Molecular Cloning Of Reproductive Gene And The Expression Patterns During Ovary Development And Their Responses To Temperature Stress In Yellow Catfish (Pelteobagrus Fulvidraco)

This paper mainly study on cultured female yellow catfish, from which the testis,hemadenology, FSHR/LHR, P450c17I, aromatase and Sox9are taken and studied bythe methods of molecular methods in details. The main results and conclusions are asfollows:1Molecular cloning of FSHR and LHR expression analysis duringthe reproductive cycle in female yellow catfish (Pelteobagrusfulvidraco)The complete cDNA sequence of LHR and FSHR were cloned throughdegenerate primer PCR amplification and SmartTMRace technology. We cloned thecomplete cDNA sequence of FSHR, the length is2340bp, encoding a protein of661amino acids and. LHR is2524bp and encode the protein of701amino acids. Thesegene belongs toGPCR family, which contains the conserved seven TM helix.Throughthe analysis we found that the amino acids sequence contains nine Leucine-richrepeats(LRRs), five potential N-linked glycosylation sites of FSHR. And that only oneprotein kinase C phosphorylation site is403Y in ELⅠand three protein kinase Cphosphorylation site in C-terminal domain. There are four potential N-linkedglycosylation sites in LHR and phosphorylation site predictions identified24Ser,6Thr and5Tyr potential phosphorylation sites. Predicted the only one protein kinase Cphosphorylation site is400S. The tissue distribution pattern showed highly expressionof FSHR in ovary and brain, and trace amounts was detected weakly in other tissues.The relative mRNA expression level of FSHR in ovary at different stages ofreproductive cycle suggested it was relative high from Ⅲ to Ⅳ phase. Andexpression level of FSHR in brain at different stages of reproductive cycle the highlyexpression in Ⅵ stage. Tissue distribution pattern showed preferential expression ofLHR in ovary, and trace amounts was detected in brain, kidney, heart, liver andintestine. The High transcript level of LHR in ovary was revealed in the reproductivephase of ovarian cycle, which was the relative high expression from Ⅲ to Ⅴ phase and the highest level in stageⅤ. And we also analyzed the brain expression level inreproductive cycle, found the highest level in stage Ⅳ consistent with the level of E2.We inferred the gene of FSHR in ovary might also be involved in the processes whichregulate ovulation maturation and ovulation. And preferentially promote vitellogeninaccumulation.And the gene of LHR in brain and ovary might also be involved in theprocesses which preferentially promote vitellogenesis and regulate ovulationmaturation and ovulate either indirectly or directly.2Molecular cloning of P450c17I and the expression Patterns duringOvary Development and Their Responses to Temperature Stress inFemale Yellow Catfish (Pelteobagrus fulvidraco)P450c17I, a key steroidogenic enzyme, play important roles in the production ofsex steroid and cortisol. The complete cDNA sequence of P450c17I was clonedthrough degenerate primer PCR amplification and SmartTMRace technology. Wecloned the complete cDNA sequence of P450c17I gene and the length is2071bp,encoding a protein of514amino acids.The GenBank number is HQ832642.3. Regionsimportant to enzymatic function, which include the transmembrane region, conservedP450c17-specific region, Ozols tridecapeptide region and cytochrome P450cysteineheme-iron ligand signature regions were identified in the yellow catfish P450c17I.P450c17-I mRNA was expressed at high levels in the ovary and head kidney, atmoderate levels in the brain, liver and stomach, and at low levels in the kidney, gilland spleen. Changes in serum T and CYP17-I expression levels corresponding withovarian developmental stage The High transcript levels of CYP17-I in ovary wasrevealed in the reproductive phase of ovarian cycle, which were the highest levels instage Ⅴ. While the levels of CYP17-I in brain were significantly dropped from stageⅤ,opposited to the trand of ovary. Except the stage Ⅵ, the expression of CYP17-I inovary corresponded with serum testosterone (T) levels by heat stress. But there wasno correlation with the expression of CYP17-I in brain.It has been shown that duringoocyte development and final maturation/ovulation, CYP17-I expression plays acentral role in the biosynthesis of sex-steroids.3Expression Patterns of Cytochrome P450Aromatase Genes duringOvary Development and Their Responses to Temperature Stress inFemale Yellow Catfish (Pelteobagrus fulvidraco) Cytochrome P450aromatase (P450arom) plays a pivotal role in ovarydevelopment. In this study, we used semi-quantitative reverse transcription PCR(RT-PCR) to analyze spatiotemporal expressions of two P450arom genes (CYP19Aand CYP19B) and their responses to temperature stress in female yellow catfish(Pelteobagrus fulvidraco). Tissue distribution pattern of CYP19showed that CYP19Bwas abundantly expressed in fish brain and ovary (brain> ovary), but weakly inintestines, whereas CYP19A was exclusively expressed in ovary. Semi-quantitativeRT-PCR analyses showed high transcript abundance of both CYP19A and CYP19B inthe ovarian reproductive cycle, corresponding with serum estradiol-17β (E2) levels.Increases in aromatases, serum E2and testosterone (T) levels in fish exposed to highertemperature indicated stimulation of ovarian maturation and recrudescence by heatstress in stages Ⅱ and Ⅴ during the ovarian cycle, whereas associated decreases instage Ⅲ suggested vitellogenesis inhibition by heat stress. Gene expression of CYP19was closely related to levels of serum E2. Results demonstrated CYP19played acrucial role in the reproductive cycle of female yellow catfish. Different temperaturestress affected CYP19gene expression in the fish ovarian reproductive cycle.Associated P450arom genes could be useful for studying physiological aspects ofyellow catfish.4The expression of Sox9during ovarian development and heat stressin yellow catfish（Pelteobagrus fulvidraco）Since the Sox9play a pivotal role during ovary development, in this study, theimportance of two forms of Sox9in the process of ovarian recrudescence in yellowcatfish (Pelteobagrus fulvidraco) was analyzed. The cloned of Sox9a1and Sox9a2have been submitted in NCBI. Tissue distribution pattern showed preferentialexpression of Sox9a2in brain and ovary (brain>ovary), and trace amounts wasdetected in stomach, while the extensive expression of Sox9a1was observed in themany tissue. A semi-quantitative RT-PCR was developed to measure the mRNA levels.The High transcript levels of both isoforms in ovary were revealed in the reproductivephase of ovarian cycle, which were the highest levels in stage Ⅲ(about Sox9a1) andⅤ(about Sox9a2).While the levels of Sox9in brain were significantly dropped fromstage Ⅱ. The phase-dependent rising of Sox9a2and Estradiol-17β (E2) after exposingfish to higher temperature revealed the stimulatory role of heat stress. Gene expression of Sox9a1in ovary can not corresponding with serum E2levels in fishexposed to higher temperature. Interesting Sox9expression in brain was contrary tothe level of E2in stage Ⅱ. The expression level of Sox9a1and Sox9a2were closelyrelated to level of serum E2in stage Ⅲ-Ⅵ. Results demonstrated Sox9a2in ovaryplayed a crucial role in the reproductive cycle of female yellow catfish. And Sox9a1inovary are not involved in controlling the reproductive cycle. In addition, we inferedtemperature can affect the negative regulation between Sox9in brain and E2, while themechanism needed to be further research.