中华绒螯蟹CYP2和CPR基因的表达及其对胆固醇含量的影响

发布时间：2018-04-22 16:46

本文选题：中华绒螯蟹 + CYP2　；参考：《大连工业大学》2016年硕士论文

【摘要】：中华绒螯蟹(Eriocheir sinensis)在长江、辽河、黄河等我国淡水江河流域分布较为广泛,其风味独特、营养物质含量丰富、味道又特别鲜美,深受广大消费者喜爱。近年来,随着人们生活水平的不断提高,追求更高的生活品质和健康饮食理念的深入人心,人们对吃什么更美味、更有营养、更健康非常重视。在我国中华绒螯蟹养殖中“生长慢、规格小、胆固醇含量高和因蜕皮不完整导致的残肢断臂等问题”直接对中华绒螯蟹品质和效益带来影响,给其生产和加工也带来了巨大的挑战。中华绒螯蟹的蜕皮贯穿整个生命周期,与其生长、发育及繁殖息息相关。因此对中华绒螯蟹蜕皮相关基因以及在蜕皮过程中胆固醇含量的变化的研究,有助于我们解决蟹类“生长慢、规格小、胆固醇含量高和残肢断臂等”的问题,从而更好的指导中华绒螯蟹的养殖及其深加工。本文从已构建的甲壳动物cDNA文库中获得了2个ESTs序列,并设计相关的特异性引物,结合RACE技术克隆得到了这2个蜕皮相关基因的完整cDNA序列,并对获得的全长序列进行了结构分析和功能预测,然后进一步分析了两个基因在螃蟹蜕皮各个时期的表达量及螃蟹蜕皮各个时间段胆固醇含量的相关性。本论文针对这2个蜕皮相关基因及胆固醇含量的相关性所做的主要研究内容如下:1.CYP2基因克隆、序列分析及表达特性研究CYP2家族是目前已知的CYP450同工酶中最大、物种之间保守性最低的家族。本文通过使用分子克隆技术得到了中华绒螯蟹CYP2 cDNA基因的全序列(GenBank登录号为KT159166),并且利用Real-Time PCR方法分析了CYP2基因在中华绒螯蟹各个组织中的表达以及蜕皮不同周期在各组织中的相对表达量。实验结果显示,中华绒螯蟹CYP2的cDNA全长1772 bp,编码491个氨基酸序列,包括一个84 bp的5’端非编码区,一个212 bp的3’端非编码区、一个1475 bp的开放阅读框。实时荧光定量PCR结果显示,中华绒螯蟹蜕皮前,CYP2在鳃中的表达量相对最高;在肝胰脏、肌肉中表达量略低于鳃;在Y器官、眼柄、胸神经节、脑神经节、肠中少量表达;在心和胃中表达量最低。中华绒螯蟹在不同的蜕皮时期中,通过荧光定量实验得出:肝胰脏中CYP2基因表达量在退票前期蜕皮前期(D期)和蜕皮中期(E期)最高;眼柄中CYP2基因表达量从蜕皮中期(E期)到蜕皮后期(AB期)有上升趋势;鳃中CYP2基因表达量在蜕皮前期(D期)和蜕皮后期(AB期)最高;Y器官、脑神经节和肠中CYP2基因表达量在蜕皮中期(E期)最高;肌肉中CYP2基因表达量在蜕皮前期(D期)最高;胸神经节和胃中CYP2基因表达量在蜕皮后期(AB期)最高;Y器官中CYP2基因在蜕皮前期(D期)表达量高于蜕皮后期(AB期)和蜕皮中期(E期)。以上实验结果表明:CYP2基因对中华绒螯蟹蜕皮生长中的调控可能起到非常重要的作用。2.CPR基因克隆、序列分析及表达特性研究CPR是细胞色素氧化还原酶系的重要组成部分,在细胞色素介导的氧化还原反应中起到限速作用。以CPR基因的转录组序列为基础来设计该基因的特异引物,并使用RACE技术成功克隆得到中华绒螯蟹CPR cDNA全序列(GenBank登录号为KT159167)。其cDNA序列全长为3717 bp,包括一个42 bp的5’端非编码区,一个1268 bp的3’端非编码区,一个2046 bp的开放阅读框共编码681个氨基酸。因为氨基酸序列所构建的系统进化树和传统物种的进化规律基本保持一致。通过实时荧光定量PCR结果表示,CPR在各组织部位均有表达,肝胰脏和胃的表达量最高,在蜕皮周期中也可以清楚的看出蜕皮前期(D期),在肝胰脏和胃中CPR的表达量显著高于蜕皮中期(E期)和蜕皮后期(AB期)。说明该基因在中华绒螯蟹蜕皮生长中起到非常重要的作用。3.蜕皮周期中各组织中胆固醇含量与蜕皮基因表达量的相关性分析胆固醇被大家所熟知,是性激素、胆汁酸、肾上腺皮质激素、维生素D以及蜕皮激素的前体,在生理功能上具有十分重要的地位。基于对中华绒螯蟹蜕皮周期中,蜕皮D、E、AB期的胆固醇含量与蜕皮基因CPR和CYP2基因的相对表达量分析表明:中华绒螯蟹在肌肉、鳃三个组织中胆固醇的含量为:蜕皮前期(D期)蜕皮中期(E期)蜕皮后期(AB期)。CYP2基因在蜕皮过程中肌肉中的相对表达量为:蜕皮前期(D期)蜕皮中期(E期)蜕皮后期(AB期);而CPR基因的在蜕皮过程中的在肌肉、鳃和心中的相对表达量均是:蜕皮前期(D期)蜕皮中期(E期)蜕皮后期(AB期)。说明蜕皮周期中胆固醇含量与CYP2和CPR基因在组织中的的相对表达量,在某种程度上可能会具有协调作用。
[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.

【学位授予单位】：大连工业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4

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