吉富罗非鱼硬脂酰辅酶A去饱和酶（SCD）基因的克隆及温度与饲料脂肪源对其表达的研究

发布时间：2018-05-02 19:53

本文选题：吉富罗非鱼 + 硬脂酰辅酶A去饱和酶　；参考：《南京农业大学》2016年硕士论文

【摘要】：硬脂酰辅酶A去饱和酶(stearoyl-CoA desaturates,SCD)是合成单不饱和脂肪酸(monounsaturated fatty acid,MUFA)的限速酶,是调节肝脏脂肪生成和脂类氧化的关键控制点。经联合国粮农组织(FAO)调查数据显示,2014年全球100多个罗非鱼养殖地区的年产量总和达5446800t,其中中国罗非鱼产量在2014年增长至1450000t。由于罗非鱼对低温环境耐受力较差以及养殖过程中的不当投喂,使罗非鱼产业受到重大的经济损失。吉富罗非鱼是我国南方各省主要的淡水经济养殖鱼类,研究吉富罗非鱼的SCD基因对于提高抗低温能力及提高商品鱼的营养价值具有重要意义。本实验是国内首次从吉富罗非鱼体内克隆得到SCD基因的cDNA序列,同时对基因结构和表达时序特征进行分析。并结合饲料中的脂肪酸组成和水温对吉富罗非鱼生长、生理、脂肪酸组成与SCD表达的影响,从营养、环境及分子水平较为深入研究了 SCD的表达调控网络。为罗非鱼的脂肪酸调控以及培育高品质、高营养价值的罗非鱼提供了一定的理论依据。1.吉富罗非鱼SCD基因的克隆与序列分析本研究利用实时荧光定量PCR(real-time PCR,RT-PCR)和cDNA末端快速扩增法(rapid-amplificationof cDNAends,RACE)技术克隆获得了吉富罗非鱼的SCD基因的cDNA全长序列。结果显示,SCD基因的cDNA总长为1333bp,由173bp5'非编码区、152bp 3'非编码区以及编码335个氨基酸的1008bp开放阅读框组成。与GenBank中,尼罗罗非鱼、莫桑比克罗非鱼相似度较高,基因序列同源性分别达99%和98%,氨基酸序列同源性分别为99%和97%;与其他鱼类SCD基因序列相似度达83%以上,氨基酸序列同源性达79%以上。与其他鱼类的SCD基因相似度很高,说明该基因在物种间高度保守。2.吉富罗非鱼SCD基因组织及时空表达规律本研究通过RT-PCR检测了 SCD基因在吉富罗非鱼受精卵及胚胎发育阶段(受精后 1h、12h、24h、48h、72h、96h)、仔稚鱼阶段(出膜后 1d、5d、10d、15d、20d、25d、30d)及成鱼组织(血、肝脏、脾脏、肾脏、心脏、前肠、后肠、性腺、脑、皮肤、肌肉、鳃)中的表达。在胚胎发育的早期阶段1h和12h时,SCD基因的表达量较高,24h时出现明显下降的趋势,48h时有所升高,随后逐渐下降,96h最低。在受精卵及胚胎的早期阶段(1-15d)时,SCD表达量较低,并呈现逐步上升的趋势。20d时SCD的表达量显著升高,随后呈先下降后升高的趋势。表明了罗非鱼在发育各个阶段所需的脂肪酸营养不同,从而导致脂肪酸代谢酶的表达水平也有所不同,为吉富罗非鱼繁殖阶段及鱼苗培育阶段的脂肪酸营养的提供了理论依据。分别对雄鱼和雌鱼组织的SCD表达量进行检测,结果表明该基因在吉富罗非鱼雄性和雌性的各组织表达量的高低有所不同,在肝脏中表达量均达到最高,其次在雄鱼的精巢中表达量较高,而雌鱼的肌肉中的表达量仅次于肝脏。在雌鱼和雄鱼的脾脏中表达量均为最低。证明了吉富罗非鱼雌鱼与雄鱼的脂肪酸代谢机制具有一定差异,为进一步研究罗非鱼UFA的合成途径及调控机理提供了一定理论基础。3.温度对吉富罗非鱼SCD基因表达的研究本实验对吉富罗非鱼在22℃、28℃和34℃三种不同养殖温度下对SCD基因及肌肉脂肪酸相关性进行分析,同时对生长指标、血常规及血液生理指标进行了测定。28℃终体重显著高于22和34℃(P0.05),FCR和PER均高于22和34℃。不同组间的肌肉脂肪酸组成具有显著性差异(P0.05),SFA含量随温度的升高而降低而UFA含量呈相反的变化趋势。22℃时罗非鱼含较高的PUFA,尤其是n-3PUFA。SCD基因的表达及活性在低温时显著性升高(P0.05),并且增加吉富罗非鱼SCD的表达及活性可提升肌肉MUFA的合成。此外,降低水温可降低机体TC和TG的含量,对于提高吉富罗非鱼抗低温能力具有一定帮助。4.脂肪源对吉富罗非鱼SCD基因表达的研究日粮中添加不同脂肪源对鱼类的生长、生理等均具有一定影响,主要是由于饲料中脂肪酸组成不同,及不同鱼类对脂肪酸利用不同引起的。本研究分别以椰子油(主要含SFA)、橄榄油(主要含MUFA)、鱼油(脂肪酸比例均衡)及鱼油+豆油(1:1)(主要含PUFA)为脂肪源,进行60d的养殖实验。通过分析生长指标,发现饲料中添加脂肪酸比例较均衡的鱼油组生长效果最好。添加不同脂肪源对血液和肝脏的生理指标具有一定影响,添加SFA会引起血液与肝脏中TC、TG含量上升,而添加PUFA可以有效地降低体脂的沉积。并且添加PUFA可以增强机体免疫力,提高抗氧化酶活性,降低氧化损伤。此外,饲料中不同脂肪酸组成对肌肉和肝脏脂肪酸的组成具有显著性影响,鱼体脂肪酸组成中的SFA及UFA分别于饲料脂肪酸组成的SFA及UFA呈正相关。并且饲料中高水平的PUFA会抑制SCD的表达与活性并降低体脂沉积而增加SFA会促进SCD的表达与活性。因此,均衡日粮中的脂肪酸组成,适当提高多不饱和脂肪酸含量有助于增加吉富罗非鱼生长性能,降低血清脂肪沉积。
[Abstract]:The stearyl coenzyme A desaturase (stearoyl-CoA desaturates, SCD) is the speed limiting enzyme for the synthesis of monounsaturated fatty acids (monounsaturated fatty acid, MUFA). It is the key control point for regulating liver adipose formation and lipid oxidation. The annual output of more than 100 global tilapia in 2014 by the United Nations Food and agriculture Organization (FAO) survey data The total amount of Chinese tilapia increased to 5446800t in 2014 to 1450000t., due to the poor tolerance of tilapia to low temperature environment and the improper feeding in the breeding process, the tilapia industry suffered major economic losses. The gene is of great significance to improve the ability