【作者】 李楠；

【导师】 杨弘；

【作者基本信息】 南京农业大学 ， 水产养殖， 2021， 硕士

【摘要】 鱼类的繁殖活动受到环境因子和自身脑-垂体-性腺轴（brain-pituitary-gonad,BPG）的共同调控。在鱼类的BPG轴中,促性腺激素释放激素（Gonadotropin-releasing hormone,Gn RH）是在大脑生成并分泌,作用于垂体,垂体受到刺激,释放出促性腺激素（gonadotropic hormone,GtH）,GtH和性腺上的促性腺激素受体（gonadotropic hormone receptors,GtHR）结合后会开启下游通路来参与鱼类的繁殖活动。本文的研究对象为尼罗罗非鱼（Oreochromis niloticus）,使用RACE（rapid amplification of c DNA ends）技术,获得了尼罗罗非鱼促黄体生成素受体（LHR）和促卵泡激素受体（FSHR）基因全长;使用实时定量荧光PCR对其进行组织表达、外源激素注射和温度处理后,FSHR和LHR基因及生殖有关基因的表达以及酶联免疫法（ELISA）测定血清中性类固醇类激素含量的变化进行了一系列研究。1.尼罗罗非鱼FSHR和LHR基因克隆与序列分析尼罗罗非鱼FSHR基因cDNA全长2577 bp（Gene Bank:MW353713）,开放阅读框长2106 bp,共编码701个氨基酸,5’UTR长174 bp,3’UTR长297 bp。尼罗罗非鱼LHR的c DNA全长3177 bp（Gene Bank:MW353714）,开放阅读框长2082 bp,共编码693个氨基酸,5’UTR长221 bp,3’UTR长874 bp。生物信息学分析显示,尼罗罗非鱼FSHR和LHR在进化阶段相对保守。2.尼罗罗非鱼FSHR和LHR基因组织表达分析RT-PCR检测FSHR和LHR在尼罗罗非鱼肝脏、脾脏、头肾、肾脏、鳃、肠、肌肉、脑、心脏、血液、皮肤、精巢、卵巢等13个组织中的表达情况。结果显示:尼罗罗非鱼FSHR和LHR主要在精巢、卵巢中表达。3.外源激素注射对尼罗罗非鱼FSHR和LHR及相关基因表达影响和血清中性类固醇类激素含量变化分析在注射不同外源激素（DOM、LRH-A₂、DOM+LRH-A₂）后的72 h内,在卵巢中,注射组FSHR表达量先上升后下降,在12 h达到最大值（P<0.05）;LHR在注射后72 h达到最大（P<0.05）。在雌鱼的脑中,注射组FSHR表达量在12 h达到最大值（P<0.05）,在注射后72 h表达量降至最低（P<0.05）;LHR在注射后72 h表达量将至最低（P<0.05）。注射组E₂和P均呈现出先上升后下降的趋势,并分别在24 h和36 h达到最大值（P<0.05）。在卵巢中,注射组VTG表达量呈现先上升后下降的趋势,并在48 h达到最大（P<0.05）;在雌鱼的肝脏中,注射组VTG的表达量呈现先上升后下降的趋势,并在36 h达到最大（P<0.05）。卵巢中Amh的表达量呈现出先下降后上升的趋势,在注射后的24 h和72 h Amh的表达量显著低于对照组（P<0.05）。在精巢中,注射组FSHR在注射后呈现先上升后下降的趋势,在24 h达到最大（P<0.05）;LHR表达量在注射后72 h表达量达到最大（P<0.05）。注射组雄鱼血清中的T均呈现出先上升后下降的趋势,并都在注射后的24 h达到最大值（P<0.05）。在雄鱼的大脑中,注射组FSHR呈现出先上升后下降的趋势,并在24 h达到最大值（P<0.05）;LHR表达量呈现出先上升后下降的趋势,在36 h时表达量达到最大（P<0.05）。精巢中Amh的表达量呈现出先下降后上升再下降的趋势,在注射后的36 h和72 h Amh的表达量显著低于对照组（P<0.05）。4.温度对尼罗罗非鱼FSHR和LHR及相关基因表达影响表达和血清中性类固醇类激素含量变化分析在不同温度（24℃、28℃、32℃）处理28 d内,结果显示:雌鱼血清E₂均呈现出不断上升的趋势,在第28 d,各个温度处理组E₂含量与0 d各个温度处理组E₂含量相比差异显著（P<0.05）。血清P含量呈现出先上升后下降再上升的趋势,在第28 d时,28℃处理组的P含量与24℃处理组P含量差异显著（P<0.05）。雌鱼脑中FSHR呈现出先下降后上升再下降的趋势,在第28 d时,不同温度处理组脑中的FSHR表达量与0 d不同温度处理组脑中的FSHR表达量差异显著（P<0.05）;LHR呈现出先下降后上升的趋势,在第28 d时,28℃处理组的LHR表达量与24℃处理组的LHR表达量差异显著（P<0.05）。卵巢中FSHR呈现出先上升后下降又上升的趋势,在第7 d、21 d,不同温度处理组卵巢中的FSHR表达量与0 d不同温度处理组卵巢中的FSHR表达量相比差异显著（P<0.05）;LHR呈现出先下降后上升再下降的趋势。在卵巢中,各个温度处理组VTG表达量均呈现先上升的趋势,在第14 d,28℃和32℃组中VTG的表达量与24℃组中VTG的表达量相比差异显著（P<0.05）;在雌鱼的肝脏中,不同温度组VTG表达量均呈现出下降的趋势,在第28 d,不同温度组VTG表达量相较于0 d VTG表达量显著下降（P<0.05）。雌鱼卵巢中Amh的表达量基本呈现出下降的趋势,在14 d,不同温度处理组Amh的表达量与0 d相比显著下降（P<0.05）。雄鱼血清T均呈现出不断上升的趋势,在第28 d时,24℃处理组T含量与28℃处理组相比差异显著（P<0.05）。雄鱼脑中FSHR呈现出先下降后上升再下降的趋势,在第28 d,不同温度处理组脑中的FSHR表达量与0 d不同温度处理组脑中的FSHR表达量相比差异显著（P<0.05）;LHR呈现出先下降后上升再下降的趋势。精巢中FSHR呈现下降的趋势,在第7 d后,FSHR表达量相对于0 d显著下降（P<0.05）,LHR呈现出先下降后上升的趋势,在第14 d后,各个温度处理组精巢中的LHR表达量对比0 d均显著降低（P<0.05）。精巢中Amh的表达量呈现出下降的趋势,在第21d,不同温度处理组Amh的表达量与0 d相比显著下降（P<0.05）。更多还原

【Abstract】 The reproductive activity of fish is affected by environmental factors and its own brain pituitary gonad（BPG）axis.In the BPG axis of fish,gonadotropin releasing hormone（Gn RH）is produced and excreted in the brain and acts on the pituitary gland.The pituitary gland is stimulated to excrete gonadotropic hormone（GTH）.The combination of GTH and gonadal receptor（GtHR）will open the downstream pathway to participate in the reproductive activities of fish.The