青蛤抗菌肽基因的克隆及其在组织间的表达分析

Molecular Cloning and Expression of Mytimacin Gene from Cyclina sinensis

利用构建的SMART-cDNA文库及高通量测序方法,获得了青蛤抗菌肽macin家族相关基因(mytimacin)的全长序列,采用荧光定量PCR方法分析了mytimacin在青蛤各组织的表达情况,并在鳗弧菌胁迫下分析了mytima-cin在外套膜中的时序表达关系。结果表明,mytimacin基因全长461bp,开放阅读框为261bp,编码86个氨基酸,具有24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普遍表达,其中外套膜表达水平最高,在鳃中表达最低;在鳗弧菌刺激后6～24h,青蛤外套膜中mytimacin的表达量出现明显上调的趋势且与对照组差异显著(P<0.05),说明mytimacin抗菌肽基因在青蛤的免疫反应中具有重要作用。

The macin family of antibacterial peptide gene （mytimacin） of Cyclina sinensis were cloned with SMART-cDNA library and large scale EST sequencing method, and based on RT-PCR, we described the constitutive expression in different tissues of the mytimacin gene and analyzed the expression level of mytimacin gene in mantle after injection of Vibrio anguil- larum. Result showed that the full length of mytimacin was 461 bp, with a 261 bp open reading frame encoding a 86-amino acid peptide and the deduced peptide contained a 24-amino acid signal peptide. Reverse transeriptase PCR analysis showed that mytiraacin transcripts were constitutively expressed in hemoeytes, liver, gill, adducter and mantle, with the highest level in mantle and the lowest level in gill. Mytimaein mRNA levels were found increasing within 6 - 24 h after challenge with Vibrio anguillarum, and there was significant difference with the control group （ P 〈 0.05 ）. Above indicated that mytimaein play a fundamental role in the immune response of Cyclina sinensis.