Chang Yaqing

Chemokine receptor genes CCR3 and CCR9 in turbot (Scophthalmus maximus):Cloning and tissue distribution

Turbot is an important aquaculture species in the eastern North Atlantic and Mediterranean regions,China, and Korea. Research on the molecular mechanisms of the turbot immune system could contribute to improving the economic performance of turbot aquaculture. In this study, we cloned and characterized the full-length cDNA of CCR3 and CCR9 in turbot(Scophthalmus maximus). The full-length cDNAs were obtained by the 5′ and3′-RACE method. The CCR9 cDNA was 1 441 bp in length, consisting of a 59 bp 5′ UTR, a 278 bp 3′ UTR, and a1 104 bp ORF encoding a 367 amino acid polypeptide. The CCR3 cDNA was 1 451 bp in length and contained a92 bp 5′ UTR, a 267 bp 3′ UTR, and a 1 083 bp ORF encoding 360 amino acids. The TMHMM(TransMembrane prediction using Hidden Markov Models) analysis confirmed that they were seven-transmembrane-spanning proteins. A phylogenetic tree based on the amino acid sequences was constructed by the neighbor-joining(NJ) method using Mega4. The results of phylogenetic analysis revealed that turbot CCR3 and CCR9 were more similar to the cDNAs of other teleosts than to each other. The relationships exhibited in the tree are consistent with their evolutionary relationships. The CCR9 and CCR3 mRNA expression levels in various tissues was measured by quantitative RT-PCR(qRT-PCR). Both gene transcripts were expressed in all of the tissues analyzed, with the highest expression being in the spleen, head kidney, and heart. The lipopolysaccharide(LPS) challenge results suggest that CCR9 expression in liver was more sensitive than in the other three tissues, while CCR3 expression in spleen and liver was more sensitive than in head kidney or blood. CCR3 and CCR9 expression levels were both high in immune-related tissues and after induction by LPS. Our results indicate that both genes play a role in the turbot immune system.This work further elucidates the functions of these genes in immune responses, which will help to better understand the important relationship between fish disease resistance and innate immunity.

The development of BAC-end sequence-based microsatellite markers and analysis on population genetic diversity in Zhikong scallop (Chlamys farreri)

In this study, microsatellite markers were developed from the BAC-end sequences and used to analyze the genetic structure and genetic differentiation in two populations in Zhikong scallop (Chlamys farreri). A total of 3 374 microsatellites were identified from the 17 477 BAC-end sequences (BESs), of which tetra-nucleotide motifs were most abundant (26.6%), followed by penta-nucleotide motifs (17.7%) and hexa-nucleotide motifs were the least (12.0%). 77.3% SSRs were successfully amplified(99/128), and 43 SSRs(33.6%) were polymorphic in the parentage of mapping population. In order to apply these BES-SSR markers, 14 polymorphic SSRs were chosen to amplify and then analyze the genetic diversity in Dalian population and Qingdao population. A total of 395 alleles were obtained at the fourteen microsatel-lite markers and the number of alleles in each locus ranged from 8 to 38 in the two populations. The aver-age number of alleles (Na) was 18.928 6 and 26.214 3 respectively. The average effective number of alleles (Ne) was 11.750 5 and 17.089 1 respectively. The mean observed heterozygosity (Ho) was 0.510 0 and 0.420 4. The mean expected heterozygosity (He) was 0.915 6 and 0.945 0. The data suggested both popula-tions have high genetic diversities. The mean polymorphic information content (PIC) was 0.894 0 and 0.930 2, which were both greater than 0.5, indicating the fourteen loci were highly polymorphic. The un-biased genetic identity index was 0.487 9, and the genetic distance was 0.717 7. The coefficient of gene differentiation (FST) and gene flow (Nm) between two populations were 0.024 3 and 10.017 9 respectively. Low genetic differentiation was observed between the two populations, and the variance mainly came from individual difference. Significant deviation was detected by Hardy-Weinberg equilibrium test. There was heterozygote deficiency at all loci. The results showed BAC-end sequences were an effective resource for development of SSR markers for genetic and genomic researches.

Triploidy induction in Pacific oysterCrassostrea gigas by caffeine with thermal shock

Zygotes of Pacific oyster (Crassostrea gigas) were treated for triploid induction with caffeine (10 mmol/L, 15mmol/L and 20mmol/L) in combination with thermal shocks (at 40 minute post-fertilization) lasting for 5 and 10 minutes. The highest yield of triploids, 41.5%, was obtained from the treatment with 20 mmol/L caffeine at 34°C lasting 10 minutes. The triploid levels were less than 30.0% in other treatments. Triploid induction was more effective in treatment with 15–20 mmol/L caffeine at 34–38 °C than with lower concentrations of caffeine at temperatures below 32 °C. Our results suggest that triploid induction with caffeine in combination with thermal shocks is less efficient than some other methods reported previously.