多代近交对三疣梭子蟹能量代谢影响的研究

发布时间：2018-07-14 14:31

【摘要】：三疣梭子蟹(Portunus trituberculatus)是我国重要的大型海洋经济蟹类,经济价值高,已经成我国重要的沿海地区渔业养殖对象,2015年三疣梭子蟹养殖产量达117,772吨。由于三疣梭子蟹野生苗种的减少,为满足养殖需要苗种不得不依靠人工蓄养的亲本提供。因此可不避免地造成了三疣梭子蟹群体内近交。然而近交增加了子代基因纯合度和隐形有害基因突显的机会,导致三疣梭子蟹重要经济性状的衰退。本研究系统地研究了近交对三疣梭子蟹氧化磷酸化代谢和碳水化合物代谢关键酶及其基因表达的影响,为进一步理解近交衰退的机制提供重要的基础信息。具体研究内容如下:1.三疣梭子蟹氧化磷酸化代谢在家系近交中的变化分析为探究近交对三疣梭子蟹氧化磷酸化代谢的影响,首先采用RACE技术克隆获得三疣梭子蟹线粒体呼吸链5个复合体关键亚基基因的c DNA全长序列,并对这些基因序列进行生物信息学分析。三疣梭子蟹线粒体呼吸链复合体Ⅰ亚基基因c DNA序列全长为1005bp,包含705bp的ORF,编码234个氨基酸,该蛋白是复合体Ⅰ的核心亚基Ndufv2,命名为pt Ndufv2;复合体Ⅱ亚基基因c DNA序列全长为915bp,包含540bp的ORF,编码由179个氨基酸组成的复合体Ⅱ关键亚基SDHC,命名为pt SDHC;复合体Ⅲ亚基基因c DNA序列全长为2371bp,包含942bp的ORF,编码由313个氨基酸组成的Cytc1亚基,命名为pt Cytochrome c1;复合体Ⅳ亚基基因c DNA序列全长为1171bp,包含318bp的ORF,编码由105个氨基酸组成的COX6B亚基,命名为pt COX6B;复合体Ⅴ亚基基因F-ATPaseβ基因的c DNA序列全长为1965bp,包含1053bp的ORF,编码一个由350个氨基酸组成的ATP合酶β亚基,命名为pt F-ATPaseβ。这些基因与相近的物种同源性较高,在进化上也表现出较高的保守性,可为其他海洋生物相关的研究提供参考。分析了不同近交世代家系三疣梭子蟹的肝胰腺和心脏中线粒体呼吸链5个复合体活力,并采用实时荧光定量PCR技术分析这些复合体基因在不同近交世代家系三疣梭子蟹的肝胰腺和心脏中的表达情况。结果表明近交造成了三疣梭子蟹肝胰腺中全部5个复合体活力及其亚基基因的衰退,心脏中除复合体II之外4个复合体活力及其亚基基因的衰退,这一结果证实了近交衰退出现在了三疣梭子蟹氧化磷酸化代谢通路之中。2.三疣梭子蟹碳水化合物代谢在家系近交中的变化分析为分析三疣梭子蟹碳水化合物代谢在家系近交中的变化,本研究检测了不同近交世代家系的三疣梭子蟹碳水化合物代谢关键酶的活力及糖原含量,分析近交对其代谢酶活力的影响。利用RT-PCR的方法检测了这些关键酶m RNA的相对表达的变化,分析近交对其基因表达的影响。本实验结果显示随近交系数的增加,肝胰腺和肌肉中的己糖激酶(HK)活力显著下降,丙酮酸脱氢酶(PK)在鳃和血清中也呈现下降趋势,同时两者基因相对表达量表现出和酶活同样的下降趋势,这表明了三疣梭子蟹糖酵解代谢的衰退;肌肉和鳃中琥珀酸脱氢酶(SDH)活力随着近交系数的增加不断增强,这说明三疣梭子蟹有氧代谢水平随近交不断增强。然而SDH基因相对表达量还是呈现衰退趋势,这表明近交不可避免的造成了三疣梭子蟹有氧代谢关键基因(SDH)的衰退;乳酸脱氢酶(LDH)酶活力随近交系数的增加没有显著变化,而LDH基因相对表达量却呈现下降趋势,说明近交对三疣梭子蟹无氧代谢有着不利的影响。该结果证实了近交造成了三疣梭子蟹碳水化合物代谢的衰退。3.三疣梭子蟹同家系大小个体之间碳水化合物代谢的差异为分析体重分化与碳水化合物代谢之间的关系,本研究通过酶活检测与RTPCR技术,检测了三疣梭子蟹同一近交家系中大小个体中碳水化合物代谢关键酶的活力及其基因相对表达情况。结果显示三疣梭子蟹大小个体在碳水化合物代谢方面有着显著的差异:F6代中大个体肝胰腺和肌肉中及F10代肌肉中的HK活力显著高于小个体;F6代大个体中肝胰腺、肌肉和鳃及F10代肝胰腺和鳃的PK活力显著显著高于小个体。这表明,无论是F6还是F10代中,三疣梭子蟹大个体的糖酵解代谢相对于小个体要旺盛,分解利用碳水化合物的能力强。F6代大个体肝胰腺和鳃中及F10代的鳃中的SDH活力显著高于小个体,表明三疣梭子蟹大个体的肝胰腺和鳃中的有氧代谢水平相对于小个体较高。F6代中大个体肝胰腺和鳃中的LDH活力显著高于小个体,这说明大个体无氧代谢的水平较小个体强。大个体的肝糖原和肌糖原含量均显著高于小个体,这说明大个体贮存的糖原较多,推测其血糖水平也较高,揭示了其生长较快的原因。4.三疣梭子蟹酵母双杂交c DNA文库的构建及Pt F-ATPaseβ互作蛋白的初步筛选本实验在Clontech公司构建了三疣梭子蟹酵母双杂交三框读码c DNA文库,该文库各质量参数均达标,且插入片段大小分布广泛,多样性良好,可用于利用酵母双杂交技术筛选关键基因的互作蛋白。为解析三疣梭子蟹ATP合酶β亚基蛋白F-ATPaseβ的功能,本实验运用In-Fusion Cloning技术和Yeastmaker Yeast Transformation技术,构建了p GBKT7-Pt ATPaseβ诱饵载体。并利用酵母双杂交技术在三疣梭子蟹酵母双杂交三框读码c DNA文库中初步筛选出ATP合酶β亚基蛋白的互作蛋白。经过高度缺陷培养基的筛选、三框读码、回复杂交、测序及生物信息学分析,获得三疣梭子蟹ATP合酶β亚基蛋白的互作蛋白如下:延伸因子2(elongation factor 2)、法尼酸甲基转移酶(Farnesoic acid O-methyltransferase)、60S核糖体蛋白L37-A(Probable 60S ribosomal protein L37-A)、核酸反转录酶样蛋白(endonuclease-reverse transcriptase-like protein)、动力蛋白激活蛋白亚基3(dynactin subunit 3-like)、细胞色素c氧化酶亚基VIb/cox12(putative cytochrome c oxidase subunit VIb/cox12)、蛋白磷酸酶1调节亚基3C(Protein phosphatase 1 regulatory subunit 3C)、线粒体NADH脱氢酶[辅酶]1β亚基2(NADH dehydrogenase[ubiquinone]1 beta subcomplex subunit 2,mitochondrial)、S-methyl-5-thioribose激酶(S-methyl-5-thioribose kinase)、伪装样蛋白(masquerade-like protein)、假定蛋白(hypothetical protein)。推测,三疣梭子蟹F-ATPaseβ蛋白的功能可能涉及到氧化磷酸化、蛋白质合成、糖原代谢、有丝分裂及渗透压调节等生物学过程。
