Ye Xing

An outer membrane protein, OmpK, is an effective vaccine candidate for Vibrio harveyi in Orange-spotted grouper (Epinephelus coioides).

The outer membrane proteins of the fish pathogen, Vibrio harveyi, have a role in interaction between bacterium and host and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein, OmpK, which serves as the receptor for broad-host-range vibriophage KVP40 in V. harveyi, was isolated and characterized. Then the OmpK gene coding for mature peptide was subcloned into prokaryotic expression vector pBV220 and transformed into Escherichia coli DH5 alpha strain. After temperature induction, a recombinant protein was detected about 28 kDa in molecular weight and accounted for 24.8% of total proteins of whole cell as estimated by SDS-PAGE and scanning analysis of gel image. Polyclonal antibodies were raised in rabbits against the purified protein and the reaction of the antibody was confirmed by western blotting using the purified protein and crude extract of V. harveyi. Orange-spotted groupers (Epinephelus coioides) vaccinated with recombinant OmpK produced specific antibodies, and were highly resistant to infection by virulent V. harveyi. These results indicate that the OmpK is an effective vaccine candidate against V. harveyi in Orange-spotted groupers.

Cloning and characterization of the tiger shrimp lysozyme

Lysozymes are key proteins to invertebrates in the innate immune responses against bacterial infections. A lysozyme gene isolated from tiger shrimp, Penaeus monodon, was cloned, sequenced and characterized. The cDNA consists of a signal peptide of 18 amino acids and a mature peptide of 140 amino acids. The lysozyme is presumed to be a chicken-type lysozyme for it possesses two catalytic sites and eight cysteine residues which are highly conserved across species of chicken-type lysozymes. The lysozyme cDNAs of Penaeus semisulcatus, Litopenaeus vannamei, Macrobrachium nipponense and Macrobrachium rosenbergii were also cloned. High similarities existed among shrimp and prawn lysozymes but phylogenetic relationship of shrimps and prawns based on lysozyme molecules did not quite consistent with traditional taxonomic classification. High mRNA expression was detected in hepatopancreas, haemocytes and gill of tiger shrimp. Recombinant lysozyme exhibited potent lytic activities against fish pathogens providing evidence of the involvement of lysozyme in shrimp immunity.

Rapid identification of Streptococus agalactiae and Streptococus iniae with duplex PCR assay.

Streptococus agalactiae and S. iniae are two common pathogens of fish Streptococcus diseases. Symptoms of disease caused by both Streptococcus pathogens were quite similar, and it was difficult to distinguish the two pathogens from each other directly from the symptoms. Therefore it is necessary to develop a rapid identification method in order to control the disease effectively and rapidly. According to the cfb gene sequence of S. agalactiae and 16S rRNA gene sequence of S. iniae, two pairs of primers were designed and synthesized, and a rapid duplex PCR assay for identification of S. agalactiae and S. iniae was established by optimization of amplification conditions. A 474 bp fragment specific for S. agalactiae and a 296 bp fragment for S. iniae were produced, but no product was amplified from the other common pathogenic bacteria of fish such as Aeromonas hydrophila and Pseudomonas aeruginosa. The method is quite sensitive, and can detect a template concentration as low as 3.2×10-3 ng/μL for S. agalactiae and 3.0×10-2 ng/μL for S. iniae, respectively. A total of eleven pathogen samples collected from pond-cultured tilapia in Guangdong and Hainan provinces were analyzed by duplex PCR. Sequencing and BLAST analysis revealed that all the sequences were cfb gene of S. agalactiae. Results of molecular identification were consistent with the previous results of biochemical assays. The double PCR method would provide a sensitive and accurate method for rapid identification of S. agalactiae and S. iniae.

Identification of microsatellite markers associated with growth traits in largemouth bass (Micropterus salmoides L.)

Forty microsatellite loci were selected to amplify the genomic DNA of cultured stock of largemouth bass (Micropterus salmoides L.) in China. Genotypic differences of these microsatellites loci were analyzed using 2-test between the maximal weight group and minimal weight group. Sixteen microsatellites showing significantly difference (P<0.1) were used in genotyping 121 individuals, and the associations between their genotypes and growth traits were examined. Microsatellite loci of JZL60, JZL67, JZL72, JZL124, MiSaTPW76, MiSaTPW117 and MiSaTPW173 were significantly associated with body weight, body length, and body height (P < 0.05 or P < 0.01). The most favorable genotypes for growth traits were AA at JZL60, BB at JZL67, AC at JZL72, BB at MiSaTPW76 and BC at MiSaTPW117. In addition, a total of 47 alleles for the 16 loci were detected (2 approximately 5 alleles for each locus). The average effective number of alleles (Ae), observed heterozygosity (Ho), expected heterozygosity (He) and mean polymorphic information content (PIC) was 2.938, 0.515, 0.500 and 0.445, respectively, both of which indicated that the population genetic diversity was medium.

