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Cloning and Expression Analysis of Scavenger Receptor Class B Type Ⅰ and Antifreeze Proteinstype Ⅱ Genes in Lutjanus Sanguineus

Author: ZhangZuo
Tutor: JianJiChang
School: Guangdong Ocean University
Course: Marine biology
Keywords: Lutjanus sanguineus SR-BⅠ AFPⅡ RACE Prokaryotic Expression Real-time PCR
CLC: S917.4
Type: Master's thesis
Year: 2011
Downloads: 9
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Abstract


The Scavenger Receptor Class B TypeⅠ(SR-B I) and Antifreeze Proteins TypeⅡ(AFP II) genes of Lutjanus sanguineus were cloned using degenerate primers designed from the conserved domains of related genes presenting in NCBI. Sequence analysis showed that the open reading frame of SR-BⅠcDNA is 966 bp, putatively coding 233 amino acid residues with a theoretical molecuLar weight of 36.735 kDa and an estimated isoelectric point of 8.75. The open reading frame of AFPⅡcDNA is 609bp, putatively coding 203 amino acid residues with a theoretical molecuLar weight of 22.45 kDa and an estimated isoelectric point of 5.52.Based on the full-length of AFPⅡ, A recombinant of prokaryotic expression vector pET-AFPⅡwas constructed and transformed to E.coli. BL21 .The optimum expression condition was 0.7mmol/L IPTG, 37℃3 hours .After injection of Vibrio alginolyticus, it was found the expression of SR-BⅠsignificantly increased in liver, head kidney, spleen, intestine, heart and thymus of Lutjanus sanguineus by Real-time PCR. The highest expression of SR-BⅠwas found in intestine, following in liver, head kidney, spleen, heart and the lowest in thymus.The expression patterns of AFPⅡunder the challenge of low temperature were examined by Real-time PCR assays. Three healthy Lutjanus sanguineus were kept in the 17℃water 30min for the experimental group and three healthy Lutjanus sanguineus were kept in the temperature of 25℃as the control group, Total RNA of the test group and control group individuals were extracted from head kidney, thymus, liver, spleen, gill and intestine respectively. Real-time PCR showed that the expression of AFP II in intestine and liver of test group are higher than that of the control group, while the gene expression in head kidney, thymus, spleen and gill in the control group are higher than that of the test group. The study supplied theoretic base for the prevention of Lutjanus sanguineus lipid metabolism diseases,Furthermore, the paper established the foundation for the study on the antifreeze in Transgenic fish.

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