[Abstract]:Portunus trituberculatus (Portunus) is an important large marine economic crab in China and has high economic value. It has become an important fishery culture object in the coastal areas of China. In 2015, the production of three warts of the shuttle crab was 117772 tons. Because of the decrease of the wild seedling of the three verruca crabs, it has to rely on artificial storage for the cultivation of the seedlings to meet the needs of the seedlings. In this study, the acidification and carbohydrate metabolism and carbohydrates of the three Clostridium crabs were systematically studied in this study. The inbreeding resulted in the inbreeding of the three Clostridium crabs. However, inbreeding increased the chances of the homozygosity of the progeny and the invisibility of the hidden harmful genes, which resulted in the decline of the important economic characters of the three Clostridium crabs. The effects of metabolic key enzymes and their gene expression provide important basic information for further understanding of the mechanism of inbreeding. The specific content of the study is as follows: 1. analysis of the changes in oxidative phosphorylation in the pedigree of the portunus verruca three in order to explore the effect of inbreeding on the oxidative phosphorylation of the three shuttle crab, first of which was cloned by RACE Technology The C DNA full length sequence of the key subunit gene of the 5 complex of the mitochondrial respiratory chain of the portunus verruca was obtained, and the sequence of these genes was analyzed by bioinformatics. The total length of the C DNA sequence of the mitochondrial respiratory chain complex I gene of the mitochondrial respiratory chain complex of the portunus verruca was 1005bp, including the ORF of 705bp and the coding of 234 amino acids, which was the nucleus of the complex I. The heart subunit Ndufv2, named Pt Ndufv2, the C DNA sequence of the complex II subunit of the complex is 915bp, contains 540bp ORF, encodes the key subunit of the complex of 179 amino acids, SDHC, named Pt SDHC; the complex III subunit gene contains the entire length of the sequence, encodes the subunit consisting of 313 amino acids. Pt Cytochrome C1, C DNA sequence of complex IV subunit, 1171bp, ORF of 318bp, and COX6B subunit of 105 amino acids, named Pt COX6B. The genes are named Pt F-ATPase beta. These genes have high homology with similar species and exhibit higher conservatism in evolution. They can provide reference for other marine related studies. The activity of 5 complexes of mitochondrial respiratory chain in the hepatopancreas and heart of three Portunus verruca from different inbred families was analyzed, and real-time fluorescence was used. Quantitative PCR technique was used to analyze the expression of these complex genes in the hepatopancreas and heart of the portunus verruca three in different inbred lines. The results showed that the activity of all 5 complexes in the hepatopancreas and the subunit genes in the hepatopancreas of three Portunus crabs were caused by inbreeding, and the activity of 4 complexes except the complex II in the heart and its