to resist low temperature and to improve the nutritional value of commercial fish. This experiment is the first time that the cDNA sequence of SCD gene has been cloned from the fishes of fishes in China, and the structure of the gene and the temporal characteristics of the expression are analyzed. The effect of fatty acid composition and SCD expression, the expression regulation network of SCD was studied in depth from nutrition, environment and molecular level. It provides a certain theoretical basis for the regulation of fatty acids and the cultivation of high nutritional value of tilapia and high nutritive value of tilapia. The cloning and sequence analysis of the SCD gene of Gefu tilapia,.1., is used in real time. PCR (real-time PCR, RT-PCR) and cDNA terminal rapid amplification (rapid-amplificationof cDNAends, RACE) were cloned to obtain the cDNA full length of the SCD gene of June tilapia. The results showed that the cDNA total length of the SCD gene was that of the non coding region, the non coding region and the encoding of the 335 amino acids. In GenBank, the similarity of Nile tilapia and Mozambique tilapia is higher, the homology of the gene sequence is 99% and 98% respectively, the homology of amino acid sequence is 99% and 97%, and the similarity of the SCD gene sequence of other fishes is more than 83% and the homology of the amino acid sequence is above 79%. The similarity of the SCD gene of other fishes is very similar. SCD gene tissue and spatio-temporal expression of.2. Junfu tilapia were highly conserved between species. The SCD gene was detected by RT-PCR in the fertilized egg and embryonic development stage of tilapia (1H, 12h, 24h, 48h, 72h, 96h). The expression of spleen, kidney, heart, foregut, hindgut, hindgut, gonadal, brain, skin, muscle, gill. At the early stage of embryonic development 1H and 12h, the expression of SCD gene is higher, the trend of 24h decreases obviously, 48h increases, and then gradually decreases, and 96h is the lowest. The expression of SCD is low in the early stage of fertilized eggs and embryos (1-15d). The expression of SCD increased significantly at.20d, and then decreased and then increased. It showed that the fatty acid nutrition of the tilapia at various stages of development was different, and the expression level of fatty acid metabolizing enzymes was also different, which was the fatty acid nutrition in the breeding stage and the cultivation stage of the fish. A theoretical basis was provided. The expression of SCD expression in male and female tissues was detected. The results showed that the expression of the gene in the male and female tissues of the male and female was different, the expression in the liver was the highest, followed by the high amount of the sperm in the male's spermary, and the amount of expression in the female's muscles was only the only time. The expression of the spleen in the female and male was the lowest. It was proved that the metabolic mechanism