生長阻滯羅氏沼蝦相關(guān)激素含量及其基因的表達
發(fā)布時間：2018-07-27 10:47
【摘要】：近年來,揚州市高郵區(qū)域部分羅氏沼蝦(Macrobrachium rosenbergii)養(yǎng)殖塘口出現(xiàn)“生長緩慢”癥狀,蝦農(nóng)統(tǒng)稱為“鐵殼蝦”,“鐵殼蝦”現(xiàn)象凸顯,給蝦農(nóng)帶來了一定的經(jīng)濟損失和巨大的精神壓力。國內(nèi)學者從多方面對其進行了研究,但是,迄今為止,對于引起羅氏沼蝦生長阻滯現(xiàn)象的主要原因仍無定論。羅氏沼蝦作為甲殼動物,其生長伴隨著蛻殼的過程,而蛻殼受到蛻殼激素的影響,生長阻滯的羅氏沼蝦有可能存在激素水平的異常,而與激素相關(guān)的基因表達也可能表現(xiàn)出不同。因此,本論文假定,生長阻滯羅氏沼蝦的激素含量及其相關(guān)基因的表達完全不同于生長正常的羅氏沼蝦。為了驗證上述假說,本論文以高郵區(qū)域人工飼養(yǎng)的羅氏沼蝦為研究對象,首先測定采集樣本個體的形態(tài)學參數(shù),采用不同的數(shù)學統(tǒng)計方法對各形態(tài)學參數(shù)進行分析,確定鑒定羅氏沼蝦生長阻滯個體的最佳統(tǒng)計學方法和最佳度量的參數(shù);然后按照以上確定的最佳參數(shù)對不同的樣本進行分類,對分類個體進行蛻皮時期的判別,選擇蛻皮間期的羅氏沼蝦,通過酶聯(lián)免疫法測定各類樣本中蛻皮激素、蛻皮抑制激素和甲基法尼酯的含量,通過熒光定量PCR測定各類樣本的肝胰腺和眼柄中生長相關(guān)基因的mRNA表達水平,通過Western Blot測定各類樣本的肝胰腺和眼柄中EcR蛋白的表達,探討各類激素及其相關(guān)基因在生長阻滯現(xiàn)象中的作用,揭示影響水生甲殼類動物生長的生理調(diào)控機制,為廣大養(yǎng)殖戶的健康養(yǎng)殖提供可能的新措施和新方法。研究結(jié)果如下:1)聚類分析中能夠?qū)颖緞澐譃檎Ｎr、正常幼蝦和阻滯蝦的參數(shù)有體重、肥滿度和第一尾節(jié)寬度/體長;主成分分析中能夠區(qū)分三類蝦的參數(shù)有體長、腹尾長、螯長、第一尾節(jié)寬度、體長/體重、腹尾長/體重;相關(guān)性分析中能夠區(qū)分三類蝦的參數(shù)有體重、體長、腹尾長、第一尾節(jié)寬度、體長/體重、腹尾長/體重。綜合分析認為,聚類分析是最佳統(tǒng)計學方法,體重、肥滿度和第一尾節(jié)寬度/體長3個參數(shù)是最佳統(tǒng)計學參數(shù)。區(qū)分正常蝦、正常幼蝦和阻滯蝦的參數(shù)標準為:雄性體重參數(shù)范圍22.1668～25.1034、17.0517～17.6608和 13.4706～14.214,雄性肥滿度參數(shù)范圍 1.1598～1.2127、1.2541～1.2640 和 1.1274～1.1556,雄性第一尾節(jié)寬度/體長比值參數(shù)范圍0.1294～0.1360、0.1363～0.1384和0.1435～0.1494;雌性體重參數(shù)范圍 18.1526～19.9768、13.2198～14.0242 和 11.1046～11.9157,雌性肥滿度參數(shù)范圍1.1332～1.1563、1.1873～1.2075和1.1010～1.1107;雌性第一尾節(jié)寬度/體長比值參數(shù)范圍 0.1327～0.1376、0.1392～0.1414 和 0.1415～0.1441。2)2015年8月采集的樣本中,雄性阻滯蝦20E水平顯著低于正常蝦(P0.05),雌性阻滯蝦20E水平顯著低于正常蝦和正常幼蝦(P0.05);雌性阻滯蝦MIH水平顯著高于正常蝦和正常幼蝦(P0.05);雌雄阻滯蝦MF水平均顯著低于正常蝦(P0.05)。2016年6月采集的樣本中,雄性阻滯蝦20E水平顯著低于正常蝦和正常幼蝦(P0.05),雌性阻滯蝦20E水平顯著低于正常蝦(P0.05);雌雄阻滯蝦MIH水平均顯著高于正常蝦和正常幼蝦(P0.05);雌性阻滯蝦MF水平顯著低于正常蝦和正常幼蝦(P0.05)。