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Cloning and Analysis of Disease-resistance Genes from Large Yellow Croaker

Author: XieFangJing
Tutor: WangYiLei
School: Jimei University
Course: Aquaculture
Keywords: Large yellow croaker RT-PCR Quantitative PCR ACP Function of disease resistance gene
CLC: S917.4
Type: Master's thesis
Year: 2007
Downloads: 209
Quote: 2
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Abstract


Vibrio parahaemolyticus, intraperitoneal injection of large yellow croaker (experimental group) or 0.9% sodium chloride solution (control group), the injection 4h, 1d, 2d, 4d, 8d, 12d and 16d after tail blood serum was determined nitric oxide synthase, lysozyme and tyrosinase activity changes. The results show that injection of Vibrio parahaemolyticus after large yellow croaker serum lysozyme activity in the 2d and 12d significantly higher than that of the control group was significantly lower than the control group in the 16d; tyrosinase activity was significantly higher in the 4d and 16d, significantly higher than the control group; extremely 8d but no significant increase of nitric oxide synthase activity in vivo in large yellow croaker. That large yellow croaker serum lysozyme and tyrosinase involved in the body's immune defense of Vibrio parahaemolyticus. This study on the basis of the above results, the use of the RT-PCR technique to clone large yellow croaker TNFα, Mx, IgL and IgH gene, and quantitative PCR analysis of the expression of these four genes. The results are as follows: (1 the) TNFαcDNA sequence of full-length 1339 bp 5'UTR 68 bp ORF of 759 bp 3'UTR is 512 bp, encoding 252 amino acids, the molecular weight of about 28kDa. The polypeptide derived from the sequence containing the tag sequence of the transmembrane domain of the TNF family. Homology analysis showed that the sequence of other bony fish or mammals TNFα with high similarity. Evolutionary analysis shows that the large yellow croaker TNFα and the other teleost TNFα occupy a separate branch of the evolutionary mammalian TNFα formation of another branch. Quantitative PCR analysis found that the 2d and 4d Vibrio group of large yellow croaker TNFα gene expression in the head kidney very significantly higher than the control; 2d vibrio group of large yellow croaker TNFα gene expression in the blood was significantly higher than that in the control group, while the 4d, the Vibrio group of large yellow croaker TNFα gene expression very significantly higher than that of the control group. (2) obtained 2209bp MX full-length cDNA contains a 1890bp ORF of encoding 629 amino acids, molecular weight of about 72 kDa. May be encoded in the sequence, has a characteristic structure of the Mx protein, such as GTP enzyme motifs, the GTPase dynamic protein superfamily label sequence (LPRGSGIVTR) and the C-terminal region of the leucine zipper structure. Large yellow croaker by intraperitoneal injection of Vibrio parahaemolyticus 2d, head kidney and blood Mx gene expression was significantly up-regulated and peak in 2d. (3) cloned into the the a 977bp IgL full - length cDNA sequence and a 1621bp IgH cDNA fragment. IgL coding region composed by the two domains of the variable region and a constant region; changes in the complementarity determining region is large, low similarity with other fish. Phylogenetic analysis indicated that the large yellow croaker fish IgL independent class clustered into one. Quantitative PCR showed that injection of Vibrio parahaemolyticus 8d and 12d, the rhubarb head kidney IgL and IgH gene expression were significantly higher than that in the control group. The annealing control primer (ACP) technology is a new method developed on the basis of the DDRT-PCR for cloning of differentially expressed genes. Annealing control primer 3 'side of the core portion and the 5' end of the universal primer sequence portion regulating portion from the middle of the poly deoxyinosine [Poly (DI)] connections, can significantly increase the specificity of primer annealing. The method has high repeatability, low false positive and wide distribution of rapid economic and PCR products. Improvement of this method, a large yellow croaker disease resistance 1360bp fibrinogen beta chain gene fragment was cloned into.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Aquatic basic science > Aquatic Biology > Aquatic Zoology
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