饲料中添加二氢吡啶和注射三碘甲腺原氨酸（T3）对尼罗罗非鱼机体脂肪沉积及相关脂解基因表达的影响

发布时间：2017-12-27 08:08

本文关键词：饲料中添加二氢吡啶和注射三碘甲腺原氨酸（T3）对尼罗罗非鱼机体脂肪沉积及相关脂解基因表达的影响　出处：《华东师范大学》2017年硕士论文　论文类型：学位论文

【摘要】：本论文以尼罗罗非鱼(Oreochromis niloticus)为研究对象,应用营养学、生物化学和分子生物学等方法,较为详细地研究了饲料脂肪水平、饲料二氢吡啶以及体外注射三碘甲状腺原氨酸(T3)对尼罗罗非鱼生长和机体脂肪沉积的影响,具体研究结果如下:(1)尼罗罗非鱼激素敏感脂肪酶(HSL)基因、单甘油酯脂肪酶(MGL)基因的克隆与相关生物信息学分析为探究HSL和MGL基因在尼罗罗非鱼脂肪分解代谢中的调控作用,本实验首先克隆得到尼罗罗非鱼HSL基因和MGL基因的全长,并探讨了 HSL基因和MGL基因组织特异性表达,以期进一步研究上述基因的功能和作用机制。结果表明,尼罗罗非鱼HSL基因全长是3147bp,开放阅读框长度是2142bp,可以编码714个氨基酸残基,蛋白质的分子量为69.70 kDa,等电点(pi)为7.64。MGL基因全长是7448bp,开放阅读框长度是918bp,可以编码306个氨基酸残基,MGL蛋白质的分子量为34 kDa,等电点(pi)为5.88。荧光定量分析发现,HSL基因和MGL基因在脑、鳃、心脏、皮肤、白肌、红肌、肝、腹腔脂肪、脾、肾脏和肠组织中均有表达,其中HSL基因在脂肪组织中表达量最高,其次是红肌、白肌和脑等组织。MGL基因在脑中表达量最高,红肌,白肌等组织次之。这说明了 HSL和MGL基因对尼罗罗非鱼体内脂肪的水解起着重要的作用,但具体的功能和作用机制尚需进一步的实验验证。(2)饲料脂肪水平以及饥饿处理对尼罗罗非鱼脂解基因表达的影响为探究饲料脂肪水平,以及饥饿处理对尼罗罗非鱼脂解基因表达的影响。本实验设计了脂肪水平为5%(正常脂)、15%(高脂)的两种饲料,饱食投喂初重为6.13±0.20g的尼罗罗非鱼6周,实验结束后分别在最后一次饱食1、5、12、24和168小时后取脂肪、肝脏和白肌组织样品,分析不同时间点不同组织中ATGL、HSL、MGL基因的表达情况,结果表明:高脂组(15%)的脂体比高于正常组,但随着饥饿时间的延长,差异趋于减小。脂肪组织中,随着饥饿时间的延长,高脂组ATGL基因的表达呈先下降后上升的趋势,正常组基因表达呈上升趋势;脂肪组织中高脂组HSL基因表达呈先上升后下降的趋势,在24h时表达量显著升高(P0.05),中脂组基因表达在5h和24h组高于其他组;脂肪组织中各时间点高脂组MGL基因表达无显著变化(P0.05),正常组基因表达呈先下降后上升的趋势,在168h组基因表达显著上调(P0.05)。以上结果表明高脂处理和饥饿均能够促进脂解基因的表达,从而促进脂肪水解。但在正常养殖条件下,饥饿虽然可以促进脂肪的水解,但是不利于鱼类的正常生长。(3)高脂饲料中添加二氢吡啶对尼罗罗非鱼生长、体成分、脂肪沉积以及脂解相关基因表达的影响将235尾健康无伤,初始体重为5.91 ±0.43g的尼罗罗非鱼随机分成5个处理组,分别投喂脂肪水平5%,脂肪水平15%的两种饲料,其中15%脂肪水平的饲料分别添加Omg/kg、50mg/kg、100mg/kg和150mg/kg二氢吡啶,共5种饲料对鱼进行投喂,试验周期为6周。结果表明:高脂饲料中添加二氢吡啶对尼罗罗非鱼的生长起到了促进作用,但是同高脂饲料组相比,高脂饲料中添加50mg/kg和100mg/kg二氢吡啶可以显著降低鱼体粗脂肪含量(P0.05),而150mg/kg添加组则无显著变化(P0.05)。组织学的结果显示,同高脂组相比,50mg/kg二氢吡啶添加组肝脏组织脂肪空泡与高脂组相比显著减少,但150mg/kg二氢吡啶组脂滴积累与高脂组相比则无差异。高脂饲料抑制了脂肪酶活性以及T3的分泌,而添加二氢吡啶缓解了抑制作用,且二者变化趋势一致,与二氢吡啶的添加量呈正相关。饲料中添加50mg/kg二氢吡啶显著提高了脂解基因以及β氧化基因的表达量,且显著降低脂合成基因的表达。同时,高脂饲料会增加炎症基因(IL-lβ、TNF-α)的表达,但添加50mg/kg二氢吡啶可以显著抑制炎症基因的表达(P0.05),与正常组炎症基因表达无显著差异(P0.05),随着二氢吡啶添加量的增加,炎症基因的表达呈上升趋势。提示高脂饲料中二氢吡啶添加量为50mg/kg时能显著降低鱼体、肝脏组织以及肌肉组织内脂肪的沉积,但过量添加(150mg/kg)则无明显效果。同时,高脂饲料中添加50mg/kg的二氢吡啶能抑制炎症相关基因的表达,缓解炎症反应,有利于促进鱼类的健康生长,且添加二氢吡啶后T3含量的变化趋势与脂肪酶活性的变化趋势一致。因此,猜测二氢吡啶可能通过T3发挥促生长和脂解的作用。(4)高脂饲料投喂尼罗罗非鱼后注射T3对脂解基因表达的影响为深入探究T3对尼罗罗非鱼体内脂肪沉积的影响,本研究采用脂肪水平为15%的饲料进行投喂,八周后注射浓度分别为0.5、1和1.5nM/g的T3,对照组注射生理盐水。并在注射1,3和6小时后取样,结果显示注射T3组脂体比下降,且随着T3浓度的增加以及作用时间的延长,脂体比下降幅度变大。同对照组相比,注射0.5nM/g后,鱼体脂肪组织中ATGL基因表达在1h和3h显著下调(P0.05);1.5nM/gT3注射1h和1nM/g注射3h后,脂肪组织中HSL基因表达显著上调(P0.05),其他处理组无显著变化(P0.05);1nM/gT3注射3h后脂肪组织中MGL基因表达显著上调(P0.05),其他处理组无显著变化(P0.05)。实验结果表明,注射T3能够有效降低尼罗罗非鱼体内的脂肪沉积,在注射后六小时,脂体比下降,却对脂肪组织中脂解相关基因表达无显著的影响,因此T3或不是通过促进脂解基因的表达来促进体内的脂解,而可能是通过其他方面,如自噬等来促进脂解。
[Abstract]:In this paper, the Nile tilapia (Oreochromis niloticus) as the research object, the application of nutrition, biochemistry and molecular biology methods, a more detailed study of the dietary fat level, feed two hydrogen pyridine and in vitro injection three triiodothyronine (T3) effect on the growth of Nile tilapia and body fat deposition, the specific research results are as follows: (1) the Nile tilapia hormone sensitive lipase (HSL) gene, monoglyceride lipase (MGL) gene cloning and bioinformatics analysis to explore the HSL and MGL genes in Nile tilapia