The full length of major histocompatibility complex (MHC) class IIB cDNA was cloned from a Chinese population of Paralichthys olivaceus by homology cloning and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The MHC IIB genomic sequence is 1,864 bp long and consists of 34-bp 5'UTR, 741-bp open reading frame, 407-bp 3'UTR, 96-bp intron1, 392-bp intron2, 85-bp intron3, and 109-bp intron4. Phylogenetic analysis showed that the putative MHC class IIB amino acid of the Chinese P. olivaceus shared 28.3% to 85.4% identity with that of the reported MHC class IIB in other species. A significant association between MHC IIB polymorphism and disease resistance/susceptibility was found in Chinese P. olivaceus. Thirteen different MHC IIB alleles were identified among 411 clones from 84 individuals. Among the 280 (268) nucleotides, 32 (11.4%) nucleotide positions were variable. Most alleles such as alleles a, b, c, d, e, f, j, k, i, m were commonly found in both resistant and susceptible stock. Via chi2 test, allele d was significantly more prevalent in individuals from susceptible stock than from resistant stock, and their percentages were 23.80% and 7.14%, respectively. In addition, allele g occurred in 9 and allele h in 4 of 42 resistant individuals that were not present in the susceptible stock; their percentages were 21.4% and 9.52%, respectively. Although allele l was found only in 8 individuals from the susceptible stock, its percentage is 19.05%.

ABSTRACT:To improve the culture system of bovine somatic cell nuclear transfer (SCNT) embryos, we studied the effects of activin A on developmental competence of bovine SCNT embryos during the early development stage based on the traditional culture method, and analyzed the expression level of the genes related to blastocyst hatching (Na/K-ATPase, Glut-1) and related to activin A signalling pathway (ActRII and Smad2). We generated the bovine SCNT embryo using a Holstein cow oocyte as recipient cytoplasm and a foetal ear fibroblast (Holstein cow, 120 days) as donor cell. The embryos were cultured as follows: experiment 1, the addition of activin A at the concentrations of 0 (control), 20 (M1-20), 40 (M1-40) or 80 ng/ml (M1-80) to the media during the first three days and no addition during the subsequent 5 days; experiment 2, no addition of activin A to the media during the first 3 days and the addition of activin A at the concentrations of 0 (control), 20 (M2-20), 40 (M2-40) or 80 ng/ml (M2-80) during the subsequent 5 days. The results indicated that the blastocyst formation rate and hatching rate, and total blastomere numbers as well as ICM/TE obtained in experiment 1 were not significantly different from the control group (P > 0.05). In contrast, these values obtained in experiment 2 were significantly higher than in the control group (P < 0.05). In addition, the relative abundance (ratio to GAPDH mRNA) of each gene (Glut-1, ActR II and Smad2) was not significantly different among the treatments in the experiment. The expression levels of 4 genes (Na/K-ATPase, Glut-1, ActR II and Smad2) in blastocysts obtained in experiment 2 were higher than those obtained in experiment 1. In conclusion, the present study suggests that the addition of activin A to the culture media from day 4 to day 8 can enhance the developmental competence of bovine SCNT embryos.