Studies On Spermatogenesis-related Genes In Half Smooth Tongue Sole(cynoglossus Semilaevis) And Screening Of Immune-related Ests In Turbot(Scophthalmus Maximus)

Half smooth tongue sole (Cynoglossus semilaevis) is China’s unique high-qualitymariculture fishes. Tongue sole exhibits sexually dimorphic growth, with malesgrowing much slower than females, and the male individual is two to four times lessthan the female. In addition, tongue sole employ the female heterogamety sexdetermination system ZZ/ZW with a heteromorphic W chromosome. Femaleindividuals can reverse to male under field conditions. All of these reasons led to maleproportion is too high under the condition of natural breeding, which seriouslyhindered the tongue sole’s further development of the aquaculture industry. In order toimprove the proportion of female, we tried to use the pseudo-males as male parents inthe breeding. But the proportion of female does not improve. So we tested thegenotype of pseudo-male sperm, surprisingly, only Z sperm can be found. Combinedwith tongue sole genome sequencing results, we found that though W chromosomemuch larger than Z chromosome, but the number of genes in W chromosome far lessthan that in Z chromosomes, the genetic information is also contained a lot less. Inthis paper, three tongue sole Z chromosome specific genes, which related tospermatogenesis has carried on the preliminary study, and speculated the function ofthese genes during the process of spermatogenesis, for revealing tongue sole lackingof the W sperm, and provide theoretical basis for high female ratio and even theall-female breeding. The main results were as follows:1. Full length cDNA and the whole genomic sequence of tesk1in tongue solewas isolated and identified. Using quantitative PCR, we tested the expression pattern of tesk1in tongue sole. The results showed that, highest expression of tesk1mRNAwas detected in testis, and a small amount in the brain, liver, kidney. We detectedtesk1mRNA expression in the testis of male and pseudo-male individuals, but not intriploid males. We measured tesk1mRNA expression in testis at differentdevelopmental stages. There was no or very low levels of expression between16and116d after hatch. Between5–8months after hatch, the level of tesk1mRNAexpression increased significantly. The tesk1mRNA expression level in the testesincreased significantly1year after hatch. In situ hybridization results showed that thetesk1mainly in spermatid and spermatozoa of tongue sole. So we speculated thattesk1may play a role in spermiogenesis phase. The tesk1was located on the tonguesole Z chromosome by chromosome fluorescence in situ hybridization. By GenomeWalking, part of the upstream regulation sequence was obtained. And the HSF, SRY,C/EBP, CRE-BP transcription factor binding sites were discovered on it. Methylationanalysis showed that there was no relation between the gene expression level and themethylation level.2. We cloned the full length cDNA and the whole genomic sequence of neurl3intongue sole. Using quantitative PCR, we tested the expression pattern of neurl3. Theresults of quantitative PCR determined that neurl3expressed highly in testis. Theresults of quantitative PCR in different stage determined that the expression of neurl3was little before116day and was sharply increased at the age of116day, and theexpression at116day was the highest. In situ hybridization results showed that theneurl3mainly in spermatid and spermatozoa of tongue sole. The neurl3was locatedon the tongue sole Z chromosome by chromosome fluorescence in situ hybridization.We constructed prokaryotic expression vector pET28A-E3, recombinant protein withexpected molecular weight can obtained by IPTG induction.3. We obtained the full length cDNA and the whole genomic sequence of strbp intongue sole. We tested the expression pattern of strbp by quantitative PCR. We foundthat this gene is mainly expressed in mature tongue sole gonad (both testis and ovary),although in the brain and spleen also detected the expression of this gene, but muchlower than the gonads. In situ hybridization results showed that the tongue sole STRBP mainly expressed in testis in the stage from spermatid to spermatozoa. So wespeculated that strbp may play a role in this period.Turbot (Scophthalmus maximus) is an important species in aquaculture and itcontributes enormously to the economic development of coastal provinces in China.However, frequent outbreaks of disease serious impact on the development of turbotaquaculture industry. Using drugs can reduce the loss that causes the disease, but notonly make the pathogen resistance, more importantly, drug residues can causeenvironmental pollution, and pose a threat to human health. Therefore, the use ofmolecular biological technique to improve the disease resistance of farmed fish isimperative. We constructed the cDNA library of turbot spleen and head-kidney, andthe library storage was2.26x106and1.45x106respectively. A total of3656ESTs wereobtained by sequencing. Through bioinformatics analysis, we identified149genes inturbot, including45immune-related genes, which were found for the first time in theturbot.