lipolysis regulation of metabolism, the first cloned full-length Nile tilapia HSL gene and MGL gene, and to explore the the HSL gene and MGL gene tissue-specific expression, in order to investigate the function and mechanism of the gene. The results showed that the full length of HSL gene of Nile tilapia was 3147bp, and the length of open reading frame was 2142bp, which could encode 714 amino acid residues. The molecular weight of the protein was 69.70 kDa and the isoelectric point (PI) was 7.64. The full length of the MGL gene is 7448bp, the length of the open reading frame is 918bp, which can encode 306 amino acid residues, the molecular weight of the MGL protein is 34 kDa, and the isoelectric point (PI) is 5.88. Fluorescent quantitative analysis showed that HSL gene and MGL gene were expressed in brain, gill, heart, skin, white muscle, red muscle, liver, abdominal fat, spleen, kidney and intestinal tissue. The expression level of HSL gene in adipose tissue was the highest, followed by red muscle, white muscle and brain tissue. The expression of MGL gene in the brain is the highest, and the red and white muscles are the next. This indicates that HSL and MGL genes play an important role in the hydrolysis of fat in Nile tilapia, but the specific function and mechanism still need further experimental verification. (2) effects of dietary lipid levels and starvation treatment on the expression of genes to explore the lipolysis of Nile tilapia feed fat level, and the effects of starvation on expression of lipase gene in Nile tilapia. The experimental design of fat level was 5% (normal fat), 15% (high fat) two kinds of feed, satiation feeding initial weight was 6.13 + 0.20g of Nile tilapia 6 weeks after the end of the experiment respectively in the last 1, 5, 12, with 24 and 168 hours after taking the fat, liver and white muscle tissue samples of different time points in different tissues of ATGL, HSL, MGL gene expression, the results show that the high fat group (15%) of the body fat ratio is higher than the normal group, but with the increase of the starvation time difference tends to decrease. In adipose tissue, with the prolonging of starvation, the expression of ATGL gene in high fat group decreased first and then increased, normal gene expression increased in adipose tissue of high fat group; HSL gene expression was first increased and then decreased, 24h expression increased significantly (P0.05), lipid group gene expression is higher than that of other groups in 5h and 24h group; each time point in adipose tissue of high fat group MGL gene expression had no significant change (P0.05), normal group gene expression decreased first and then increased. The expression was significantly higher in group 168h (P0.05) gene. The above results show that high fat and hunger could promote the expression of lipolytic genes, so as to promote the hydrolysis of fats. But under normal conditions, hunger can promote the hydrolysis of fat, but it is not conducive to the normal growth of fish. (3) high fat diet added two hydrogen pyridine on Nile tilapia growth