生长阻滞罗氏沼虾相关激素含量及其基因的表达
发布时间：2018-07-27 10:47
【摘要】：近年来,扬州市高邮区域部分罗氏沼虾(Macrobrachium rosenbergii)养殖塘口出现“生长缓慢”症状,虾农统称为“铁壳虾”,“铁壳虾”现象凸显,给虾农带来了一定的经济损失和巨大的精神压力。国内学者从多方面对其进行了研究,但是,迄今为止,对于引起罗氏沼虾生长阻滞现象的主要原因仍无定论。罗氏沼虾作为甲壳动物,其生长伴随着蜕壳的过程,而蜕壳受到蜕壳激素的影响,生长阻滞的罗氏沼虾有可能存在激素水平的异常,而与激素相关的基因表达也可能表现出不同。因此,本论文假定,生长阻滞罗氏沼虾的激素含量及其相关基因的表达完全不同于生长正常的罗氏沼虾。为了验证上述假说,本论文以高邮区域人工饲养的罗氏沼虾为研究对象,首先测定采集样本个体的形态学参数,采用不同的数学统计方法对各形态学参数进行分析,确定鉴定罗氏沼虾生长阻滞个体的最佳统计学方法和最佳度量的参数;然后按照以上确定的最佳参数对不同的样本进行分类,对分类个体进行蜕皮时期的判别,选择蜕皮间期的罗氏沼虾,通过酶联免疫法测定各类样本中蜕皮激素、蜕皮抑制激素和甲基法尼酯的含量,通过荧光定量PCR测定各类样本的肝胰腺和眼柄中生长相关基因的mRNA表达水平,通过Western Blot测定各类样本的肝胰腺和眼柄中EcR蛋白的表达,探讨各类激素及其相关基因在生长阻滞现象中的作用,揭示影响水生甲壳类动物生长的生理调控机制,为广大养殖户的健康养殖提供可能的新措施和新方法。研究结果如下:1)聚类分析中能够将样本划分为正常虾、正常幼虾和阻滞虾的参数有体重、肥满度和第一尾节宽度/体长;主成分分析中能够区分三类虾的参数有体长、腹尾长、螯长、第一尾节宽度、体长/体重、腹尾长/体重;相关性分析中能够区分三类虾的参数有体重、体长、腹尾长、第一尾节宽度、体长/体重、腹尾长/体重。综合分析认为,聚类分析是最佳统计学方法,体重、肥满度和第一尾节宽度/体长3个参数是最佳统计学参数。区分正常虾、正常幼虾和阻滞虾的参数标准为:雄性体重参数范围22.1668～25.1034、17.0517～17.6608和 13.4706～14.214,雄性肥满度参数范围 1.1598～1.2127、1.2541～1.2640 和 1.1274～1.1556,雄性第一尾节宽度/体长比值参数范围0.1294～0.1360、0.1363～0.1384和0.1435～0.1494;雌性体重参数范围 18.1526～19.9768、13.2198～14.0242 和 11.1046～11.9157,雌性肥满度参数范围1.1332～1.1563、1.1873～1.2075和1.1010～1.1107;雌性第一尾节宽度/体长比值参数范围 0.1327～0.1376、0.1392～0.1414 和 0.1415～0.1441。2)2015年8月采集的样本中,雄性阻滞虾20E水平显著低于正常虾(P0.05),雌性阻滞虾20E水平显著低于正常虾和正常幼虾(P0.05);雌性阻滞虾MIH水平显著高于正常虾和正常幼虾(P0.05);雌雄阻滞虾MF水平均显著低于正常虾(P0.05)。2016年6月采集的样本中,雄性阻滞虾20E水平显著低于正常虾和正常幼虾(P0.05),雌性阻滞虾20E水平显著低于正常虾(P0.05);雌雄阻滞虾MIH水平均显著高于正常虾和正常幼虾(P0.05);雌性阻滞虾MF水平显著低于正常虾和正常幼虾(P0.05)。3)2015年8月采集的样本中,雄性阻滞虾肝胰腺中的EcR mRNA表达量显著低于正常虾(P0.05),MIH mRNA表达量显著高于正常虾和正常幼虾(P0.05),RxRmRNA表达量显著低于正常虾和正常幼虾(P0.05),雄性阻滞虾眼柄中的MIH mRNA表达量显著高于正常幼虾(P0.05);雌性阻滞虾肝胰腺中的EcR mRNA表达量显著低于正常虾和正常幼虾(P0.05),JHEH mRNA表达量显著高于正常幼虾,MIH mRNA表达量显著高于正常虾和正常幼虾(P0.05)。2016年6月采集的样本中,雄性阻滞虾肝胰腺中的EcR和RxR mRNA表达量均显著低于正常虾和正常幼虾(P0.05),GIH、MIH和JHEHmRNA表达量均显著高于正常虾和正常幼虾(P0.05),雄性阻滞虾眼柄中的GIH和MIH mRNA表达量显著高于正常虾和正常幼虾(P0.05);雌性阻滞虾肝胰腺中的EcR和RxRmRNA表达量显著低于正常虾和正常幼虾(P0.05),GIH和MIH mRNA表达量显著高于正常虾和正常幼虾(P0.05),JHEH mRNA表达量显著高于正常幼虾(P0.05),雌性阻滞虾眼柄中的GIH和MIH mRNA表达量显著高于正常虾和正常幼虾(P0.05)。4)2015年8月采集的样本中,雌性阻滞虾和正常幼虾眼柄中的EcR含量显著低于正常虾(P0.05)。2016年6月采集的样本中,雄性阻滞虾肝胰腺中的EcR含量显著低于正常虾(P0.05);雌性阻滞虾眼柄中的EcR含量显著低于正常虾(P0.05)。以上结果表明,聚类分析、主成分分析和相关性分析均可区分生长阻滞罗氏沼虾,聚类分析是区分的最佳统计学方法,而体重、肥满度和第一尾节宽度/体长是区分生长阻滞罗氏沼虾的最佳参数;生长阻滞虾中20E和MF的含量较低,而MIH的含量较高;生长阻滞虾中EcR和RxR基因表达量和蛋白水平较低,而MIH基因表达量较高。说明阻碍罗氏沼虾生长的原因可能是EcR和RxR的表达量下降和MIH表达量升高降低了 20羟蜕皮激素和甲基法尼酯的水平、提高了蜕皮抑制激素水平所致。
[Abstract]:In recent years, the culture pond mouth of the regional part of the Gaoyou region of Yangzhou City, Yangzhou, has the symptoms of "slow growth". The shrimp farmers are called "iron shell shrimp", "iron shell shrimp" phenomenon is prominent, which brings a certain economic loss and great spiritual pressure to the shrimp farmers. So far, the main reason for the growth block of the growth of the Macrobrachium roseneus is still unsettled. As a crustaceans, the growth of the Macrobrachium Roche is accompanied by the process of molting, and the molt is affected by the molting hormone, and the growth block of the Macrobrachium Roche may have abnormal hormone levels, and the gene expression related to the hormone may also show. Therefore, this thesis assumes that the hormone content and related genes of growth block of Macrobrachium rosenii are completely different from that of normal Roche marsh. In order to verify the hypothesis, the morphological parameters of the individuals in the collected samples were measured and the different numbers were used in this paper to study the artificial rearing marsh Roche prawns in Gaoyou. A statistical method is used to analyze the morphological