Cloning, characterization and expression analysis of coagulation factor II gene in grass carp (Ctenopharyngodon idella).
Cloning, characterization and expression analysis of coagulation factor II gene in grass carp (Ctenopharyngodon idella).
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DOI：
10.4238/2015.october.28.39
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发表时间：
2015
期刊：
影响因子：
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通讯作者：
B. Xu;K. Chen;Y. Yao;Q. L. Liu;T. Xiao;J. Su;H. Peng
中科院分区：
文献类型：
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作者：
B. Xu;K. Chen;Y. Yao;Q. L. Liu;T. Xiao;J. Su;H. Peng
本文研究了草鱼凝血因子II （FII）基因的结构和功能，并确定了其在凝血机制中的作用。FII基因EST通过构建的脾转录组数据库获得；3′和5′RACE分别获得FII基因的全长序列。克隆出FII的开放阅读框（ORF），全长为1718 bp， ORF为1572 bp；该基因含有25 bp的5'-未翻译区（UTR）和108 bp的3'-未翻译区。ORF编码524种氨基酸，包括74种碱性氨基酸（精氨酸和赖氨酸）和69种酸性氨基酸（天冬氨酸和谷氨酸）。理论上的圆周率是6.22。计算的不稳定性指数（II）为39.81，表明FII是一种稳定的蛋白；氨基酸序列比较表明，草鱼的FII与红鳍龙鱼的FII最相似（71%），其次是筋膜Oplegnathus fasciatus（48%）和crocea Larimichthys（47%）。实时反转录PCR分析显示，正常情况下，FII在肝脏中表达量最高，其次是鳃、脾脏、胸腺和头肾（P < 0.001）。注射草鱼呼肠孤病毒873 （GCRV873）后，FII的表达模式显著改变（P < 0.001）；注射后基因表达高，提示与血小板和补体系统联合启动凝血系统和机体防御有关的反应。
Here, we characterized the structure and function of the coagulation factor II (FII) gene in grass carp and determined its role in coagulation mechanisms. The FII gene EST was obtained using a constructed splenic transcriptome database; the full-length FII gene sequence was obtained by 3' and 5' RACE. The open reading frame (ORF) of FII was cloned and the full-length gene was found to be 1718 bp, with an ORF of 1572 bp; the gene contained a 25 bp 5'-untranslated region (UTR) and 108 bp 3'-UTR. The ORF encoded 524 amino acids, including 74 alkaline amino acids (arginine and lysine) and 69 acidic amino acids (aspartic acid and glutamic acid). The theoretical pI was 6.22. The calculated instability index (II) was 39.81, indicating that FII was a stable protein; the half-life period was predicted to be approximately 30 h. Amino acid sequence comparisons indicated that grass carp FII showed most similarity (71%) to FII of Takifugu rubripes, followed by Oplegnathus fasciatus (48% similarity) and Larimichthys crocea (47% similarity). A real-time reverse transcription PCR analysis showed that under normal circumstances, FII was most highly expressed in the liver, followed by the gill, spleen, thymus, and head-kidney (P < 0.001). After injection of the grass carp reovirus 873 (GCRV873), the pattern of FII expression was significantly altered (P < 0.001); gene expression was high after injection, suggesting a response involving the initiation of the coagulation system and defense of the body in combination with the platelet and complement system.