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Full-length Cloning and Expression Analysis of Blood Coagulation Factor Ⅱ Gene cDNA in Ctenopharyngodon Idellus

Author: YaoYiBin
Tutor: XiaoDiaoYi
School: Hunan Agricultural University
Course: Fishery resources
Keywords: Ctenopharyngodon idellus Blood Coagulation Factor Ⅱ cDNA end rapidamplification (RACE) Fluorescence quantitative
CLC: S965.112
Type: Master's thesis
Year: 2013
Downloads: 4
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Abstract


Grass Carp (Ctenopharyngodon idellus) commonly known as Wan, White Wan, Deep black carp, etc. Carp shape orders cyprinidae jarraud fish species of Grass Carp, mostly plain region of rivers and lakes, as a typical vegetarian fish. Because of its delicious, high nutritional value, the advantages of wide and rapid growth, feed sources, production become China’s largest freshwater economic fishes. But with Grass Carp, artificial breeding germplasm resources recession environmental degradation and other reasons, for various disease of Grass Carp in the very great degree restricted the expanding its aquaculture industry scale and density, of which im call arc virus (Grass Carp Reovirus, GCRV) of Grass Carp caused by hemorrhagic disease such as disease had the greatest influence, it season long, transmission is fast, the risk is high, to Grass Carp aquaculture caused huge economic losses. Studies have reported that in the vertebrate in the blood, there are thirteen kinds of clotting factor (factor Ⅰ-ⅩⅢ), when the bacteria or virus infection of the body cause tissue damage is activated. Thirteen kinds of clotting factors in enzymatic cascade, the soluble protein hydrolysis is not soluble protein, and from liquid state into the blood flow of the gel state, finally, reach the role of hemostatic.Given the clotting factors in the animal body tissue Coagulation, anticoagulation and fibrinolysis plays a very important role in the process of this study in Grass Carp spleen transcriptome data found in the Grass Carp ests Ⅱ gene clotting factors (UNIGENE39671), using cDNA end Rapid Amplification (Rapid Amplification of cDNA Ends, RACE) technology, Amplification Grass Carp clotting factors Ⅱ (Blood Coagulation Factor Ⅱ)3’end and5’end sequences, and length of the Grass Carp Blood Coagulation Factor Ⅱ gene sequence. Grass Carp by RT-PCR technique, Grass Carp Open Reading Frame(ORF) of clotting factor Ⅱ gene amplification, and transduction into e. coli DH5alpha cells. By microbial preli minary identification, detection, sequencing, the PCR after validation into pET32a (+)-Ther expression plasmid. At37℃, with a tendency for8h IPTG induction, when the OD600=0.8using SDS-PAGE electrophoresis detection. Results show that the Grass Carp clotting factor Ⅱ gene sequence is1718bp, among them, the5’end sequences of the translation section25bp and3’end sequences of the translation section108bp [not including poly (A)], PCR amplification of Open Reading Frame(ORF) size of1572bp, encoding524amino acids. By protein sequence homology comparison results show that Grass Carp clotting factor Ⅱ protein’s primary structure are similar to those of most species of prothrombin. Grass Carp Ⅱ protein coagulation factors and Danio rerio prothrombin precursors of homology, the highest is98%; Followed by Oncorhynchus mykiss prothrombin, homology was68%; With Larimichthys crocea, Oreochromis niloticus, Oplegnathus fasciatus, Takifugu rubripes clotting factors Ⅱ are65%,64%,64%,65%respectively; With Taeniopygia guttata prothrombin homology is the lowest,60%. Due to primary structure defines the structure of the senior, in turn, affects the function of the protein, which can be concluded that:Grass Carp clotting factor Ⅱ protein has the basic properties of prothrombin, belongs to the clotting proteins.At the same time, the use of fluorescent quantitative PCR technology, the artificial injection of0.65%of the fish with physiological saline and Grass Carp hemorrhagic disease of Grass Carp virus of liver, spleen, thymus, head kidney and the gill tissues of Grass Carp clotting factor Ⅱ gene mRNA expression level detection. The result shows:the clotting factor Ⅱ gene in Grass Carp body gill, spleen, head kidney, liver and thymus tissues were expressed, and the expression difference significant (P<0.05). In gill, head kidney, liver and thymus tissues, the expression of clotting factor Ⅱ is raised; Only in the spleen tissue, in the form of expression. Compared to the same point in time change regulation of the expression of different organizations, in the Grass Carp in the body, the expression of clotting factor Ⅱ quantity significant difference (P<0.05). After tapping poison, assumes the rise and fall and rise again, to reduce the line rule. In the lower expression of spleen tissue,0h expression quantity is relatively high, then decreased expression within48h,48h to express low,72h about restoring to the original level after the falling again, to138h reached the highest level. In the first rise in kidney and liver tissue expression, have similar laws. Namely0h express quantity is relatively high, and the decreased expression within48h,72h after reaching the highest level, when once again fall, then reaches the lowest level. In thymus and rise in gill tissue expression, with the head kidney and liver tissue expression of law is very different, as shown in figure3.4and figure3.5. Namely0h express quantity is relatively high, in the expression amount within24h to72h has risen sharply, express the amount in a long time at a higher level, then down, then reaches the lowest level. Therefore, clotting factor Ⅱ receptor for Grass Carp hemorrhage virus is more sensitive, vulnerable to the virus induced activation. This study using fluorescence quantitative study Grass Carp clotting factor Ⅱ gene expression in Grass Carp different organizations, aims to provide theoretical basis for the study of the disease-resistant breeding of Grass Carp and the scientific research materials, and lays the foundation for further study on the Grass Carp of hemorrhagic disease resistance.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Aquaculture technology > A variety of fish farming > Freshwater fish > Grass carp ( grass carp )
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