parameters and determine the best statistical methods and parameters for identifying the growth block of the Macrobrachium macrobrachicus, and then classify the different samples according to the best parameters above, the classification of the molt period for the classified individuals, and the selection of the molt interphase Roche prawns, through enzyme linked The content of ecdysone, ecdysone and methylfarnyl in all kinds of samples was determined by immunoassay. The expression of mRNA expression of growth related genes in hepatopancreas and eye handle of various samples was measured by fluorescence quantitative PCR. The expression of EcR protein in hepatopancreas and eye handle of various samples was measured by Western Blot, and the various hormones and their correlation were discussed. The role of gene in growth retardation reveals the physiological regulation mechanism affecting the growth of aquatic crustaceans and provides possible new measures and new methods for the healthy breeding of the farmers. The results are as follows: 1) in the cluster analysis, the samples can be divided into normal prawns, and the parameters of normal juvenile and retarded shrimp are weight, fullness and first. One tail width / body length; principal component analysis can distinguish three kinds of shrimp with length, length, length, body length / body weight, abdominal tail length / body weight. In correlation analysis, the parameters of three types of prawns can be distinguished from weight, body length, tail length, first tail width, body length / weight, abdominal tail length / weight. Cluster analysis thinks clustering Analysis is the best statistical method. The 3 parameters of weight, fullness and first tail width / body length are the best statistical parameters. The parameter criteria for distinguishing normal prawns, normal young prawns and retarding shrimps are: the range of male body weight parameters is 22.1668 ~ 25.1034,17.0517 to 17.6608 and 13.4706 ~ 14.214, and the range of male fertility parameter ranges from 1.1598 to 1.2127,1.2541. To 1.2640 and 1.1274 to 1.1556, the width of the width / length ratio of the male first tail was 0.1294 ~ 0.1360,0.1363 to 0.1384 and 0.1435 to 0.1494; the range of female body weight parameters was 18.1526 ~ 19.9768,13.2198 to 14.0242 and 11.1046 ~ 11.9157. The range of female fertilizer full degree parameters ranged from 1.1556 to 1.2075 and 1.1010 to 18.1526. The 20E level of male block shrimp was significantly lower than that of normal shrimp (P0.05), and the 20E level of female block shrimp was significantly lower than normal shrimp and normal young shrimp (P0.05), and the MIH level of female block shrimp was significantly higher than normal prawns and normal prawns in the samples collected from 0.1327 to 0.1376,0.1392 to 0.1414 and 0.1415 ~ 0.1441.2 in August 2015. Chang Youxia (P0.05); the MF level of male and male retarding shrimp was significantly lower than that of normal shrimp (P0.05).2016 June. The 20E level of male block shrimp was significantly lower than that of normal and normal shrimp (P0.05), and the 20E level of female block shrimp was significantly lower than that of normal shrimp (P0.05), and the MIH level of male and male retarding shrimp was significantly higher than that of normal and normal young prawns (P0.05). The MF level of sexual block shrimp was significantly lower than that of normal shrimp and normal young shrimp (P0.05).3). In the samples collected in August 2015, the expression of EcR mRNA in the hepatopancreas of male