level was not significantly higher than that in females even at 9 and 12 months of age. The present results provide important clues for understanding the sexual growth dimorphism mechanisms in half-smooth tongue sole.

Growth differences and dimorphic expression of growth hormone (GH) in female and male Cynoglossus semilaevis after male sexual maturation

Half-smooth tongue sole, Cynoglossus semilaevis, is an ideal model to investigate the regulatory mechanisms of sexual growth dimorphism in fish species. The aim of the study was to investigate the effect of differential age of sexual maturity for females and males on growth and GH mRNA expression in C. semilaevis. The body weight differences between the sexes were not significant in C. semilaevis at age 5 months when females and males were all immature. Significant differences in body weight between the sexes were found after early sexual maturation of males at the age of 9 months. The body weight of 21-month-old females (621.4 ± 86.4g), still not immature, was even 3.28 times higher than that of the males (189.7 ± 14.4g). The cDNAs encoding GH in C. semilaevis was cloned. The GH gene is 2924bp long and consists of six exons and five introns. The results of qRT-PCR showed that GH mRNA levels of the immature females were not significantly different from that of immature males at age 5 months. However, GH mRNA levels of the immature females were significantly higher compared with those of the mature males at age 9 months (P<0.05). At age 11 months, GH mRNA levels of females were even 6.4-fold higher than that of males. In conclusion, for the first time we show that early sexual maturity of males is the main cause of sexual growth dimorphism in C. semilaevis and exert significant effect on GH mRNA expression.

Isolation and characterization of polymorphic microsatellite DNA markers in the rock bream (Oplegnathus fasciatus)

From a (GT)13-enriched genomic library of Oplegnathus fasciatus, 14 polymorphic microsatellite were isolated and characterized in a test population with alleles ranging from two to nine, the observed and expected heterozygosities from 0.0000 to 1.0000, and from 0.1726 to 0.8507, respectively. Five loci deviated from the Hardy–Weinberg equilibrium, and linkage disequilibrium between two loci was significant. Two loci were also polymorphic in Pagrosomus major assessed for cross-species amplification. These polymorphic microsatellites will be useful for genetic diversity analysis and molecule-assisted breeding for O. fasciatus.

Ten polymorphic microsatellite loci for the Atlantic halibut (Hippoglossus hippoglossus) and cross-species application in related species

In the present study, 10 polymorphic microsatellite DNA loci from Atlantic halibut (Hippoglossus hippoglossus) were isolated and characterized. The number of alleles for these loci ranged from 2 to 4 in tested 24 individuals. Observed and expected heterozygosities per locus varied from 0.21 to 0.70 and from 0.31 to 0.65, respectively. Most of these 10 microsatellite loci were successfully amplified and showed polymorphic in five related species. These loci will be useful for the assessment of genetic diversity and population structure of Atlantic halibut.

Isolation and characterization of polymorphic microsatellite loci from black snapper Sebastodes fuscescens (Houttuyn)

Black snapper Sebastodes fuscescens (Houttuyn) is an economocally important species. Fourty-five microsatellite loci were isolated from an enriched genomic library of S. fuscescens. Ten of these loci were polymorphic in a test population with alleles per locus ranging from 2 to 5, and observed and expected heterozygosities per locus from 0.21 to 1.00 and from 0.19 to 0.86, respectively. Three loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was found between pairs of loci. These polymorphic microsatellite loci would be useful for investigating genetic diversity of S. fuscescens and other related species.