and body composition, fat deposition and lipolysis related gene expression in the 235 tail health without injury, the initial weight was 5.91 + 0.43g of Nile tilapia were divided into 5 groups, were fed fat level 5%, two kinds of dietary fat level 15% the 15% level of fat feed were added to Omg/kg, 50mg/kg, 100mg/kg and 150mg/kg two hydrogen pyridine, a total of 5 kinds of feed on fish feeding, the test period was 6 weeks. The results showed that adding two hydrogen pyridine in the high fat diet of Nile tilapia played an important role to promote growth, but compared with the high fat diet group, the addition of 50mg/kg and 100mg/kg two hydrogen pyridine in the high fat diet can significantly reduce the fat content of fish body (P0.05), and the addition of 150mg/kg had no significant changes (P0.05). Histological results showed that compared with the high-fat group, the fat vacuoles in liver tissue of 50mg/kg two pyridine group increased significantly compared with those in high fat group, but there was no difference in lipid droplet accumulation in 150mg/kg two hydrogen pyridine group compared with those in high fat group. High fat diet inhibited lipase activity and T3 secretion, while adding two hydropyridine alleviated the inhibitory effect, and the two had the same trend of change, which was positively correlated with the dosage of two hydropyridine. Add 50mg/kg two hydrogen pyridine feed significantly increased the expression of Lipolytic Gene and beta oxidation and significantly reduced the expression of genes, low-fat synthetic gene. At the same time, the high fat diet will increase the inflammatory gene (IL-l beta, TNF- alpha) expression, but the expression of 50mg/kg two add hydrogen pyridine can significantly inhibit the inflammatory gene (P0.05), there was no significant difference between normal group and gene expression of inflammation (P0.05), with the increasing of the amount of two hydrogen pyridine, the expression of inflammatory genes were increased trend. It is suggested that the amount of two hydropyridine in high fat diet can significantly reduce the deposition of fat in fish body, liver tissue and muscle tissue, but excessive addition (150mg/kg) has no obvious effect on 50mg/kg. At the same time, adding two 50mg/kg hydropyridine to high fat diet can inhibit the expression of inflammation related genes, alleviate inflammatory reaction, and promote the healthy growth of fishes. After adding two hydropyridine, the trend of T3 content is consistent with the trend of lipase activity. Therefore, guess two DHPs may play a role in promoting the growth and lipolysis by T3. (4) high fat feed effect on lipolysis of gene expression after injection of T3 feed of tilapia in order to explore the effect of T3 on Tilapia body fat deposition, this study uses fat levels of 15% diets were fed eight weeks after injection, the concentrations were 0.5, 1 and 1.5nM/g T3, the control group was injected with physiological brine
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S965.125

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