block shrimp was significantly lower than that of normal prawns (P0.05). The expression of MIH mRNA was significantly higher than that of normal and normal young prawns (P0.05). The expression of RxRmRNA was significantly lower than that of normal and normal young prawns (P0.05). The expression of MIH mRNA in the eye handle of retarding shrimp was significantly higher than that of normal young shrimp (P0.05), and the expression of EcR mRNA in the hepatopancreas of female block shrimp was significantly lower than that of normal and normal young prawns (P0.05). The expression of JHEH mRNA was significantly higher than that of normal young prawns. The expression of MIH mRNA was significantly higher than that of normal shrimp and normal young shrimp (P0.05) in the samples collected in the.2016 year of.2016 year June. The expression of EcR and RxR mRNA in the hepatopancreas of sexual block shrimp were significantly lower than that of normal and normal young prawns (P0.05). The expressions of GIH, MIH and JHEHmRNA were significantly higher than those of normal and normal shrimp (P0.05). The GIH and MIH mRNA expression in the eye handle of male retarding shrimp was significantly higher than that of normal and normal young shrimps (P0.05). The expression of and RxRmRNA was significantly lower than normal and normal young prawns (P0.05). The expression of GIH and MIH mRNA was significantly higher than normal and normal young prawns (P0.05). The expression of JHEH mRNA was significantly higher than that of normal young shrimp (P0.05). The expression of GIH and MIH mRNA in the eye handle of female block shrimp was significantly higher than that of normal and normal juvenile shrimp. The EcR content in the eye handle of the female block shrimp and the normal juvenile shrimp was significantly lower than the normal shrimp (P0.05) samples collected in June.2016. The EcR content in the hepatopancreas of the male retarding shrimp was significantly lower than that of the normal shrimp (P0.05), and the EcR content in the eye handle of the female block shrimp was significantly lower than that of the normal shrimp (P0.05). The results showed that the cluster analysis and principal component analysis were found. The cluster analysis was the best statistical method to distinguish the growth block, and the weight, the fullness and the first tail width / body length were the best parameters to distinguish the growth block. The content of 20E and MF in the growth retarded shrimps was lower, and the content of MIH was higher; the EcR and RxR gene table in the growth retarded prawns The amount and protein level were low, and the expression of MIH gene was higher. It was suggested that the decrease of EcR and RxR expression and the increase of MIH expression decreased the level of 20 HYDROXYECDYSONE and methylfarnyl, and increased the level of ecdysone.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4
[Abstract]:In recent years, the culture pond mouth of the regional part of the Gaoyou region of Yangzhou City, Yangzhou, has the symptoms of "slow growth". The shrimp farmers are called "iron shell shrimp", "iron shell shrimp" phenomenon is prominent, which brings a certain economic loss and great spiritual pressure to the shrimp farmers. So far, the main reason for the growth block of the growth of the Macrobrachium roseneus is still unsettled. As a crustaceans, the growth of the Macrobrachium Roche is accompanied by the process of molting, and the molt is affected by the molting hormone, and the growth block of the