| The half-smooth tongue sole (Cynoglossus semilaevis) is one of the important economic marine fish species in north China, and is highly valued because of its outstanding taste. But factually in nature, females grow two to three times faster and bigger than males, and this specie is sexually dimorphic, so low proportion of physiological female limited the rapid development and large-scale production. Furthermore, it is important to exactly understander the gonad differentiation and sex determination mechanism of half-smooth tongue sole, which is of great significance to improve the quality of breeding and single sex breeding. Currently, sex determination mechanism ofhalf smooth tongue soleis unclear, and their gender differentiation decisions were not found. So study of gender related geneesand analysis of their functions in half smooth tongue sole is artificial for the control half smooth tongue sole gender and provide basic data.Hedgehog (HH) signaling plays crucial roles in embryonic development and adult tissue homeostasis, and its abnormalleads to many diseases including cancer. In this study, according to the information of genomic sequencing of Cynoglossus semilaevis, the complete cDNA of csshh、csdhh、csptchl x1、csptchl x2、 cssmo in Hedgehog signaling pathway were cloned and named csshh, csdhh, csptchl x1, csptchl x2, cssmo and csglil.The temporal expression of csshh、csdhh、csptchl x1、 csptchl x2、cssmo and csglil in different embryonic and gonad developmental stages and adult tissue distribution was analyzed by RT-qPCR. The methylation degrees of csdhh, csshh, csptchl x1 of promoter region in gonad tissues were analyzed. In addition, In Situ Hybridization of the csdhh, csptchl x1 and csptchl x2 in gonad tissues were analyzed. By Fluorescence In Site Hybriditzation(FISH), we located the position of csptchl x1 and csptchl x2 in chromosome.The construction of eukaryotic expression vector of csshh amd csdhh, and prokaryotic csptchl x1 and csptchl x2 were recombined, which will promote the depth function research in future.The results as follows.After RT-PCR and RACE, a 1922bp csshhcDNA was obtained. The complete cDNA sequence contains a 266 bp 5’UTR, a 1296 bp open reading frame (ORF), along with a 360 bp 3’UTR. The ORF encodes a putative protein with 432 amino acid residues, with a predicted molecular weight of 47.28 kDa and an isoelectric point (pI) of6.95.The full length of csdhh cDNA is 2473bp, along with a 475 bp 5’UTR, a 612bp 3’UTR and a 1386 bp ORF, encoding 432 aa, with a predicted molecular weight of 51.38kDa and an isoelectric point (pI) of 8.17.The full length of csptchl x1 cDNA is 5212bp, along with a 298 bp 5’UTR, a 282 bp 3’UTR and a 4269 bp ORF, encoding 1543 aa, the molecular weight of csptchl x1 is 171.5KDa, the isoelectric point is 6.29.The full length of csptchl x2 cDNA is 5773bp, along with a 66 bp 5’UTR, a 769 bp 3’UTR and a 4638 bp ORF, encoding 1543 aa. The full length of cssmo cDNA is 2535bp, along with a 98 bp 5’UTR, a 232 bp 3’UTR and a 2205 bp ORF, encoding 736 aa, with a predicted molecular weight of 83.14kDa and an isoelectric point (pI) of8.35.All of the five cloned gene has the classical structures with their family. By the Phylogenetic analysis, we found they are highly conserved during evolution.2. In the period of embryonic development stages, the relative quantitative results showed that:in this study, the expression of the 6 genes in HH signaling pathways of half smooth tongue sole showed a rise-fall trend. From blastocysts they began to rise. Csdhh gene, cssmo and csglil gene in the neural stage had a highest expression, which is significantly higher than other period. While csshh gene, csptchl x1 and csptchl x2 relatively high expressedin the blastocyst, articulate and foramen closed period, respectively. These stages are critical period of cell differentiation, suggesting that the HH signaling pathway play an important role in embryo differentiation and the formation of tissues and organs.3. The relative expression in eight organs of adult female and male wereanalyzed. Results showed that the the six genes has certain expression in alleight kinds of organs, indicated they all closely related to the organs’development. Csdhh csptchl x1, cssmo and csglil had a common expressionstyle, expressed high in male testis, so we speculation csdhh--csptch 1 x1 --cssmo--csglil signaling pathways is important for the development of the testis. While csshh gene and csptchl x2 expressed higher in liver and brain,and female gland expression is higher than male, so we speculated that transmembrane protein csptchl x2 gene is membrane receptor of csshh outside the cell, had an important role in the occurrence of a variety of tissues and organs, especially the development of female gonad.4. The relative expression in different development periods of gonad were analyzed. The results showed that the expression of csdhh genes in male from 35 days to 65 days was extremely low, and an uptrend since 80 days, a peak in 150 days, from one-age to two-age fell, and at 95 days, the relative expression in the testis is significantly higher than that of ovary, suggested csdhh gene play an important role in differentiation and development of the testis. csdhh, csptchl x1, cssmo and csglil genes had a rapid rise in 95 days in male gonad, and the testis expression in male and pseudo male is significantly higher than the expression in ovarian of one-year, which illustrated csdhh--csptchl x1--cssmo--csglil signaling pathways is important for the development of the testis. As to the csshh gene expression, we found that from 50 days to 65 days, the stage female gonad started to differentiate; the relative expression of csshh in female gonad was higher than that of males. While from 80 days to 95 days, the stage male sex started to differentiate, the relative expression of csshh gene in male was higher than that of female. The relative expression csshh gene in 8-month, one and two-year in female gonad was significantly higher than that of male and pseudo-male, which suggested that csshh gene has a certain relationship with ovarian differentiation and development. While csptchl x2 specifically expressed in the female gonad and a marked increase was turned in the gonad differentiated of 50 days, a low expression from 65 days to 65 days, the expression pattern is same as csSHH, suggesting csptchl x2 may be membrane receptor of csshh gene in the process of signaling pathways, regulating the gonad differentiated and development in the female gonad development.5. The methylation degree of csdhh, csshh, csptchl x1 of promoter region in gonad tissues were analyzed. The results of the methylation degree of csdhh, csshh, csptchl x1 was higher in female than that of male. The gene expression was positive correlation with the methylation degree.6. The csdhh, csptchl x1 and csptchl x2 in situ hybridization in gonad were analyzed. According to the results of csdhh and csptchl x1, hybridization signals mainly expressed in the primary spermatocyte, secondary spermatocyte and sperm cells and expressed support cells of one-year testis, while weak signals expression in ovarian; while the results of csptchl x2, Hybridization signals mainly expressed in primitive germ cells, oogonium, oocyte and follicular cells of ovary, and the primary spermatocyte, secondary spermatocyte of testit. And the expression in ovarian is extremely weak. And the expression of one-age male and female gonad is higher than the expression of 8-month, which further intuitived that HH signaling pathways play an important role in germ cell development.7. By Fluorescence In Site Hybriditzation (FISH), we concluded that csptchl x1 and csptchl x2 were located in normal chromosome not in sexchromosome.8. The construction of eukaryotic expression vector of csshh amd csdhh, and prokaryotic csptchl x1 and csptchl x2 were recombined, which will promote the depth function research in future. |
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