本文選題：中華絨螯蟹 + CYP2��； 參考：《大連工業(yè)大學》2016年碩士論文

【摘要】：中華絨螯蟹(Eriocheir sinensis)在長江、遼河、黃河等我國淡水江河流域分布較為廣泛,其風味獨特、營養(yǎng)物質含量豐富、味道又特別鮮美,深受廣大消費者喜愛。近年來,隨著人們生活水平的不斷提高,追求更高的生活品質和健康飲食理念的深入人心,人們對吃什么更美味、更有營養(yǎng)、更健康非常重視。在我國中華絨螯蟹養(yǎng)殖中“生長慢、規(guī)格小、膽固醇含量高和因蛻皮不完整導致的殘肢斷臂等問題”直接對中華絨螯蟹品質和效益帶來影響,給其生產(chǎn)和加工也帶來了巨大的挑戰(zhàn)。中華絨螯蟹的蛻皮貫穿整個生命周期,與其生長、發(fā)育及繁殖息息相關。因此對中華絨螯蟹蛻皮相關基因以及在蛻皮過程中膽固醇含量的變化的研究,有助于我們解決蟹類“生長慢、規(guī)格小、膽固醇含量高和殘肢斷臂等”的問題,從而更好的指導中華絨螯蟹的養(yǎng)殖及其深加工。本文從已構建的甲殼動物cDNA文庫中獲得了2個ESTs序列,并設計相關的特異性引物,結合RACE技術克隆得到了這2個蛻皮相關基因的完整cDNA序列,并對獲得的全長序列進行了結構分析和功能預測,然后進一步分析了兩個基因在螃蟹蛻皮各個時期的表達量及螃蟹蛻皮各個時間段膽固醇含量的相關性。本論文針對這2個蛻皮相關基因及膽固醇含量的相關性所做的主要研究內容如下:1.CYP2基因克隆、序列分析及表達特性研究CYP2家族是目前已知的CYP450同工酶中最大、物種之間保守性最低的家族。本文通過使用分子克隆技術得到了中華絨螯蟹CYP2 cDNA基因的全序列(GenBank登錄號為KT159166),并且利用Real-Time PCR方法分析了CYP2基因在中華絨螯蟹各個組織中的表達以及蛻皮不同周期在各組織中的相對表達量。實驗結果顯示,中華絨螯蟹CYP2的cDNA全長1772 bp,編碼491個氨基酸序列,包括一個84 bp的5’端非編碼區(qū),一個212 bp的3’端非編碼區(qū)、一個1475 bp的開放閱讀框。實時熒光定量PCR結果顯示,中華絨螯蟹蛻皮前,CYP2在鰓中的表達量相對最高;在肝胰臟、肌肉中表達量略低于鰓;在Y器官、眼柄、胸神經(jīng)節(jié)、腦神經(jīng)節(jié)、腸中少量表達;在心和胃中表達量最低。中華絨螯蟹在不同的蛻皮時期中,通過熒光定量實驗得出:肝胰臟中CYP2基因表達量在退票前期蛻皮前期(D期)和蛻皮中期(E期)最高;眼柄中CYP2基因表達量從蛻皮中期(E期)到蛻皮后期(AB期)有上升趨勢;鰓中CYP2基因表達量在蛻皮前期(D期)和蛻皮后期(AB期)最高;Y器官、腦神經(jīng)節(jié)和腸中CYP2基因表達量在蛻皮中期(E期)最高;肌肉中CYP2基因表達量在蛻皮前期(D期)最高;胸神經(jīng)節(jié)和胃中CYP2基因表達量在蛻皮后期(AB期)最高;Y器官中CYP2基因在蛻皮前期(D期)表達量高于蛻皮后期(AB期)和蛻皮中期(E期)。以上實驗結果表明:CYP2基因對中華絨螯蟹蛻皮生長中的調控可能起到非常重要的作用。2.CPR基因克隆、序列分析及表達特性研究CPR是細胞色素氧化還原酶系的重要組成部分,在細胞色素介導的氧化還原反應中起到限速作用。以CPR基因的轉錄組序列為基礎來設計該基因的特異引物,并使用RACE技術成功克隆得到中華絨螯蟹CPR cDNA全序列(GenBank登錄號為KT159167)。其cDNA序列全長為3717 bp,包括一個42 bp的5’端非編碼區(qū),一個1268 bp的3’端非編碼區(qū),一個2046 bp的開放閱讀框共編碼681個氨基酸。因為氨基酸序列所構建的系統(tǒng)進化樹和傳統(tǒng)物種的進化規(guī)律基本保持一致。通過實時熒光定量PCR結果表示,CPR在各組織部位均有表達,肝胰臟和胃的表達量最高,在蛻皮周期中也可以清楚的看出蛻皮前期(D期),在肝胰臟和胃中CPR的表達量顯著高于蛻皮中期(E期)和蛻皮后期(AB期)。說明該基因在中華絨螯蟹蛻皮生長中起到非常重要的作用。3.蛻皮周期中各組織中膽固醇含量與蛻皮基因表達量的相關性分析膽固醇被大家所熟知,是性激素、膽汁酸、腎上腺皮質激素、維生素D以及蛻皮激素的前體,在生理功能上具有十分重要的地位。基于對中華絨螯蟹蛻皮周期中,蛻皮D、E、AB期的膽固醇含量與蛻皮基因CPR和CYP2基因的相對表達量分析表明:中華絨螯蟹在肌肉、鰓三個組織中膽固醇的含量為:蛻皮前期(D期)蛻皮中期(E期)蛻皮后期(AB期)。CYP2基因在蛻皮過程中肌肉中的相對表達量為:蛻皮前期(D期)蛻皮中期(E期)蛻皮后期(AB期);而CPR基因的在蛻皮過程中的在肌肉、鰓和心中的相對表達量均是:蛻皮前期(D期)蛻皮中期(E期)蛻皮后期(AB期)。說明蛻皮周期中膽固醇含量與CYP2和CPR基因在組織中的的相對表達量,在某種程度上可能會具有協(xié)調作用。
[Abstract]:Eriocheir sinensis (Eriocheir sinensis) is widely distributed in the Yangtze River, Liaohe River and the Yellow River River Basin. Its flavor is unique, its nutrient content is rich, and the taste is very delicious. It is deeply loved by the vast majority of consumers. In recent years, with the improvement of people's living standards, the pursuit of higher quality of life and the deep concept of healthy diet is deep. In the culture of Chinese Eriocheir sinensis, the problems of "slow growth, small size, high cholesterol content and broken limbs caused by incomplete molt" have a direct effect on the quality and benefit of Eriocheir sinensis, which has brought great challenge to the production and processing of Chinese mitten crab. The molting of Eriocheir sinensis throughout the whole life cycle is closely related to its growth, development and reproduction. Therefore, the study on the related genes and the changes in the cholesterol content during the molting process of the Chinese Mitten Crab Eriocheir crabs will help us to solve the problem of "slow growth, small size, high cholesterol content and broken arm broken arm, etc." Better guidance on the culture and processing of Eriocheir sinensis. In this paper, 2 ESTs sequences were obtained from the constructed crustaceans cDNA library, and the specific primers were designed. The complete cDNA sequences of these 2 molting related genes were cloned by RACE technique, and the structure analysis and function prediction of the full length sequences were also carried out. The correlation between the expression of two genes at each period of crabs molting and the cholesterol content in each period of crabs molting was further analyzed. The main contents of this paper on the correlation of these 2 molt related genes and cholesterol content were as follows: 1.CYP2 gene clon, sequence analysis and expression characteristics of the CYP2 family The family of the largest CYP450 isozymes and the lowest conservatism among species is known. In this paper, the whole sequence of the CYP2 cDNA gene of Eriocheir sinensis (GenBank logon number KT159166) was