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钝缀锦蛤TXNDC5基因克隆及溶藻弧菌胁迫下表达规律研究
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Abstract:
为探究钝缀锦蛤(Tapes dorsatus) TXNDC5基因特征，本实验采用梯度PCR技术扩增获得TdTXNDC5基因的开放阅读框序列，其长度为1236 bp，可编码411个氨基酸残基。预测得知，钝缀锦蛤TXNDC5蛋白等电点为5.05，理论分子量为46.40 kD，分子中具有3个典型的PDI结构域，N末端具有1段长度为20 aa的信号肽。同源性分析结果显示，钝缀锦蛤TXNDC5蛋白与其它3种海水贝类的同源性为37.9%~70.8%，表明TXNDC5蛋白在不同种类的海水贝类物种中具有一定的保守性。基于TXNDC5蛋白的氨基酸序列构建系统发育树，结果显示钝缀锦蛤仅与硬壳蛤聚为一支。实时荧光定量PCR结果显示，TdTXNDC5基因在钝缀锦蛤的8种组织中均有表达，鳃的表达量最高，外套膜次之。溶藻弧菌胁迫下，TdTXNDC5基因在钝缀锦蛤鳃和内脏团组织中的表达量均在第3 h时达到峰值，外套膜在第6 h达到峰值。上述结果表明，TdTXNDC5基因可能参与了钝缀锦蛤应对细菌感染的过程，在钝缀锦蛤先天免疫防御中具有重要作用。
In order to explore the characteristics of TXNDC5 gene in Tapes dorsatus, gradient PCR was used to amplify the open reading frame sequence of TdTXNDC5 gene which was 1236 bp in length and could encode 411 amino acid residues. The isoelectric Point (pI) of TdTXNDC5 protein was 5.05 and the theoretical molecular weight was 46.40 kD. There were three typical PDI domains in TdTXNDC5 and a signal peptide composed of 20 amino acid residues at the N-terminal. The results of homology analysis showed that TXNDC5 protein of T. dorsatus had 37.9%~70.8% homology with the other three marine mollusks, indicating that TXNDC5 protein was conserved in different marine shellfish species. The phylogenetic tree was constructed based on the amino acid sequence of TXNDC5 protein, and the results showed that T. dorsatus was only clustered with Mercenaria mercenaria. Quantitative Real-time PCR results showed that TdTXNDC5 gene was expressed in all the 8 tissues of T. dorsatus, and the highest expression level was found in gill, followed by mantle. The expression of TdTXNDC5 gene in gill and visceral mass of T. dorsatus reached the peak at 3 h and in mantle at 6 h under the stress of Vibrio alginolyticus. These results suggest that TdTXNDC5 gene may be involved in the response to bacterial infection and play an important role in innate immune defense.
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