of fatty acid in the female and male fish was different, which provided a theoretical basis for the further study of the synthesis and regulation mechanism of UFA, and the study on the expression of Ji Fuluo non fish SCD gene by.3. temperature was a study of the experiment. The correlation between SCD gene and muscle fatty acids was analyzed at 22, 28 and 34 degrees centigrade. At the same time, the growth index, blood routine and blood physiological index were determined to be higher than 22 and 34 centigrade (P0.05), FCR and PER were higher than 22 and 34. The composition of fatty acid between different groups was significant. P0.05, the content of SFA decreased with the increase of temperature, while the UFA content was in the opposite direction. The higher PUFA in the tilapia at.22 C, especially the expression and activity of n-3PUFA.SCD gene increased significantly at low temperature (P0.05), and the expression and activity of SCD of the fish of Gefu tilapia could increase the synthesis of muscle MUFA. In addition, the water decreased water. Temperature can reduce the content of TC and TG. It has certain help to improve the anti low temperature ability of the fishes of Gefu tilapia, which can help the.4. fat source to express the SCD gene of GF riloticus. The diet of different fats has a certain effect on the growth and physiology of fish. It is mainly due to the difference of fatty acid composition in the feed and the fat of different fish. In this study, we used coconut oil (mainly SFA), olive oil (mainly containing MUFA), fish oil (fatty acid proportion equilibrium) and fish oil + bean oil (1:1) as the fat source, and carried out the culture experiment of 60d. By analyzing the growth index, it was found that the growth effect of fatty acid in the feed was the best. Adding different fat sources has a certain effect on the physiological indexes of blood and liver. Adding SFA can cause the increase of TC and TG in the blood and liver, and the addition of PUFA can effectively reduce the deposition of body fat. And adding PUFA can enhance the immunity of the body, improve the activity of antioxidant enzymes and reduce the oxidative damage. In addition, the composition of different fatty acids in the feed is made up. The composition of fatty acids in the muscle and liver has a significant effect. The SFA and UFA in the fatty acid composition of the fish body are positively related to the SFA and UFA of the fatty acids in the feed. And the high level PUFA in the feed inhibits the expression and activity of SCD and reduces the body fat deposition, and increases the expression and activity of SCD by increasing the SFA. Therefore, the balanced diet is balanced in the diet. Fatty acid composition and proper increase of polyunsaturated fatty acids are beneficial to increase growth performance and reduce serum fat deposition.

【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4

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