The Polymorphism Of Serine Protease Ⅰ-1 And C-type Lysozyme In Japanese Flounder (Paralichthys Olivaceus)

Japanese flounder (Paralichthys olivaceus) is an important flatfish in China. Its delicious flavor and high nutrition are adored by many customers, and it has important economic value. Industrial culture of Japanese flounder in China has been booming quickly since 1980s. The rush in flounder’s cultivation also raises serial problems, such as deteriorating culture environment, high frequency of diseases, low yield and antibiotic misuse. All these problems cause a drop of the genetic diversity in the breeding groups, making the flounders vulnerable to diseases, and the industry is suffering from the massive death of the flounders. There is a great urge in breeding high disease-resistance and quick-growing flounders to save the industry. Our studies are as follows:1. The Japanese flounders were injected by Vibrio anguillarumHealthy mixed group of Japanese flounders was infected by Vibrio anguillarum. They are divided into control group and treatment group. The control group is injected saline and the treatment group is injected 109cfu/ml bacteria suspension, used for intraperitoneal injection. The flounders died in the early period of the challenge experiment are graduated as susceptible individuals and the flounders survived through the challenge are classified resistant individuals.2. The polymorphism of serine protease I-1 (SPI-1) in Japanese flounderSerine protease is a group of proteolytic enzymes that are important for its potential applications. In this study, we identify nine single nucleotide polymorphisms (SNPs) in the coding sequence of serine protease I-1 gene in Japanese flounder (Paralichthys olivaceus), PoSP I-1. Moreover, we also amplify 2605bp promoter sequence of PoSP 1-1 and identify 28 polymorphic sites. All SNPs are genotyped not only the individuals susceptible to Vibrio anguillarum but also the individuals resistance. The genotype frequency of SNP365A/G in the coding sequence of PoSP 1-1 is characterized to be significantly different between the resistant group and susceptible group. As a non-synonymous mutation, the frequency of AG genotype in the resistant group is significantly higher (60%) than that in the susceptible group (40%) (P=0.01). The relative expression of PoSP I-1 in the resistant group is also higher than that in the susceptible group (p=0.0003). This implies that POSP 1-1 is involved in the resistance of Japanese flounder to Vibrio anguillarum.365A/G of PoSP 1-1 is a potential marker associated with the resistance. Thus, PoSP I-1 365A/G may be a candidate marker that could apply to disease resistant selective-breeding of Japanese flounder in the future.3. The polymorphism of c-type lysozyme in Japanese flounderLysozyme is a crucially widely spread shydrolase in organisms. This important defender can defend against bacterial pathogenic infection in innate immunity. In this study, we identify nine single nucleotide polymorphisms (SNPs) in the coding sequence of chicken-type lysozyme gene in Japanese flounder. Moreover, we also amplify 2077bp promoter region of lysozyme and identify two sites of insert-deletion (ins-del) polymorphisms and nine sites of SNPs. All SNPs are genotyped not only the individuals susceptible to Vibrio anguillarum but also the individuals resistance. SNP140 in the coding sequence of PolysC is characterized to be a significant marker via analyzing its distribution in the resistant individuals and susceptible individuals, which are distinguished on the basis of the survival time afterwards Vibrio anguillarum challenge. As a non-synonymous mutation, the frequency of SNP140GC genotype in the resistant group is extremely higher (70.0%) than that in the susceptible group (33.0%). The quantitative expression of lysozyme gene in liver, kidney and spleen in the resistant group is also higher than that in the susceptible group. This could imply that 140G/C may be a fairly association with the resistance of Japanese flounder to Vibrio anguillarum. This result can be further proved that chicken-type lysozyme gene may be associated with the resistant and susceptible individuals in Japanese flounder. These consequences imply that 140G/C may be a potential marker that could put into use in the disease resistant selective-breeding of Japanese flounder in the future.4. Cloning and sequencing of lysozyme and its promoter regionThe promoter region of PoSP I-1 and PolysC are searched from our genomic library sequenced by the 454 FLX Titanium sequencing platform using the cDNA sequence of serine protease I-1 and lysozyme.10 Japanese flounders are used in amplifying promoter region. By sequencing, twenty-eight polymorphisms are identified in the amplified promoter region of PoLysC. Two ins-del polymorphisms and nine SNPs are identified in the amplified promoter region of PoSP I-1. The transcription Factor Binding Sites of upstream regulatory region of PoSP I-1 and PolysC are analyzed using TFSEARCH.