The genetic structure of four populations of Oreochromis aureus by microsatellite DNA analysis.

Genetic diversity and phylogenetic relationships among four breed varieties of Oreochromis aureus (83), O.aureus (02), O.aureus (05) and red O.aureus were studied using microsatellite analysis technology.19 pairs of microsatellite DNA primers of Oreochromis niloticu registered in GenBank which were selected and proved to amplify successfully on Oreochromis aureus by pre-experiment were used.The PCR reaction condition especially concentration of Mg2+ and annealing temperature were adjusted for each pair of primers.Amplified PCR products were electrophoresed in PAGE and stained according to the silver stain procedures and then imagined by Alphalmager.A total of 82 alleles were obtained from the four populations.The number of alleles for each locus ranged from 2-8, and the average alleles was 4.3 each locus.Average observed heterozygosity and average expected heterozygosity were calculated by POPGENE3.2 software.The results showed that the observed value of average heterozygosity of the four O.aureus populations ranged from 0.154 to 0.391.The expected value of average heterozygosity ranged from 0.181 to 0.428, and the average polymorphism information contents ranged from 0.1513 to 0.3882, respectively.The lowest genetic diversity level was found in the O.aureus (02).The highest genetic similarity index (0.9422) and the lowest genetic distance (0.0596) were found between O.aureus (05) and O.aureus (83), which indicated a closer phylogenetic relationship between these two populations.On the other hand, the lowest genetic similarity index (0.6977) and the highest genetic distance (0.3599) existed between O.aureus (83) and red O.aureus, which suggested a farthest phylogenetic relationship between these two populations.The evaluation results of the genetic deviation index D revealed that there were loci departed from Hardy-Weinberg equilibrium in all the four O.aureus populations.Results of null allele analysis by MicroChecker showed that there were loci showing evidences for null alleles in the populations except red O.aureus population, including one locus for O.aureus (83), two for O.aureus (02) and seven for O.aureus (05).Such results suggested that locus with evidence for null alleles in the populations except red O.aureus might be one of the reasons causing deviation of Hardy-Weinberg equilibrium.Phylogenetic relationship of the four populations was analyzed by using UPGMA methods based on their genetic distances and the results showed that O.aureus (83) and O.aureus (05) populations were clustered in the first group, the O.aureus (02) in the second group, while the red O.aureus was in a separate group.

SNPs detection in largemouth bass myostatin gene and its association with growth traits

Myostatin or GDF-8,a member of the transforming growth factor-β(TGF-β) surperfamily,has been demonstrated to be a negative regulator of myogenesis and inhibited myoblast proliferation during cell cycle and myogenic differentiation in animals.In present research,two SNPs sites(C-1453T and T+33C) were identified from total MSTN gene in larghmouth bass by using PCR-SSCP technique and sequencing;The mutational site of C-1453 was between the regions of E8 box and Octamer(+).the mutation of T+33C was situated in the first extron of the MSTN gene,however the mutation of the first extron has not changed the encoded amino which belonged to the synonymy mutation.The three kinds of genotypes per mutation site were obtained by PCR-RFLP method and the genotype frequency of the mutation was caculated in the population of 128 individuals.A general linear model was used to statistically analyze associations of MSTN gene polymorphisms with major growth traits respectively,and the results indicated that there was no significant difference(P0.05) by all genotypes of single mutation site.Six diplotypes,with the minor allelic frequencies of above 3%,were constructed based on two SNPs in the experiment population.The means of diplotype D2 were over other diplotypes and the means of diplotype D5 were lower other diplotypes in terms of body width,body height,body length,body weight,and snout length therfore associations analysis indicated that there were significant associations between diplotype D2 and D5 in the five growth traits of largemouth bass.The study implies that diplotypes D2 and D5 of MSTN gene could be used as the moleculer genetic marker for selection breeding.

Correlation analysis of microsatellite DNA markers with major growth traits of tilapia (Oreochromis niloticus).

In order to obtain more molecule markers linked to growth-related traits of tilapia and evaluate their accuracy,65 microsatellites loci were amplified in two Nile tilapia populations (Luye and Panyu),and the marker-trait association was analyzed by GLM(general linear model)procedure of SPSS software. The correlation analysis results showed that 8 loci(UNH130,UNH183,UNH911,GM558,UNH211,UNH176,UNH914 and UNH974) had a significant impact on major growth traits (weight,body length and body height) of Luye population(P0.05 or P0.01). In Panyu tilapia population,UNH914 and UNH974 had a significant impact on major growth traits (P0.05 or P0.01),and UNH176 just had a significant impact on body weight (P0.05). The favorable genotypes or allele linked to growth traits including weight,body length and body height were determined by multiple comparison analysis between the phenotypic value and different genotypes of microsatellites markers linked to major growth traits. In this study,the microsatellite loci with a significant effect on growth traits were found,and they were potential valuable genetic markers for marker-assisted selection of tilapia.