subunit genes in the heart. The results showed that the change of carbohydrate metabolism in.2. three of.2. three of shuttle crabs was analyzed to analyze the changes of carbohydrate metabolism in the pedigrees in the pedigree of the portunus verruca crabs. The results of this study were to detect three warts in the families of different inbred lines. The activity of the key enzymes of carbohydrate metabolism and the content of glycogen were analyzed. The changes of the relative expression of these key enzymes, m RNA, were detected by RT-PCR, and the effect of inbreeding on the gene expression was analyzed. The results showed that with the increase of inbreeding coefficient, the hexose in the hepatopancreas and the muscles. The kinase (HK) activity decreased significantly, and the pyruvate dehydrogenase (PK) also showed a downward trend in the gills and serum, and the relative expression of the two genes showed the same downward trend as the enzyme activity, which indicated the decline of glycolysis metabolism of the three warts; the activity of succinic dehydrogenase (SDH) in the muscles and gills increased with the increase of inbreeding coefficient. This indicates that the oxygen metabolism level of the three wart crabs increases with the inbreeding. However, the relative expression of SDH gene presents a decline trend, which indicates that inbreeding inevitably leads to the decline of the key gene of oxygen metabolism (SDH) of the portunus crabs, and the activity of lactate dehydrogenase (LDH) enzyme does not change significantly with the increase of the inbreeding coefficient, while the LDH gene is not significantly changed. The relative expression showed a downward trend, indicating that inbreeding had an adverse effect on the anaerobic metabolism of three wart crabs. The results confirmed that inbreeding resulted in a decline in carbohydrate metabolism of the shuttle three of the portunus verruca.3.. The difference in carbohydrate metabolism between the same family size individuals of the portunus verruca three was the analysis of body weight differentiation and carbohydrate metabolism. The relationship between the enzyme activity detection and RTPCR technique was used to detect the activity of key enzymes of carbohydrate metabolism and the relative gene expression of the carbohydrate metabolism in the same inbred lines of the three Portunus verruca. The results showed that there were significant differences in carbohydrate metabolism between the size individuals of the three Portunus verruca and the large individual liver in the F6 generation. The HK activity in the pancreas and muscle and the F10 generation muscle was significantly higher than that in the small individuals. The PK activity of the hepatopancreas, the muscles and gills and the gills of the F10 generation and the gill of the F6 generation was significantly higher than that of the small individuals. This indicates that the glycolysis phase of the large individuals of the three wart crabs is exuberant to small individuals in the F6 and F10 