本文選題：紅羅非魚(yú) + 耐寒性　； 參考：《南京農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：體色是魚(yú)類一種較獨(dú)特的表型性狀,有重要的生物多樣性價(jià)值和遺傳學(xué)理論研究?jī)r(jià)值。魚(yú)類體色變化是一個(gè)復(fù)雜的過(guò)程,遺傳因素以及水溫、光照、pH值、溶氧等眾多外界環(huán)境因素都會(huì)造成魚(yú)類體色的變化。在紅羅非越冬養(yǎng)殖期間發(fā)現(xiàn),經(jīng)歷過(guò)低溫的紅羅非魚(yú)通常會(huì)發(fā)生體色變化,出現(xiàn)紅色或者黑色斑紋,這樣的變化會(huì)使紅羅非魚(yú)的經(jīng)濟(jì)價(jià)值下降。紅羅非魚(yú)是暖水性魚(yú)類,具有不耐低溫的特性。推斷低溫會(huì)影響紅羅非魚(yú)體色變化。本文從血清生理生化指標(biāo)、抗氧化酶及兩個(gè)抗寒候選基因角度分析比較了以色列、臺(tái)灣和馬來(lái)西亞三個(gè)紅羅非魚(yú)品系的耐寒性差異。為了進(jìn)一步深入探討紅羅非魚(yú)體色變化的機(jī)制,從黑色素合成通路的分子角度比較了6個(gè)體色相關(guān)基因(MC1R、TYRP1、ASIP、DCT、SOX10和S1C7A11)在三個(gè)紅羅非魚(yú)品系中的表達(dá)差異。主要研究結(jié)果如下:1.三個(gè)不同品系紅羅非魚(yú)耐寒性能比較實(shí)驗(yàn)選取平均體重為500g左右的以色列品系、臺(tái)灣品系和馬來(lái)西亞品系的紅羅非魚(yú)作為實(shí)驗(yàn)對(duì)象,設(shè)定19℃的常溫對(duì)照組和從19℃開(kāi)始每天降低1℃直到8℃的冷應(yīng)激實(shí)驗(yàn)組,每組設(shè)定2個(gè)平行組,每個(gè)平行組中6尾魚(yú)。對(duì)照組在實(shí)驗(yàn)開(kāi)始時(shí)進(jìn)行采樣,實(shí)驗(yàn)組在魚(yú)開(kāi)始出現(xiàn)死亡情況時(shí)進(jìn)行采樣。測(cè)定血清丙轉(zhuǎn)氨酶(ALT)、谷草轉(zhuǎn)氨酶(AST)、乳酸脫氫酶(LDH)、血糖(GLU)、總蛋白(TP)、K+、Na+、Cl-、總超氧化物歧化酶、丙二醛和超微量Na+-K+-ATP酶。結(jié)果表明:以色列和臺(tái)灣品系在10.3℃開(kāi)始出現(xiàn)死亡個(gè)體,馬來(lái)西亞品系的耐寒性能更強(qiáng)一點(diǎn),在9.3℃出現(xiàn)死亡個(gè)體。三個(gè)品系紅羅非魚(yú)在8.3℃全部死亡。羅非魚(yú)可在6-10℃低溫下短暫存活,但是無(wú)法存活24h以上。以色列和臺(tái)灣品系死亡溫度比馬來(lái)西亞品系高1℃。與對(duì)照組相比,三個(gè)品系的實(shí)驗(yàn)組ALT、AST、LDH、GLU含量顯著上升(P0.05),以色列和臺(tái)灣品系TP含量顯著性下降(P0.05),而馬來(lái)西亞品系TP含量顯著上升(P0.05)。以色列品系SOD含量顯著上升(P0.05),MDA含量顯著上升(P0.05),Na+-K+-ATP酶含量顯著下降(P0.05);臺(tái)灣品系和馬來(lái)西亞品系SOD含量顯著下降(P0.05),Na+-K+-ATP酶含量顯著上升(P0.05)。三種品系的耐寒性能不同,所以體內(nèi)的抗氧化酶含量以及活性也并不相同,其中馬來(lái)西亞品系經(jīng)歷的溫度更低,魚(yú)體內(nèi)臟等受損程度更加嚴(yán)重,所以體內(nèi)抗氧化酶等不能發(fā)揮其免疫的作用,而是三種品系抗氧化酶等活性表達(dá)量不同。當(dāng)受到低溫脅迫時(shí),SCD基因在以色列品系、臺(tái)灣品系肝臟中mRNA表達(dá)量顯著上升,SCD基因在馬來(lái)西亞品系肝臟中mRNA表達(dá)量顯著下降(P0.05)。HSP70基因在以色列品系、臺(tái)灣品系和馬來(lái)西亞品系肝臟中mRNA表達(dá)量顯著上升(P0.05)。可見(jiàn),急性低溫應(yīng)激可以引起紅羅非魚(yú)體的生理機(jī)能的變化,其中馬來(lái)西亞品系紅羅非的耐寒性比以色列和臺(tái)灣品系紅羅非耐寒性能可能更強(qiáng)。2、以色列、臺(tái)灣、馬來(lái)西亞品系紅羅非體色相關(guān)基因的表達(dá)分析(1)馬來(lái)西亞紅羅非6個(gè)體色相關(guān)基因的表達(dá)分析實(shí)驗(yàn)材料選擇身體健壯,健康狀況良好的馬來(lái)西亞紅羅非“紅斑”、“粉白”、“黑斑”三種體色類型的各3尾。每尾魚(yú)采集背部皮膚、腹部皮膚、肌肉、腸、心、腦共六種組織。qRT-PCR 分析表明,MC1R、ASIP、TYRP1、DCT 和 SLC7A11基因在皮膚組織中表達(dá)量顯著高于其他四個(gè)組織中的表達(dá)量(P0.05),在心臟和腸中表達(dá)量次之,在肌肉和腦中表達(dá)量最低;SOX10基因在腦中表達(dá)量顯著高于其他四個(gè)組織(P0.05),在皮膚及肌肉中表達(dá)量次之,在腸和心中表達(dá)量最低。MC1R、ASIP、TYRP1、SOX10和SLC7A11基因在紅斑體色類型皮膚中的表達(dá)量顯著地高于其他體色(P0.05)。DCT基因在粉白體色類型和黑斑體色類型的皮膚組織中表達(dá)量顯著的高于紅斑體色類型(P0.05)。由此可以得知體色類型并不是由一個(gè)基因控制的,是由一個(gè)復(fù)雜的基因網(wǎng)絡(luò)進(jìn)行調(diào)控,各基因之間相互作用最終決定與體色類型。(2)三個(gè)品系紅羅非皮膚組織中6個(gè)基因的表達(dá)差異分析實(shí)驗(yàn)材料選擇身體健壯,健康狀況良好的紅羅非魚(yú)。馬來(lái)西亞、臺(tái)灣和以色列品系各取3尾的背部和腹部皮膚組織。對(duì)這三種紅羅非品系的背部和腹部皮膚組織進(jìn)行了基因mRNA的表達(dá)差異比較。qRT-PCR分析表明,在背部皮膚組織中,MC1R、ASIP、TYRP1、DCT、SOX10和SLC7A11基因在馬來(lái)西亞品系表達(dá)量顯著的高于其他兩個(gè)品系(P0.05),ASIP和TYRP1基因在以色列品系中的表達(dá)量顯著的高于臺(tái)灣品系中的表達(dá)量(P0.05),MC1R、DCT、SOX10和SLC7A11基因在以色列和臺(tái)灣品系中的表達(dá)量沒(méi)有顯著性差異(P0.05)。