Macrobrachium Roche may have abnormal hormone levels, and the gene expression related to the hormone may also show. Therefore, this thesis assumes that the hormone content and related genes of growth block of Macrobrachium rosenii are completely different from that of normal Roche marsh. In order to verify the hypothesis, the morphological parameters of the individuals in the collected samples were measured and the different numbers were used in this paper to study the artificial rearing marsh Roche prawns in Gaoyou. A statistical method is used to analyze the morphological parameters and determine the best statistical methods and parameters for identifying the growth block of the Macrobrachium macrobrachicus, and then classify the different samples according to the best parameters above, the classification of the molt period for the classified individuals, and the selection of the molt interphase Roche prawns, through enzyme linked The content of ecdysone, ecdysone and methylfarnyl in all kinds of samples was determined by immunoassay. The expression of mRNA expression of growth related genes in hepatopancreas and eye handle of various samples was measured by fluorescence quantitative PCR. The expression of EcR protein in hepatopancreas and eye handle of various samples was measured by Western Blot, and the various hormones and their correlation were discussed. The role of gene in growth retardation reveals the physiological regulation mechanism affecting the growth of aquatic crustaceans and provides possible new measures and new methods for the healthy breeding of the farmers. The results are as follows: 1) in the cluster analysis, the samples can be divided into normal prawns, and the parameters of normal juvenile and retarded shrimp are weight, fullness and first. One tail width / body length; principal component analysis can distinguish three kinds of shrimp with length, length, length, body length / body weight, abdominal tail length / body weight. In correlation analysis, the parameters of three types of prawns can be distinguished from weight, body length, tail length, first tail width, body length / weight, abdominal tail length / weight. Cluster analysis thinks clustering Analysis is the best statistical method. The 3 parameters of weight, fullness and first tail width / body length are the best statistical parameters. The parameter criteria for distinguishing normal prawns, normal young prawns and retarding shrimps are: the range of male body weight parameters is 22.1668 ~ 25.1034,17.0517 to 17.6608 and 13.4706 ~ 14.214, and the range of male fertility parameter ranges from 1.1598 to 1.2127,1.2541. To 1.2640 and 1.1274 to 1.1556, the width of the width / length ratio of the male first tail was 0.1294 ~ 0.1360,0.1363 to 0.1384 and 0.1435 to 0.1494; the range of female body weight parameters was 18.1526 ~ 19.9768,13.2198 to 14.0242 and 11.1046 ~ 11.9157. The range of female fertilizer full degree parameters ranged from 1.1556 to 1.2075 and 1.1010 to 18.1526. The 20E level of male block shrimp was significantly lower than that of normal shrimp (P0.05), and the 20E level of female block shrimp was significantly lower than normal shrimp and normal young shrimp (P0.05), and the MIH level of female block shrimp was significantly higher than normal prawns and normal prawns in the samples collected from 0.1327 to 0.1376,0.1392 to 0.1414 and 0.1415 ~ 0.1441.2 in August 2015. Chang Youxia (P0.05); the MF level of male and male retarding shrimp was significantly lower than that of normal shrimp (P0.05).2016 June. The 20E level of male block shrimp was significantly lower than that of normal and normal shrimp (P0.05), and the 20E level of female block shrimp was significantly lower than that of normal shrimp (P0.05), and the MIH level of male and male retarding shrimp was significantly higher than that of normal and normal young prawns (P0.05). The MF level of sexual block shrimp was significantly lower than that of normal shrimp and normal young shrimp (P0.05).3). In the samples collected in August 2015, the expression of EcR mRNA in the hepatopancreas of male block shrimp was significantly lower than that of normal prawns (P0.05). The expression of MIH mRNA was significantly higher than that of normal and normal young prawns (P0.05). The expression of RxRmRNA was significantly lower than that of normal and normal young prawns (P0.05). The expression of MIH mRNA in the eye handle of retarding shrimp was significantly higher than that of normal young shrimp (P0.05), and the expression of EcR mRNA in the hepatopancreas of female block shrimp was significantly lower than that of normal and normal young prawns (P0.05). The expression of JHEH mRNA was significantly higher than that of normal young prawns. The expression of MIH mRNA was significantly higher than that of normal shrimp and normal young shrimp (P0.05) in the samples collected in the.2016 year of.2016 year June. The expression of EcR and RxR mRNA in the hepatopancreas of sexual block shrimp were significantly lower than that of normal and normal young prawns (P0.05). The expressions of GIH, MIH and JHEHmRNA were significantly higher than those of normal and normal shrimp (P0.05). The GIH and MIH mRNA expression in the eye handle of male retarding shrimp was significantly higher than that of normal and normal young shrimps (P0.05). The expression of and RxRmRNA was significantly lower than normal and normal young prawns (P0.05). The expression of GIH and MIH mRNA was significantly higher than normal and normal young prawns (P0.05). The expression of JHEH mRNA was significantly higher than that of normal young shrimp (P0.05). The expression of GIH and MIH mRNA in the eye handle of female block shrimp was significantly higher than that of normal and normal juvenile shrimp. The EcR content in the eye handle of the female block shrimp and the normal juvenile shrimp was significantly lower than the normal shrimp (P0.05) samples collected in June.2016. The EcR content in the hepatopancreas of the male retarding shrimp was significantly lower than that of the normal shrimp (P0.05), and the EcR content in the eye handle of the female block shrimp was significantly lower than that of the normal shrimp (P0.05). The results showed that the cluster analysis and principal component analysis were found. The cluster analysis was the best statistical method to distinguish the growth block, and the weight, the fullness and the first tail width / body length were the best parameters to distinguish the growth block. The content of 20E and MF in the growth retarded shrimps was lower, and the content of MIH was higher; the EcR and RxR gene table in the growth retarded prawns The amount and protein level were low, and the expression of MIH gene was higher. It was suggested that the decrease of EcR and RxR expression and the increase of MIH expression decreased the level of 20 HYDROXYECDYSONE and methylfarnyl, and increased the level of ecdysone.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4
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