三种不同品系红罗非鱼耐寒性能比较以及体色相关基因表达分析

发布时间：2018-06-28 12:06

本文选题：红罗非鱼 + 耐寒性　；参考：《南京农业大学》2016年硕士论文

【摘要】：体色是鱼类一种较独特的表型性状,有重要的生物多样性价值和遗传学理论研究价值。鱼类体色变化是一个复杂的过程,遗传因素以及水温、光照、pH值、溶氧等众多外界环境因素都会造成鱼类体色的变化。在红罗非越冬养殖期间发现,经历过低温的红罗非鱼通常会发生体色变化,出现红色或者黑色斑纹,这样的变化会使红罗非鱼的经济价值下降。红罗非鱼是暖水性鱼类,具有不耐低温的特性。推断低温会影响红罗非鱼体色变化。本文从血清生理生化指标、抗氧化酶及两个抗寒候选基因角度分析比较了以色列、台湾和马来西亚三个红罗非鱼品系的耐寒性差异。为了进一步深入探讨红罗非鱼体色变化的机制,从黑色素合成通路的分子角度比较了6个体色相关基因(MC1R、TYRP1、ASIP、DCT、SOX10和S1C7A11)在三个红罗非鱼品系中的表达差异。主要研究结果如下:1.三个不同品系红罗非鱼耐寒性能比较实验选取平均体重为500g左右的以色列品系、台湾品系和马来西亚品系的红罗非鱼作为实验对象,设定19℃的常温对照组和从19℃开始每天降低1℃直到8℃的冷应激实验组,每组设定2个平行组,每个平行组中6尾鱼。对照组在实验开始时进行采样,实验组在鱼开始出现死亡情况时进行采样。测定血清丙转氨酶(ALT)、谷草转氨酶(AST)、乳酸脱氢酶(LDH)、血糖(GLU)、总蛋白(TP)、K+、Na+、Cl-、总超氧化物歧化酶、丙二醛和超微量Na+-K+-ATP酶。结果表明:以色列和台湾品系在10.3℃开始出现死亡个体,马来西亚品系的耐寒性能更强一点,在9.3℃出现死亡个体。三个品系红罗非鱼在8.3℃全部死亡。罗非鱼可在6-10℃低温下短暂存活,但是无法存活24h以上。以色列和台湾品系死亡温度比马来西亚品系高1℃。与对照组相比,三个品系的实验组ALT、AST、LDH、GLU含量显著上升(P0.05),以色列和台湾品系TP含量显著性下降(P0.05),而马来西亚品系TP含量显著上升(P0.05)。以色列品系SOD含量显著上升(P0.05),MDA含量显著上升(P0.05),Na+-K+-ATP酶含量显著下降(P0.05);台湾品系和马来西亚品系SOD含量显著下降(P0.05),Na+-K+-ATP酶含量显著上升(P0.05)。三种品系的耐寒性能不同,所以体内的抗氧化酶含量以及活性也并不相同,其中马来西亚品系经历的温度更低,鱼体内脏等受损程度更加严重,所以体内抗氧化酶等不能发挥其免疫的作用,而是三种品系抗氧化酶等活性表达量不同。当受到低温胁迫时,SCD基因在以色列品系、台湾品系肝脏中mRNA表达量显著上升,SCD基因在马来西亚品系肝脏中mRNA表达量显著下降(P0.05)。HSP70基因在以色列品系、台湾品系和马来西亚品系肝脏中mRNA表达量显著上升(P0.05)。可见,急性低温应激可以引起红罗非鱼体的生理机能的变化,其中马来西亚品系红罗非的耐寒性比以色列和台湾品系红罗非耐寒性能可能更强。2、以色列、台湾、马来西亚品系红罗非体色相关基因的表达分析(1)马来西亚红罗非6个体色相关基因的表达分析实验材料选择身体健壮,健康状况良好的马来西亚红罗非“红斑”、“粉白”、“黑斑”三种体色类型的各3尾。每尾鱼采集背部皮肤、腹部皮肤、肌肉、肠、心、脑共六种组织。qRT-PCR 分析表明,MC1R、ASIP、TYRP1、DCT 和 SLC7A11基因在皮肤组织中表达量显著高于其他四个组织中的表达量(P0.05),在心脏和肠中表达量次之,在肌肉和脑中表达量最低;SOX10基因在脑中表达量显著高于其他四个组织(P0.05),在皮肤及肌肉中表达量次之,在肠和心中表达量最低。MC1R、ASIP、TYRP1、SOX10和SLC7A11基因在红斑体色类型皮肤中的表达量显著地高于其他体色(P0.05)。DCT基因在粉白体色类型和黑斑体色类型的皮肤组织中表达量显著的高于红斑体色类型(P0.05)。由此可以得知体色类型并不是由一个基因控制的,是由一个复杂的基因网络进行调控,各基因之间相互作用最终决定与体色类型。(2)三个品系红罗非皮肤组织中6个基因的表达差异分析实验材料选择身体健壮,健康状况良好的红罗非鱼。马来西亚、台湾和以色列品系各取3尾的背部和腹部皮肤组织。对这三种红罗非品系的背部和腹部皮肤组织进行了基因mRNA的表达差异比较。qRT-PCR分析表明,在背部皮肤组织中,MC1R、ASIP、TYRP1、DCT、SOX10和SLC7A11基因在马来西亚品系表达量显著的高于其他两个品系(P0.05),ASIP和TYRP1基因在以色列品系中的表达量显著的高于台湾品系中的表达量(P0.05),MC1R、DCT、SOX10和SLC7A11基因在以色列和台湾品系中的表达量没有显著性差异(P0.05)。在腹部皮肤组织中,MC1R、ASIP、DCT和SOX10基因在马来西亚品系表达量最高,TYRP1和SLC7A11基因在台湾品系中表达量最高。TYRP1和SLC7 A1 1基因在以色列、台湾与马来西亚品系中均有显著性差异(P0.05),MC1R、ASIP和SOX10基因在以色列与台湾品系中均存在显著性差异(P0.05)。由此可以得知体色相关基因的表达量与鱼种类有一定的相关性。(3)6个基因在各品系红罗非鱼背腹部皮肤组织中表达差异马来西亚、台湾和以色列品系各取3尾的背部和腹部皮肤组织。对每种品系红罗非品系的背部和腹部皮肤组织中的6个基因mRNA的表达差异进行比较。qRT-PCR分析表明,在以色列品系中,MC1R、TYRP1、DCT、SOX10和SLC7A11基因在背部皮肤组织的表达量显著的高于腹部皮肤(P0.05);在台湾品系中,ASIP基因在腹部皮肤组织中表达量显著的高于背部皮肤(P0.05),MC1R、DCT、SOX10和SLC7A11基因在背部的表达量显著高于腹部皮肤(P0.05)。在马来西亚品系中,MC1R、ASIP、TYRP1、SOX10和SLC7A11基因在背部的表达量显著高于腹部皮肤(P0.05)。由此可以得知,ASIP基因在红罗非鱼的背部和腹部皮肤组织表达有差异。
[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.
【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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