在腹部皮膚組織中,MC1R、ASIP、DCT和SOX10基因在馬來(lái)西亞品系表達(dá)量最高,TYRP1和SLC7A11基因在臺(tái)灣品系中表達(dá)量最高。TYRP1和SLC7 A1 1基因在以色列、臺(tái)灣與馬來(lái)西亞品系中均有顯著性差異(P0.05),MC1R、ASIP和SOX10基因在以色列與臺(tái)灣品系中均存在顯著性差異(P0.05)。由此可以得知體色相關(guān)基因的表達(dá)量與魚(yú)種類有一定的相關(guān)性。(3)6個(gè)基因在各品系紅羅非魚(yú)背腹部皮膚組織中表達(dá)差異馬來(lái)西亞、臺(tái)灣和以色列品系各取3尾的背部和腹部皮膚組織。對(duì)每種品系紅羅非品系的背部和腹部皮膚組織中的6個(gè)基因mRNA的表達(dá)差異進(jìn)行比較。qRT-PCR分析表明,在以色列品系中,MC1R、TYRP1、DCT、SOX10和SLC7A11基因在背部皮膚組織的表達(dá)量顯著的高于腹部皮膚(P0.05);在臺(tái)灣品系中,ASIP基因在腹部皮膚組織中表達(dá)量顯著的高于背部皮膚(P0.05),MC1R、DCT、SOX10和SLC7A11基因在背部的表達(dá)量顯著高于腹部皮膚(P0.05)。在馬來(lái)西亞品系中,MC1R、ASIP、TYRP1、SOX10和SLC7A11基因在背部的表達(dá)量顯著高于腹部皮膚(P0.05)。由此可以得知,ASIP基因在紅羅非魚(yú)的背部和腹部皮膚組織表達(dá)有差異。
[Abstract]:Body color is a more unique phenotypic character of fish. It has important value of biological diversity and genetic theory. The change of fish body color is a complex process. Many external environmental factors such as genetic factors and water temperature, light, pH value, dissolved oxygen and other environmental factors will cause changes in the body color of fish. Red tilapia usually changes color and appears red or black markings. This change will reduce the economic value of red tilapia. Red tilapia is warm water fish and has the characteristics of low temperature resistance. It is inferred that low temperature will affect the body color change of red tilapia. This article from serum physiological and biochemical indexes, antioxidant enzymes and two The cold tolerance difference between three red tilapia lines in Israel, Taiwan and Malaysia was analyzed by an angle of cold resistant candidate genes. In order to further explore the mechanism of the body color change of the red tilapia, 6 color related genes (MC1R, TYRP1, ASIP, DCT, SOX10 and S1C7A11) were compared in three red ROS from the molecular angle of the melanin synthesis pathway. The main results are as follows: 1. the comparison experiment of the cold resistance of three different strains of red tilapia selected the Israeli strains with the average weight of about 500g, the Taiwan strain and the red tilapia of Malaysia strain as the experimental object, setting the normal temperature control group at 19 degrees C and decreasing 1 degrees to 8 every day from 19 degrees C. In the cold stress experimental group, 2 parallel groups were set in each group, 6 fish in each parallel group. The control group was sampled at the beginning of the experiment. The experimental group was sampled at the beginning of the death of the fish. The serum prop aminotransferase (ALT), AST, LDH, GLU, TP, K+, Na+, Cl-, and total superoxide dismutase were measured. Enzyme, malondialdehyde and ultramicro Na+-K+-ATP enzyme. The results showed that the death individual began to appear in Israel and Taiwan strain at 10.3 degrees C, the cold resistance