本文選題：吉富羅非魚(yú) + 硬脂酰輔酶A去飽和酶　； 參考：《南京農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：硬脂酰輔酶A去飽和酶(stearoyl-CoA desaturates,SCD)是合成單不飽和脂肪酸(monounsaturated fatty acid,MUFA)的限速酶,是調(diào)節(jié)肝臟脂肪生成和脂類(lèi)氧化的關(guān)鍵控制點(diǎn)。經(jīng)聯(lián)合國(guó)糧農(nóng)組織(FAO)調(diào)查數(shù)據(jù)顯示,2014年全球100多個(gè)羅非魚(yú)養(yǎng)殖地區(qū)的年產(chǎn)量總和達(dá)5446800t,其中中國(guó)羅非魚(yú)產(chǎn)量在2014年增長(zhǎng)至1450000t。由于羅非魚(yú)對(duì)低溫環(huán)境耐受力較差以及養(yǎng)殖過(guò)程中的不當(dāng)投喂,使羅非魚(yú)產(chǎn)業(yè)受到重大的經(jīng)濟(jì)損失。吉富羅非魚(yú)是我國(guó)南方各省主要的淡水經(jīng)濟(jì)養(yǎng)殖魚(yú)類(lèi),研究吉富羅非魚(yú)的SCD基因?qū)τ谔岣呖沟蜏啬芰疤岣呱唐肤~(yú)的營(yíng)養(yǎng)價(jià)值具有重要意義。本實(shí)驗(yàn)是國(guó)內(nèi)首次從吉富羅非魚(yú)體內(nèi)克隆得到SCD基因的cDNA序列,同時(shí)對(duì)基因結(jié)構(gòu)和表達(dá)時(shí)序特征進(jìn)行分析。并結(jié)合飼料中的脂肪酸組成和水溫對(duì)吉富羅非魚(yú)生長(zhǎng)、生理、脂肪酸組成與SCD表達(dá)的影響,從營(yíng)養(yǎng)、環(huán)境及分子水平較為深入研究了 SCD的表達(dá)調(diào)控網(wǎng)絡(luò)。為羅非魚(yú)的脂肪酸調(diào)控以及培育高品質(zhì)、高營(yíng)養(yǎng)價(jià)值的羅非魚(yú)提供了一定的理論依據(jù)。1.吉富羅非魚(yú)SCD基因的克隆與序列分析本研究利用實(shí)時(shí)熒光定量PCR(real-time PCR,RT-PCR)和cDNA末端快速擴(kuò)增法(rapid-amplificationof cDNAends,RACE)技術(shù)克隆獲得了吉富羅非魚(yú)的SCD基因的cDNA全長(zhǎng)序列。結(jié)果顯示,SCD基因的cDNA總長(zhǎng)為1333bp,由173bp5'非編碼區(qū)、152bp 3'非編碼區(qū)以及編碼335個(gè)氨基酸的1008bp開(kāi)放閱讀框組成。與GenBank中,尼羅羅非魚(yú)、莫桑比克羅非魚(yú)相似度較高,基因序列同源性分別達(dá)99%和98%,氨基酸序列同源性分別為99%和97%;與其他魚(yú)類(lèi)SCD基因序列相似度達(dá)83%以上,氨基酸序列同源性達(dá)79%以上。與其他魚(yú)類(lèi)的SCD基因相似度很高,說(shuō)明該基因在物種間高度保守。2.吉富羅非魚(yú)SCD基因組織及時(shí)空表達(dá)規(guī)律本研究通過(guò)RT-PCR檢測(cè)了 SCD基因在吉富羅非魚(yú)受精卵及胚胎發(fā)育階段(受精后 1h、12h、24h、48h、72h、96h)、仔稚魚(yú)階段(出膜后 1d、5d、10d、15d、20d、25d、30d)及成魚(yú)組織(血、肝臟、脾臟、腎臟、心臟、前腸、后腸、性腺、腦、皮膚、肌肉、鰓)中的表達(dá)。在胚胎發(fā)育的早期階段1h和12h時(shí),SCD基因的表達(dá)量較高,24h時(shí)出現(xiàn)明顯下降的趨勢(shì),48h時(shí)有所升高,隨后逐漸下降,96h最低。在受精卵及胚胎的早期階段(1-15d)時(shí),SCD表達(dá)量較低,并呈現(xiàn)逐步上升的趨勢(shì)。