research object of this article is Nile tilapia（Oreochromis niloticus）.Using RACE（rapid amplification of c DNA ends）technology,the Nile tilapia luteinizing hormone receptor（LHR）and follicle stimulating hormone were obtained.Real time quantitative PCR and enzyme-linked immunosorbent assay（ELISA）were used to analyze the tissue expression,the expression of related genes after exogenous hormone injection and temperature treatment,and the changes of serum neutral steroid hormone concentration.1.Cloning and sequence analysis of FSHR and LHR of Nile tilapiaNile tilapia FSHR gene c DNA has a full length of 2577 bp（Gene Bank:MW353713）,of which the open reading frame is 2106 bp,and a total of 701 amino acids are encoded.The5’UTR is 174 bp and 3’UTR is 297bp.The full-length c DNA of Nile tilapia LHR is 3177 bp（Gene Bank:MW353714）,the open reading frame is 2082 bp,and a total of 693 amino acids are encoded.The 5’UTR is 221 bp,the 3’UTR is 874 bp.Bioinformatics analysis showed that FSHR and LHR of Nile tilapia were relatively conservative in the evolutionary stage.2.The tissues expression of FSHR and LHR in Nile tilapiaThe expression of FSHR and LHR in 13 tissues of Nile tilapia was detected by quantitative real-time PCR.The results showed that FSHR was mainly expressed in the testis and ovaries.2.Effects of exogenous hormone injection on the expression of FSHR,LHR and related factors in Nile tilapia and the changes of serum sex steroid hormone content.Within 72 hours after the injection of different exogenous hormones（DOM,LRH-A₂,DOM+LRH-A₂）,the expression of FSHR in ovaries of the injection group first increased and then decreased,reaching the maximum at 12 hours（P<0.05）;LHR reached the maximum at 72 h after injection（P<0.05）.In the brain of female fish,the expression of FSHR in the injection group reached the maximum at 12 h（P<0.05）,and decreased to the minimum at 72 h（P<0.05）;The expression of LHR was the lowest 72 h after injection（P<0.05）.In the injection group,E₂and P showed a trend of increasing first and then decreasing,and reached the maximum at 24 h and 36 h,respectively（P<0.05）.In the ovary,the expression of VTG in the injection group increased first and then decreased,and reached the maximum at 48 h（P<0.05）,In the liver of female fish,the expression of VTG in the injection group increased first and then decreased,and reached the maximum at 36 h（P<0.05）.The expression of AMH in ovaries decreased at first and then increased.The expression of AMH at 24 h and 72 h after injection was significantly lower than that in the control group（P<0.05）.In testis,FSHR in injection group increased first and then decreased,and reached the maximum at 24 h（P<0.05）;The expression of LHR reached the maximum at 72 h after injection（P<0.05）.T in the serum of male fish in the injection group showed a trend of increasing at first and then decreasing,and reached the maximum at 24 h after injection（P<0.05）.In the brain of male fish,FSHR in the injection group increased first and then decreased,and reached the maximum at 24 h（P<0.05）;The expression of LHR