鲫鱼TAUT和CSAD基因克隆及其表达研究

发布时间：2018-04-05 10:11

本文选题：TAUT　切入点：CSAD　出处：《湖南农业大学》2016年硕士论文

【摘要】：牛磺酸对鱼类生长发育有调控作用,TAUT和CSAD分别是动物细胞内牛磺酸跨膜转运吸收的关键载体和自主合成的主要限速酶。本研究以鲫鱼为研究对象,克隆了鲫鱼TAUT和CSAD基因,检测这两个基因mRNA在鲫鱼不同组织,昼夜节律中的相对表达,同时还研究了牛磺酸对鲫鱼生长,TAUT,CSAD表达的影响,以期为解释牛磺酸对水产动物的作用机理提供新的理论依据。1、鲫鱼TAUT和CSAD基因克隆及生物信息学分析在我们的研究中,采用RACE等技术,从鲫鱼肠道中得到了TAUT,CSAD基因的完整的ORF序列。应用生物信息学软件分析序列表明：TAUT的ORF全长为1978bp,编码625个氨基酸；氨基酸区域中存在12个跨膜区域,多重序列比对分析表明鲫鱼TAUT与鲤鱼,斑马鱼一致性高达97%。系统进化分析显示与斑马鱼的亲缘关系最近,其次是鲤鱼。CSAD的ORF全长为1503 bp,编码500个氨基酸；同源性分析表明,鲫鱼CSAD与鲤鱼CSAD的一致性高达97%；系统进化分析显示,鲫鱼CSAD与鲤鱼CSAD亲缘关系最近。2、鲫鱼TAUT和CSAD基因在不同组织和昼夜节律下的表达分析本研究采用实时荧光定量PCR技术,探讨TAUT和CSAD基因在鲫鱼不同组织分布,昼夜节律的表达差异。结果显示TAUT mRNA在肠组织中的相对表达量最高,显著高于在其它组织中的表达量,在心脏和脑中的相对表达量较低。CSAD mRNA在肌肉,心脏,肠道及肝脏中的相对表达量的差异性不显著,在脑和鳃组织中的相对表达量较低。鲫鱼肠道TAUT mRNA在昼夜节律中的相对表达量呈现的是从0：00开始逐渐上升到12：00,然后开始逐渐下降的一个过程,其中12：00表达量最高,21：00的相对表达量最低。鲫鱼肠道CSAD mRNA在昼夜节律中的相对表达量呈现的同样是从0点开始先上升后下降的过程,其中6：00时相对表达量最高,21：00的相对表达量最低。3、牛磺酸对鲫鱼TAUT和CSAD基因表达的影响本试验以480尾(初始体质量29.07±0.19g)鲫鱼(Carassius auratus)为研究对象,随机分成6组,每组设定4个重复,每个重复有鱼20尾。鲫鱼基础饲料中添加的牛磺酸的剂量分别是0.0 g/kg、1.0g/kg、2.5 g/kg、4.0g/kg、6.0g/kg以及9.0g/kg,以0 g/kg添加组为对照组,试验持续8周。试验结果表明：(1)牛磺酸对鲫鱼有促生长作用,6.0 g/kg添加组的鱼的增重率,特定生长率,蛋白质效率,饲料系数等都显著高于对照组(P＜0.05)。(2)牛磺酸提高了鲫鱼血清中T3,T4的含量,添加6.0 g/kg时显著高于对照组,且此时血清尿素氮浓度显著低于对照组和其它处理组(P＜0.05)。(3)牛磺酸能影响鲫鱼肠道TAUT, CSAD的相对表达量。在由此可见,适量添加牛磺酸能促进鲫鱼生长、肠道细胞增殖及蛋白消化吸收相关基因的表达。
[Abstract]:Taurine can regulate the growth and development of fish. Taut and CSAD are the key carriers and the main rate-limiting enzymes of taurine transmembrane transport and absorption in animal cells, respectively.In this study, the TAUT and CSAD genes of crucian carp were cloned, and the relative expression of mRNA in different tissues and circadian rhythm of crucian carp was detected. The effect of taurine on the expression of TAUTCSAD in crucian carp growth was also studied.In order to provide a new theoretical basis for explaining the action mechanism of taurine on aquatic animals, the cloning of TAUT and CSAD genes and bioinformatics analysis of crucian carp were carried out using RACE and other techniques in our research.The complete ORF sequence of taut CSAD gene was obtained from the intestine of crucian carp.The results of bioinformatics software showed that the total length of ORF was 1978bp, encoding 625 amino acids, and there were 12 transmembrane regions in the amino acid region. The results of multiple sequence alignment analysis showed that the consistency of TAUT and carp was 997%.Phylogenetic analysis showed that the ORF of carp. CSAD was 1503 BP, encoding 500 amino acids. Homology analysis showed that the identity between CSAD and CSAD was 97%.Expression of TAUT and CSAD genes in different tissues and circadian Rhythm of Carassius Carassius Carassius auratus CSAD and Carp CSAD. In this study, real-time fluorescence quantitative PCR technique was used to investigate the distribution of TAUT and CSAD genes in different tissues of crucian carp.Differences in circadian rhythm expression.The results showed that the relative expression of TAUT mRNA in intestinal tissue was the highest, significantly higher than that in other tissues, and the relative expression in heart and brain was lower. CSAD mRNA was expressed in muscle and heart.There was no significant difference in the relative expression between intestine and liver, but the relative expression in brain and Gill was lower.The relative expression of TAUT mRNA in the circadian rhythm of crucian carp was gradually increased from 0:00 to 12: 00, and then decreased gradually, and the relative expression of 12:00 was the highest and the lowest at 21: 00.The relative expression of CSAD mRNA in the circadian rhythm of crucian carp intestinal tract was similar to that of rising at 0 and then decreasing.The lowest relative expression was 21: 00 at 6:00. The effect of taurine on the expression of TAUT and CSAD genes in crucian carp was studied in 480 (initial body mass 29.07 卤0.19g) Carassius auratus.Each repeat has 20 fish.The dosage of taurine added to the basic feed of crucian carp was 0.0 g / kg ~ (-1) g / kg ~ (-1) g / kg ~ (2.5) g / kg ~ (2.5) g 路kg ~ (-1) 路kg ~ (-1) 路kg ~ (-1) 路g ~ (-1) 路kg ~ (-1), respectively.The results showed that the weight gain rate, specific growth rate, protein efficiency and feed efficiency of the group supplemented with 6. 0 g/kg of taurine were significantly higher than those of the control group (P < 0. 05, P < 0. 05, P < 0. 05, P < 0. 05). The content of T3 + T4 in the serum of Carassius auratus was significantly higher than that of the control group (P < 0. 05, P < 0. 05, P < 0. 05).The concentration of serum urea nitrogen was significantly lower than that of control group and other treatment groups (P < 0.05). Taurine could affect the relative expression of TAUTand CSAD in the intestine of crucian carp.It can be seen that appropriate amount of taurine can promote the growth of crucian carp, intestinal cell proliferation and protein digestion and absorption of related genes.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4;Q78

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