20d時(shí)SCD的表達(dá)量顯著升高,隨后呈先下降后升高的趨勢(shì)。表明了羅非魚(yú)在發(fā)育各個(gè)階段所需的脂肪酸營(yíng)養(yǎng)不同,從而導(dǎo)致脂肪酸代謝酶的表達(dá)水平也有所不同,為吉富羅非魚(yú)繁殖階段及魚(yú)苗培育階段的脂肪酸營(yíng)養(yǎng)的提供了理論依據(jù)。分別對(duì)雄魚(yú)和雌魚(yú)組織的SCD表達(dá)量進(jìn)行檢測(cè),結(jié)果表明該基因在吉富羅非魚(yú)雄性和雌性的各組織表達(dá)量的高低有所不同,在肝臟中表達(dá)量均達(dá)到最高,其次在雄魚(yú)的精巢中表達(dá)量較高,而雌魚(yú)的肌肉中的表達(dá)量?jī)H次于肝臟。在雌魚(yú)和雄魚(yú)的脾臟中表達(dá)量均為最低。證明了吉富羅非魚(yú)雌魚(yú)與雄魚(yú)的脂肪酸代謝機(jī)制具有一定差異,為進(jìn)一步研究羅非魚(yú)UFA的合成途徑及調(diào)控機(jī)理提供了一定理論基礎(chǔ)。3.溫度對(duì)吉富羅非魚(yú)SCD基因表達(dá)的研究本實(shí)驗(yàn)對(duì)吉富羅非魚(yú)在22℃、28℃和34℃三種不同養(yǎng)殖溫度下對(duì)SCD基因及肌肉脂肪酸相關(guān)性進(jìn)行分析,同時(shí)對(duì)生長(zhǎng)指標(biāo)、血常規(guī)及血液生理指標(biāo)進(jìn)行了測(cè)定。28℃終體重顯著高于22和34℃(P0.05),FCR和PER均高于22和34℃。不同組間的肌肉脂肪酸組成具有顯著性差異(P0.05),SFA含量隨溫度的升高而降低而UFA含量呈相反的變化趨勢(shì)。22℃時(shí)羅非魚(yú)含較高的PUFA,尤其是n-3PUFA。SCD基因的表達(dá)及活性在低溫時(shí)顯著性升高(P0.05),并且增加吉富羅非魚(yú)SCD的表達(dá)及活性可提升肌肉MUFA的合成。此外,降低水溫可降低機(jī)體TC和TG的含量,對(duì)于提高吉富羅非魚(yú)抗低溫能力具有一定幫助。4.脂肪源對(duì)吉富羅非魚(yú)SCD基因表達(dá)的研究日糧中添加不同脂肪源對(duì)魚(yú)類(lèi)的生長(zhǎng)、生理等均具有一定影響,主要是由于飼料中脂肪酸組成不同,及不同魚(yú)類(lèi)對(duì)脂肪酸利用不同引起的。本研究分別以椰子油(主要含SFA)、橄欖油(主要含MUFA)、魚(yú)油(脂肪酸比例均衡)及魚(yú)油+豆油(1:1)(主要含PUFA)為脂肪源,進(jìn)行60d的養(yǎng)殖實(shí)驗(yàn)。通過(guò)分析生長(zhǎng)指標(biāo),發(fā)現(xiàn)飼料中添加脂肪酸比例較均衡的魚(yú)油組生長(zhǎng)效果最好。添加不同脂肪源對(duì)血液和肝臟的生理指標(biāo)具有一定影響,添加SFA會(huì)引起血液與肝臟中TC、TG含量上升,而添加PUFA可以有效地降低體脂的沉積。并且添加PUFA可以增強(qiáng)機(jī)體免疫力,提高抗氧化酶活性,降低氧化損傷。此外,飼料中不同脂肪酸組成對(duì)肌肉和肝臟脂肪酸的組成具有顯著性影響,魚(yú)體脂肪酸組成中的SFA及UFA分別于飼料脂肪酸組成的SFA及UFA呈正相關(guān)。并且飼料中高水平的PUFA會(huì)抑制SCD的表達(dá)與活性并降低體脂沉積而增加SFA會(huì)促進(jìn)SCD的表達(dá)與活性。因此,均衡日糧中的脂肪酸組成,適當(dāng)提高多不飽和脂肪酸含量有助于增加吉富羅非魚(yú)生長(zhǎng)性能,降低血清脂肪沉積。
[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.

【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S917.4

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