青蛤Kazal型丝氨酸蛋白酶抑制剂基因的克隆及其在组织间的表达分析

Molecular Cloning and Expression of SPI Gene in Cyclina sinensis

利用构建的SMART-cDNA文库及高通量测序方法，获得了青蛤Kazal家族丝氨酸蛋白酶抑制剂基因（Serine Protease Inhibitor，SPI）的全长序列，采用荧光定量PCR方法分析了SPI在青蛤各组织的表达情况，并在鳗弧菌胁迫下分析了SPI在青蛤血液中的时序表达关系。结果表明，青蛤SPI基因全长587 bp，CDS为67-520 bp，编码151个氨基酸，具有19个氨基酸的信号肽序列；荧光定量PCR结果显示，该基因在血液、肝脏、外套膜、鳃和闭壳肌等组织中表达差异明显，其中血液中表达水平最高，在闭壳肌中表达水平较低，其他组织表达量极少；在鳗弧菌刺激后3 h和48 h青蛤血液中SPI的表达量出现明显上调的趋势且与对照组有极显著性差异（P〈0.01）,说明SPI基因在青蛤的免疫反应中具有重要的作用。

The Kazal type serine protease inhibitor gene of Cyclina sinensis were acquired by constructing SMART-cDNA library and large scale EST sequencing method. The expression of the serine protease inhibitor gene in different tissues was determined by RT-PCR, additionally, the expression of which were also determined in hemocytes after injection of Vibrio anguillarum. The results showed that the full length of SPI is 587 bp, the CDS covers a region from 67 bp to 520 bp encoding a 151-amino acid peptide including a 19-amino acid signal peptide. Reverse transcription PCR analysis showed that the express of serine protease inhibitor gene in hemocytes, liver, gills, adducter and mantle have obvious difference, and the highest expression level was observed in hemocytes, followed by in adducter. The SPI expression in hemocytes was significantly increased （P〈0.01） at 3 hours and 48 hours after the challenge of Vibrio anguillarum. Thus, SPI may play an important role in the immune response of Cyclina sinensis.