generations, and the decomposition and utilization of carbon hydration The activity of SDH in the hepatopancreas and gills and the gills of the gill and F10 generation of the large.F6 generation was significantly higher than that of the small individuals. It showed that the level of aerobic metabolism in the hepatopancreas and gills of the large individuals of the three warts was significantly higher than that of the small individuals in the larger individuals of the.F6 generation of the hepatopancreas and gills, which indicated that the anaerobic metabolism of the large individuals was unoxygened. The levels of liver glycogen and muscle glycogen in large individuals were significantly higher than those of small individuals, which indicated that the large individuals stored more glycogen and higher blood glucose levels, which revealed the construction of the.4. three hybrid C DNA library for the rapid growth of the verruca verruca and the preliminary screening of the Pt F-ATPase beta interaction protein. The three frame reading code reading code library of three crosses of Portunus verruca was constructed in Clontech company. The quality parameters of the library were all up to the standard, and the size distribution of the inserted fragment was wide and the diversity was good. It could be used to screen the intercrop protein of key genes by yeast two hybrid technique. The work was to analyze the work of ATP synthase beta subunit protein F-ATPase beta of the ATP of the portunus verruca. In this experiment, the P GBKT7-Pt ATPase beta lure carrier was constructed by using In-Fusion Cloning technology and Yeastmaker Yeast Transformation technology. The reciprocal protein of ATP synthase beta subunit protein was preliminarily screened in the three frame reading code c DNA Library of the yeast two hybrid of three verruca. Screening, three frame reading code, replying hybridization, sequencing and bioinformatics analysis, the interaction protein of ATP synthase beta subunit protein of Portunus three was obtained as follows: extension factor 2 (elongation factor 2), farnic acid methyl transferase (Farnesoic acid O-methyltransferase), 60S nucleoprotein L37-A (Probable 60S ribosomal protein), nucleic acid reaction Transcriptional enzyme like protein (endonuclease-reverse transcriptase-like protein), protein subunit 3 (dynactin subunit 3-like), cytochrome c oxidase subunit VIb/cox12 (putative cytochrome c oxidase), protein phosphatase 1 regulation subunit 1 DH dehydrogenase [coenzyme]1 beta subunit 2 (NADH dehydrogenase[ubiquinone]1 beta subcomplex subunit 2, mitochondrial), S-methyl-5-thioribose kinase (S-methyl-5-thioribose kinase), camouflage like protein (masquerade-like), hypothetical protein, presumably involved in the function of the beta protein of the portunus verruca Biological processes such as oxidative phosphorylation, protein synthesis, glycogen metabolism, mitosis and osmotic pressure regulation.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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