| Sex determination is the basis of gonad development and differentiation. In the system evolution of vertebrates, fish serves a key position. Fish as the earth’s most widely distributed, most species of vertebrates, with higher vertebrates, fish sex determination mechanism has three characteristics: primitiveness, diversity and changeability, they are all currently known type of sex determination of vertebrates. The sex of the fish are expressed as two processes: sex determination and sex differentiation, both different and interrelated. The gender is determined by the gonads form different genotypes in the direction of differentiation; Sex differentiation is programmed through a series of events, testis or ovary with secondary process. These two processes is the interaction and balance, finally show the function, form and behavior of male or female. Usually male and female genotypes which produce the phenotype of male and female. Sex differentiation in lower vertebrates, however, are susceptible to environmental factors of interference, appear different on genotype phenotype. So the diversity of fish sex determination and sex gland differentiation mechanism and instability, is the research of sex determination and sex control technology to provide the space.Paralichthys olivaceus belongs to the Pleuronectiforms, commonly known as the flounder, has more than 20 coastal natural distribution in north China, its meat is tender, fast growth, high disease resistance, high economic value, so the popularity of its breeding has been rapidly. Especially in the northern coastal in our country, has to carry out the P.olivaceus and P.olivaceus plaice factory farming. Research shows that in P.olivaceus and desert spot were P.olivaceus plaice, individual exists obvious difference between male and female, female big and fast growth. So the P.olivaceus plaice mechanism of sex determination and sex gland differentiation, to gain the whole female population, has important economic benefits and application prospects.Micrornas in eukaryotes is kind of short in length after the gene transcription level of non-coding single small RNAs that length about 20 ~ 24 nucleotides(a few less than 20 nucleotides). Widely exists in animals, plants, nematodes, including people, all kinds of creatures, the evolution of the highly conservative, tissue specificity. These mi RNA as animal growth, development, one of the key control factor in the disease, so far in P.olivaceus development related research has not been reported. Previously our laboratory has established ovarian micrornas library, and through the second generation sequencing(NGS) technology and bioinformatics methods identified 141 mature mi RNA, a number of differences in male and female gonads were screened out or specific expression of micrornas. On this basis, this chapter selected two differentially expressed great micro RNAs 26(pol-mi R-26 a, b), through a public database of target gene prediction software Pic Tar, Target Scan predict pol-mi R-26 a, 26 b have multiple target m RNA, pick out the sex determination and sex gland differentiation related emx1(empty spiracles homeobox1) as well as the important male gender dmrt1 candidate genes. According to the query NCBI databases, this chapter was designed and synthesized dmrt1 3 ’UTR fragments of primers and PCR amplification, will receive the PCR gene and psi CHECK-2 carrier to connect, build wild type recombinant plasmid. At the same time by the method of site-directed mutagenesis, dmrt1 gene site-directed mutagenesis in vitro, build mutant recombinant plasmid, which respectively transfection into 293 T cells build pol-mi R-26 a, 26 b stable cell line, using the luciferase report experimental detection pol-mi R-26 a, 26 b and its target genes dmrt1 binding sites. The results showed that the wild type plasmid psi CHECK dmrt1-3 ’UTR with pol-mi R-26 a, b mimics group 26 luciferase activity was significantly lower than psi CHECK dmrt1-3’ UTR with pol-mi R-26 a, 26 b inhibitor and blank and NC group(P < 0.01). Pol-mi R-26 a, 26 b by combining dmrt1 3 ’UTR, degradation of cutting target genes which changed the luciferase activity.To explore the function of the empty spiracles homeobox 1(emx1) gene in the development of Paralichthys olivaceus(P. olivaceus), the emx1 was cloned by PCR, the sequence feature was analyzed by Bio Edit and MEGA softwares and the expression in early development and adult tissues was detected by Real-time quantitative PCR. The obtained c DNA was 1127 bp, including a 708 bp open reading frame ORF, encoding a polypeptide of 235 amino acids. The amino acid sequence alignment analysis displayed that the emx1 from P. olivaceus shows the highest 95% homology with that of Stegastes partitus and Poecilia formosa, the sequence similarities between P. olivaceus and Danio rerio, Astyanax mexicanus, Xenopus tropicalis, Gallus gallus, Mus musculus and Homo sapiens, are 87%, 84%, 83%, 80%, 72% and 72%, respectively. The Emx1 protein was highly homologous with Emx1 in other fish through phylogency analysis. Real-time quantitative PCR revealed that the emx1 m RNA is expressed in all detected tissues of P. olivaceus. The highest level of emx1 was found in the ovary and relatively high level was in the testis, while the expression was low in other tissues. In early development of P. olivaceus, a relatively high expression of the emx1 m RNA was also found from gastrula stage to 10 d after hatching, and the highest expression occurred in the blastopore stage and heart-beating stage. The findings indicated that the emx1 plays an important role in early stage development and gonad development of P. olivaceus.To investigate the high temperature and exogenous hormones on P. olivaceus gonad differentiated pol-mi R-26 a, b and target gene expression, the influence of by high temperature induction and exogenous hormone treatment gonad differentiated impaction P. olivaceus sensitive period, and by using the fluorescent quantitative PCR technique analyzes the pol- mi R- 26 a, b, dmrt1, emx1 expression in each treatment groups were. Experiment is divided into 17β-E2 treatment group, 17α- MT treatment group, 28 ℃ high temperature induced group and the control group, the results showed that high temperature can speed up the growth of P. olivaceus, fish male and the rate of 88.72%; 17α- MT and 17β-E2 can be induced by both P. olivaceus sex reversal. Fluorescence quantitative PCR results showed that the pol-mi R-26 a and 26 b expression trends are consistent, both in the high temperature induced the expression of group and the control group, and in 17α- MT and the expression of 17β-E2 treatment group are lower. After high temperature induced emx1 expression in gram-negative gonad rise significantly(P < 0.05), but 17α-MT and 17β-E2 treatment had no significant effect on its amount of expression. Dmrt1 in high temperature induced group and 17α- MT expression is significantly higher than control group in the treatment group were the gonads, the high temperature in the group of dmrt1 expression quantity is 5.98 times in the control group(P < 0.05); 17β-E2 in the treatment group and the expression of dmrt1 is lowest. Show that high temperature induced fish to the development of male may be associated with high temperature can rise dmrt1 gene expression. Dmrt1, moreover, in the high temperature higher expression of androgen group, this group may be related to high temperature were growing rapidly and were greater than its body length weight average androgen group. |
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