of Malaysia strain was stronger and the death individual appeared at 9.3. Three strains of red tilapia died at 8.3. The tilapia could survive at 6-10 temperature, but could not survive 24h The death temperature of Israel and Taiwan strains was 1 higher than that of the Malaysia strain. Compared with the control group, the content of ALT, AST, LDH, GLU in the experimental group increased significantly (P0.05), the TP content in Israel and Taiwan decreased significantly (P0.05), while the TP content in the Malaysia strain increased significantly (P0.05). The SOD content of the Israeli strain rose significantly (P0). .05), the content of MDA increased significantly (P0.05), and the content of Na+-K+-ATP enzyme decreased significantly (P0.05); the content of SOD in Taiwan strain and Malaysia strain decreased significantly (P0.05), and the content of Na+-K+-ATP enzyme increased significantly (P0.05). The cold resistance of the three strains was different, so the content and activity of antioxidant enzymes in the body were different, among which the Malaysia strain was experienced. The temperature is lower and the damage degree of the fish viscera is more serious, so the antioxidant enzyme in the body can not play its immune function, but the active expression of antioxidant enzymes in the three strains of the three strains is different. When under low temperature stress, the expression of mRNA in the liver of the Taiwan strain increases significantly in the Israeli strain, and the SCD gene is in the Malaysia strain. The expression of mRNA in the liver decreased significantly (P0.05).HSP70 gene in the Israeli strains, the Taiwan strain and the Malaysia strain liver mRNA expression increased significantly (P0.05). The acute low temperature stress could cause the physiological function changes of the red tilapia, and the cold resistance of the Malaysia strain red rose was more red than that of the Israeli and Taiwan strains. The cold resistance of Luo Fei may be more.2, Israel, Taiwan, Malaysia and Malaysia, the expression analysis of non body color related genes (1) the expression analysis of 6 color related genes in red Rome, Malaysia, three kinds of body color, red spot, pink and black spot in Malaysia, healthy and healthy The.QRT-PCR analysis of six tissues, including back skin, abdominal skin, muscle, intestine, heart and brain, showed that the expression of MC1R, ASIP, TYRP1, DCT and SLC7A11 genes in the skin tissues was significantly higher than those of the other four tissues (P0.05), and the lowest expression in the heart and intestines, and the lowest expression in the muscles and brain; SOX1. The expression of 0 gene in the brain was significantly higher than that of the other four tissues (P0.05), the expression in the skin and muscle was the lowest, and the expression in the intestine and heart