發(fā)布時間：2017-12-27 08:08

  本文關(guān)鍵詞：飼料中添加二氫吡啶和注射三碘甲腺原氨酸（T3）對尼羅羅非魚機體脂肪沉積及相關(guān)脂解基因表達的影響　出處：《華東師范大學》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 尼羅羅非魚 基因克隆 高脂飼料 饑餓 二氫吡啶 三碘甲狀腺原氨酸

【摘要】：本論文以尼羅羅非魚(Oreochromis niloticus)為研究對象,應用營養(yǎng)學、生物化學和分子生物學等方法,較為詳細地研究了飼料脂肪水平、飼料二氫吡啶以及體外注射三碘甲狀腺原氨酸(T3)對尼羅羅非魚生長和機體脂肪沉積的影響,具體研究結(jié)果如下:(1)尼羅羅非魚激素敏感脂肪酶(HSL)基因、單甘油酯脂肪酶(MGL)基因的克隆與相關(guān)生物信息學分析為探究HSL和MGL基因在尼羅羅非魚脂肪分解代謝中的調(diào)控作用,本實驗首先克隆得到尼羅羅非魚HSL基因和MGL基因的全長,并探討了 HSL基因和MGL基因組織特異性表達,以期進一步研究上述基因的功能和作用機制。結(jié)果表明,尼羅羅非魚HSL基因全長是3147bp,開放閱讀框長度是2142bp,可以編碼714個氨基酸殘基,蛋白質(zhì)的分子量為69.70 kDa,等電點(pi)為7.64。MGL基因全長是7448bp,開放閱讀框長度是918bp,可以編碼306個氨基酸殘基,MGL蛋白質(zhì)的分子量為34 kDa,等電點(pi)為5.88。熒光定量分析發(fā)現(xiàn),HSL基因和MGL基因在腦、鰓、心臟、皮膚、白肌、紅肌、肝、腹腔脂肪、脾、腎臟和腸組織中均有表達,其中HSL基因在脂肪組織中表達量最高,其次是紅肌、白肌和腦等組織。MGL基因在腦中表達量最高,紅肌,白肌等組織次之。這說明了 HSL和MGL基因?qū)δ崃_羅非魚體內(nèi)脂肪的水解起著重要的作用,但具體的功能和作用機制尚需進一步的實驗驗證。(2)飼料脂肪水平以及饑餓處理對尼羅羅非魚脂解基因表達的影響為探究飼料脂肪水平,以及饑餓處理對尼羅羅非魚脂解基因表達的影響。本實驗設計了脂肪水平為5%(正常脂)、15%(高脂)的兩種飼料,飽食投喂初重為6.13±0.20g的尼羅羅非魚6周,實驗結(jié)束后分別在最后一次飽食1、5、12、24和168小時后取脂肪、肝臟和白肌組織樣品,分析不同時間點不同組織中ATGL、HSL、MGL基因的表達情況,結(jié)果表明:高脂組(15%)的脂體比高于正常組,但隨著饑餓時間的延長,差異趨于減小。脂肪組織中,隨著饑餓時間的延長,高脂組ATGL基因的表達呈先下降后上升的趨勢,正常組基因表達呈上升趨勢;脂肪組織中高脂組HSL基因表達呈先上升后下降的趨勢,在24h時表達量顯著升高(P0.05),中脂組基因表達在5h和24h組高于其他組;脂肪組織中各時間點高脂組MGL基因表達無顯著變化(P0.05),正常組基因表達呈先下降后上升的趨勢,在168h組基因表達顯著上調(diào)(P0.05)。以上結(jié)果表明高脂處理和饑餓均能夠促進脂解基因的表達,從而促進脂肪水解。但在正常養(yǎng)殖條件下,饑餓雖然可以促進脂肪的水解,但是不利于魚類的正常生長。(3)高脂飼料中添加二氫吡啶對尼羅羅非魚生長、體成分、脂肪沉積以及脂解相關(guān)基因表達的影響將235尾健康無傷,初始體重為5.91 ±0.43g的尼羅羅非魚隨機分成5個處理組,分別投喂脂肪水平5%,脂肪水平15%的兩種飼料,其中15%脂肪水平的飼料分別添加Omg/kg、50mg/kg、100mg/kg和150mg/kg二氫吡啶,共5種飼料對魚進行投喂,試驗周期為6周。結(jié)果表明:高脂飼料中添加二氫吡啶對尼羅羅非魚的生長起到了促進作用,但是同高脂飼料組相比,高脂飼料中添加50mg/kg和100mg/kg二氫吡啶可以顯著降低魚體粗脂肪含量(P0.05),而150mg/kg添加組則無顯著變化(P0.05)。組織學的結(jié)果顯示,同高脂組相比,50mg/kg二氫吡啶添加組肝臟組織脂肪空泡與高脂組相比顯著減少,但150mg/kg二氫吡啶組脂滴積累與高脂組相比則無差異。高脂飼料抑制了脂肪酶活性以及T3的分泌,而添加二氫吡啶緩解了抑制作用,且二者變化趨勢一致,與二氫吡啶的添加量呈正相關(guān)。飼料中添加50mg/kg二氫吡啶顯著提高了脂解基因以及β氧化基因的表達量,且顯著降低脂合成基因的表達。同時,高脂飼料會增加炎癥基因(IL-lβ、TNF-α)的表達,但添加50mg/kg二氫吡啶可以顯著抑制炎癥基因的表達(P0.05),與正常組炎癥基因表達無顯著差異(P0.05),隨著二氫吡啶添加量的增加,炎癥基因的表達呈上升趨勢。提示高脂飼料中二氫吡啶添加量為50mg/kg時能顯著降低魚體、肝臟組織以及肌肉組織內(nèi)脂肪的沉積,但過量添加(150mg/kg)則無明顯效果。同時,高脂飼料中添加50mg/kg的二氫吡啶能抑制炎癥相關(guān)基因的表達,緩解炎癥反應,有利于促進魚類的健康生長,且添加二氫吡啶后T3含量的變化趨勢與脂肪酶活性的變化趨勢一致。因此,猜測二氫吡啶可能通過T3發(fā)揮促生長和脂解的作用。(4)高脂飼料投喂尼羅羅非魚后注射T3對脂解基因表達的影響為深入探究T3對尼羅羅非魚體內(nèi)脂肪沉積的影響,本研究采用脂肪水平為15%的飼料進行投喂,八周后注射濃度分別為0.5、1和1.5nM/g的T3,對照組注射生理鹽水。并在注射1,3和6小時后取樣,結(jié)果顯示注射T3組脂體比下降,且隨著T3濃度的增加以及作用時間的延長,脂體比下降幅度變大。同對照組相比,注射0.5nM/g后,魚體脂肪組織中ATGL基因表達在1h和3h顯著下調(diào)(P0.05);1.5nM/gT3注射1h和1nM/g注射3h后,脂肪組織中HSL基因表達顯著上調(diào)(P0.05),其他處理組無顯著變化(P0.05);1nM/gT3注射3h后脂肪組織中MGL基因表達顯著上調(diào)(P0.05),其他處理組無顯著變化(P0.05)。實驗結(jié)果表明,注射T3能夠有效降低尼羅羅非魚體內(nèi)的脂肪沉積,在注射后六小時,脂體比下降,卻對脂肪組織中脂解相關(guān)基因表達無顯著的影響,因此T3或不是通過促進脂解基因的表達來促進體內(nèi)的脂解,而可能是通過其他方面,如自噬等來促進脂解。
[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
【學位授予單位】：華東師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S965.125

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