increased first and then decreased,and reached the maximum at 36 h（P<0.05）.The expression of AMH in testis showed a trend of first decreasing,then increasing,and then decreasing.The expression of AMH in testis was significantly lower than that in control group at 36 h and72 h after injection（P<0.05）.4.The effect of temperature on the expression of FSHR,LHR and related factors in Nile tilapia and the changes of serum sex steroid hormone content.In different temperatures（24℃,28℃,32℃）treatment for 28 days,the results showed that the E₂Content in serum of female fish showed a rising trend.On the 28th day,the E₂Content of each temperature treatment group was significantly different from that of each temperature treatment group on the 0 day（P<0.05）.On the 28th day,the P content of28℃treatment group was significantly different from that of 24℃treatment group（P<0.05）.On the 28th day,the expression of FSHR in the brain of different temperature treatment groups was significantly different from that in the brain of different temperature treatment groups on the 0th day（P<0.05）;LHR showed a downward trend and then an upward trend,On the 28th day,the expression of LHR in 28℃treatment group was significantly different from that in 24℃treatment group（P<0.05）.FSHR in ovaries showed a trend of first increasing,then decreasing and then increasing,On day 7 and 21,the expression of FSHR in ovaries of different temperature treatment groups was significantly different from that on day 0（P<0.05）,LHR showed a trend of first decreasing,then increasing and then decreasing.In ovaries,the expression of _VTGin each temperature treatment group was increased first,and the expression of VTG in the 14 day,28℃and32℃group was significantly different from that in 24℃group（P<0.05）.In the liver of female fish,the expression of VTG in different temperature groups showed a downward trend.On the 28th day,the expression of VTG in different temperature groups was significantly lower than that on the 0th day（P<0.05）.The expression of AMH in the ovary of female fish showed a downward trend,On day 14,the expression of AMH in different temperature treatment groups was significantly lower than that on day 0（P<0.05）.FSHR in the brain of male fish showed a trend of first decreasing,then increasing,and then decreasing,On the 28th day,the expression of FSHR in the brain of different temperature treatment groups was significantly different from that in the brain of different temperature treatment groups on the 0th day（P<0.05）,LHR showed a trend of first decreasing,then increasing and then decreasing.FSHR in testis showed a downward trend,after 7 days,the expression of FSHR decreased significantly（P<0.05）,LHR showed a downward trend and then an upward trend,after 14 days,the expression of LHR in testis of each temperature treatment group was significantly lower than that of 0 day（P<0.05）.The expression of AMH in testis showed a downward trend.On the 21st day,the expression of AMH in different temperature treatment groups was significantly lower than that on the 0 day（P<0.05）.更多还原