was lowest.MC1R. The expression of ASIP, TYRP1, SOX10 and SLC7A11 genes in the erythematous color type skin was significantly higher than that of the other body color (P0.05).DCT genes in the pink and black body color types. The expression of the skin tissue is significantly higher than that of the erythema color type (P0.05). Therefore, it can be found that the body color type is not controlled by a gene, and is regulated by a complex gene network. The interaction of each gene is ultimately determined by the type of body color. (2) the difference of the expression of 6 genes in the three strains of red non skin tissue is divided. The experimental materials selected the healthy and healthy red tilapia, healthy red tilapia, Malaysia, Taiwan, and the Israeli Department of Malaysia, each took the back and abdominal skin tissues of the 3 tails. The difference of gene expression between the back and abdominal skin tissues of the three red non strain lines was compared with.QRT-PCR analysis, in the back skin tissue, MC1R, ASIP, The expression of TYRP1, DCT, SOX10 and SLC7A11 genes in Malaysia strain was significantly higher than that of other two lines (P0.05). The expression of ASIP and TYRP1 genes in Israeli strains was significantly higher than that in Taiwan strain (P0.05), and there was no significant difference in the expression of MC1R, DCT, SOX10 and SLC7A11 genes in Israel and Taiwan strains. .05). In the abdominal skin tissue, the highest expression of MC1R, ASIP, DCT and SOX10 genes in Malaysia strain, the highest.TYRP1 and SLC7 A1 1 genes of TYRP1 and SLC7A11 genes in Taiwan, Taiwan and Malaysia are significant differences (P0.05). There was a significant difference (P0.05). It was found that the expression of body color related genes was related to the fish species. (3) the 6 genes expressed in the dorsal and abdominal skin tissues of the strains of red tilapia, Malaysia, Taiwan and Israel, each took 3 back and abdominal skin tissues. The difference in expression of 6 genes mRNA in the back and abdominal skin tissues was compared with.QRT-PCR analysis. In the Israeli strain, the expression of MC1R, TYRP1, DCT, SOX10 and SLC7A11 genes in the back skin tissues was significantly higher than that of the abdominal skin (P0.05); in the Taiwan strain, the expression of the ASIP gene in the abdominal skin tissues was significantly higher than that in the abdominal skin. The back skin (P0.05), MC1R, DCT, SOX10 and SLC7A11 genes were significantly higher in the back than in the abdominal skin (P0.05). In the Malaysia strain, the expression of MC1R, ASIP, TYRP1, SOX10 and SLC7A11 genes in the back was significantly higher than that of the abdominal skin (P0.05). There is a difference.
【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
【分類號(hào)】：S917.4

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