obtained by using molecular cloning technique, and the expression of CYP2 gene in the various tissues of Eriocheir sinensis by the Real-Time PCR method and its metamorphosis were analyzed. The experimental results showed that the cDNA of CYP2 was 1772 BP and 491 amino acid sequences, including a 84 BP 5 'terminal non coding region, a 212 BP 3' terminal non coding region, a 1475 BP open reading frame. Real time fluorescence quantitative PCR results showed that the Eriocheir sinensis molting The expression of CYP2 in the gills was the highest in the gills; in the hepatopancreas, the expression in the muscles was slightly lower than that in the gills; in the Y organs, the eye handle, the thoracic ganglion, the brain ganglion and the intestines, the expression of the CYP2 was the lowest. In the different period of the molting, the expression of the CYP2 gene in the liver and pancreas was shown before the refund of the ticket. Phase D and mid molt (phase E) were the highest in the stage of molt, and the expression of CYP2 gene in the eye handle increased from the metaphase of molt (E stage) to the late molt (AB stage); the CYP2 gene expression in the gill was highest in the prophase (D phase) and late molt (AB phase), and the Y organ, the brain ganglion and the CYP2 gene in the intestine were highest in the metaphase (E stage) of the molt, and the CYP in the muscle. The 2 gene expression was highest in the pre molting stage (D stage), and the expression of CYP2 gene in the thoracic ganglion and stomach was highest in the late molt (AB stage), and the expression of CYP2 gene in the prophase (D stage) in the Y organ was higher than that in the late molt (AB phase) and the metaphase (E phase). The results showed that the regulation of the CYP2 gene may play a role in the regulation of the growth of the ecdysone of Eriocheir sinensis. The important role of.2.CPR gene cloning, sequence analysis and expression characterization CPR is an important component of the cytochrome oxidoreductase system, which plays a speed limiting role in the redox reaction mediated by cytochrome. Based on the sequence of CPR genes, the specific primers are designed and successfully cloned using RACE technology. The whole sequence of CPR cDNA of Eriocheir sinensis (GenBank login number is KT159167). The full length of cDNA sequence is 3717 BP, including a 42 BP 5 'end non coding region, a 3' end non coding region of 1268 BP, and a 2046 BP open reading frame co coding 681 amino acids. According to the results of real time fluorescence quantitative PCR, the expression of CPR was expressed in all tissues, the expression of hepatopancreas and stomach was highest, and the prophase (D phase) could be clearly seen in the ecdysone cycle. The expression of CPR in the hepatopancreas and stomach was significantly higher than that in the metaphase of molt (E stage) and late molt (AB stage). The relationship between cholesterol content and ect gene expression in the.3. molt cycle of Eriocheir sinensis plays a very important role in the analysis of the correlation between cholesterol content and the expression of ECT gene expression. It is known to everybody that it is the precursor of sex hormone, bile acid, adrenocortical hormone, vitamin D and ecmola. It is very important in physiological function. Based on the analysis of the relative expression of cholesterol content with the CPR and CYP2 genes of the molt gene in the molt period of D, E and AB during the molt period of Eriocheir sinensis, the cholesterol content in the three tissues of the muscle and gill of the Chinese Mitten Crab Eriocheir sinensis was: the.CYP2 gene in the late molt stage (E phase) of the ecdysone (phase AB phase) of the ecdysone (stage D) in the ecdysone (stage AB) in the molt process. The relative expression in the meat is: the late molt (phase E) molt (phase AB phase) of the ecdysone (stage D phase), while the relative expression of CPR in the molt process in the muscles, gills and heart are all in the late molt (phase D stage) molt (E phase) molt (AB phase). It shows the phase of the cholesterol content in the molt cycle and the CYP2 and CPR genes in the tissue. To some extent, the amount of expression may play a coordinating role.