3)2015年8月采集的樣本中,雄性阻滯蝦肝胰腺中的EcR mRNA表達量顯著低于正常蝦(P0.05),MIH mRNA表達量顯著高于正常蝦和正常幼蝦(P0.05),RxRmRNA表達量顯著低于正常蝦和正常幼蝦(P0.05),雄性阻滯蝦眼柄中的MIH mRNA表達量顯著高于正常幼蝦(P0.05);雌性阻滯蝦肝胰腺中的EcR mRNA表達量顯著低于正常蝦和正常幼蝦(P0.05),JHEH mRNA表達量顯著高于正常幼蝦,MIH mRNA表達量顯著高于正常蝦和正常幼蝦(P0.05)。2016年6月采集的樣本中,雄性阻滯蝦肝胰腺中的EcR和RxR mRNA表達量均顯著低于正常蝦和正常幼蝦(P0.05),GIH、MIH和JHEHmRNA表達量均顯著高于正常蝦和正常幼蝦(P0.05),雄性阻滯蝦眼柄中的GIH和MIH mRNA表達量顯著高于正常蝦和正常幼蝦(P0.05);雌性阻滯蝦肝胰腺中的EcR和RxRmRNA表達量顯著低于正常蝦和正常幼蝦(P0.05),GIH和MIH mRNA表達量顯著高于正常蝦和正常幼蝦(P0.05),JHEH mRNA表達量顯著高于正常幼蝦(P0.05),雌性阻滯蝦眼柄中的GIH和MIH mRNA表達量顯著高于正常蝦和正常幼蝦(P0.05)。4)2015年8月采集的樣本中,雌性阻滯蝦和正常幼蝦眼柄中的EcR含量顯著低于正常蝦(P0.05)。2016年6月采集的樣本中,雄性阻滯蝦肝胰腺中的EcR含量顯著低于正常蝦(P0.05);雌性阻滯蝦眼柄中的EcR含量顯著低于正常蝦(P0.05)。以上結(jié)果表明,聚類分析、主成分分析和相關(guān)性分析均可區(qū)分生長阻滯羅氏沼蝦,聚類分析是區(qū)分的最佳統(tǒng)計學方法,而體重、肥滿度和第一尾節(jié)寬度/體長是區(qū)分生長阻滯羅氏沼蝦的最佳參數(shù);生長阻滯蝦中20E和MF的含量較低,而MIH的含量較高;生長阻滯蝦中EcR和RxR基因表達量和蛋白水平較低,而MIH基因表達量較高。說明阻礙羅氏沼蝦生長的原因可能是EcR和RxR的表達量下降和MIH表達量升高降低了 20羥蛻皮激素和甲基法尼酯的水平、提高了蛻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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S917.4
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5 張化俊;袁軍法;石正麗;;羅氏沼蝦極小病毒全基因組序列測定及其感染特性[A];湖北省暨武漢生物化學與分子生物學學會第八屆會員代表大會和第十五次學術(shù)年會論文摘要匯編[C];2004年
6 孫龍生;傅婷婷;李海霞;;日糧鈣、磷、鎂水平對羅氏沼蝦體組織鈣、磷、鎂含量的影響[A];2010年中國水產(chǎn)學會學術(shù)年會論文摘要集[C];2011年
7 潘曉藝;曹錚;石正麗;吳勇泉;郝貴杰;徐洋;姚嘉峗;沈錦玉;;羅氏沼蝦幼體綜合癥病原鑒定及檢測[A];2010年中國水產(chǎn)學會學術(shù)年會論文摘要集[C];2011年
8 梁賢芳;米夏蓮;;羅氏沼蝦病原檢測及模擬試驗結(jié)果分析[A];西部大開發(fā) 科教先行與可持續(xù)發(fā)展——中國科協(xié)2000年學術(shù)年會文集[C];2000年
9 陳福艷;;羅氏沼蝦的越冬管理及親蝦培育[A];廣西水產(chǎn)研究所論文集（2001—2005）[C];2006年
10 朱春華;;低鹽度條件下羅氏沼蝦人工育苗試驗[A];中國水產(chǎn)學會第五屆青年學術(shù)年會摘要集[C];2004年
相關(guān)重要報紙文章 前10條
1 陳立法;羅氏沼蝦行情看好[N];江蘇農(nóng)業(yè)科技報;2006年
2 天同 振沖 其敏 廣隸;羅氏沼蝦年出口將達1000噸[N];揚州日報;2006年
3 田野 凌愛珍 錢h嵋，
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