【學位授予單位】：大連工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S917.4

【參考文獻】

相關期刊論文 前10條

1 柳志業(yè);朱冬發(fā);謝熙;邱錫爾;周彥琦;沈錫權;;三疣梭子蟹CYP302a1基因克隆及其表達分析[J];水產(chǎn)學報;2015年05期

2 馮艷艷;李健;張德寧;劉萍;葛倩倩;呂建建;高保全;;三疣梭子蟹(Portunus trituberculatus)CYP2基因的cDNA克隆及表達分析[J];海洋與湖沼;2014年05期

3 凌淑萍;張會敏;蘇閃閃;張曉溯;劉雪艷;潘桂芳;袁藝;;洋甘菊細胞色素P450還原酶基因(CPR)的分子克隆及表達分析[J];農(nóng)業(yè)生物技術學報;2014年05期

4 王瑤;楊志剛;沈城;姚琴琴;曾奇韜;劉啟彬;成永旭;;中華絨螯蟹EcR基因全長cDNA克隆及表達分析[J];水產(chǎn)學報;2014年05期

5 張曉燕;李健;劉萍;陳萍;孫銘;楊愛國;;三疣梭子蟹(Portunus trituberculatus)CYP4C基因的cDNA克隆及表達分析[J];海洋與湖沼;2014年01期

6 曹飛飛;范永仙;陳小龍;;細胞色素P450酶系介導的甾體羥化反應及其在分子水平上的研究進展[J];國外醫(yī)藥(抗生素分冊);2013年04期

7 米智;阮成龍;李冠楠;杜文華;隆耀航;朱勇;;家蠶氟中毒后中腸NADPH-細胞色素P450還原酶和NADPH-細胞色素C還原酶活性的變化[J];蠶業(yè)科學;2012年01期

8 孫妍;張亦陳;劉逸塵;王雪惠;王宇凡;耿緒云;孫金生;楊衛(wèi)軍;;中華絨螯蟹蛻皮抑制激素基因全長cDNA克隆和重組表達[J];水生生物學報;2011年02期

9 陳立僑;李二超;;中華絨螯蟹營養(yǎng)需求的研究現(xiàn)狀和進展[J];飼料工業(yè);2009年10期

10 王燕紅;王東;李兵;趙華強;許雅香;沈衛(wèi)德;;野桑蠶細胞色素P450家族CYP4M5基因的克隆和誘導表達[J];蠶業(yè)科學;2009年01期

相關碩士學位論文 前4條

1 易勝;煙粉虱NADPH-細胞色素P450還原酶與NADH-細胞色素b5還原酶基因的克隆及表達量分析[D];湖南農(nóng)業(yè)大學;2014年

2 張曉燕;三疣梭子蟹CYP4C和EcR基因的cDNA克隆與表達分析[D];上海海洋大學;2013年

3 高艷玲;甘藍夜蛾細胞色素P450CYP4M及CYP6AB基因的克隆與表達研究[D];東北農(nóng)業(yè)大學;2012年

4 鄭海波;中華絨螯蟹的品質分析與比較[D];江南大學;2008年

，

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