三唑磷作用下翡翠贻贝抑制性消减杂交文库构建与分析

摘要

采用抑制性差减杂交技术,构建了三唑磷农药急性胁迫下翡翠贻贝雌贝性腺正反向消减杂交 cDNA 文库。正反向文库共有117个表达序列标签(expressed sequence tags, ESTs)与NCBI 数据库中已知功能蛋白具较高同源性,假定蛋白12种,另有195个ESTs未检索到同源序列。正向文库中含有细胞周期蛋白、性激素合成蛋白、DNA 修复蛋白、能量代谢相关蛋白等一系列与生殖、应激适应和代谢相关的转录本信息,而反向文库中的差异ESTs主要涉及DNA复制、转录和翻译调控基因以及骨架蛋白。结果表明三唑磷对翡翠贻贝的毒性涉及到生殖调控、能量代谢、应激适应、转录翻译调控等生理代谢的多个方面,正向文库中发现的几种生殖调控基因(细胞周期蛋白、周期蛋白依赖性蛋白激酶、合子DNA复制许可因子、精子特异性蛋白、雌激素硫酸转移酶和卵黄膜卵透明带域蛋白)可能是三唑磷生殖干扰毒性作用过程中的关键基因,这为有机磷农药的生殖毒性探索提供了基础数据。%To study the reproductive toxicity and molecular mechanism of triazophos to marine shellfishes, a suppressive subtractive hybridization (SSH) cDNA library of gonads from female Perna viridis by actue triazophos (1mg/L) stress was constructed for the first time. A total of 117expressed sequence tags (ESTs) from the forward and reverse subtraction library exhibited homology with genes of know functions, 12ESTs related to hypothetical protein, and the remaining 195 ESTs had no significant matches in the GenBank database. Among the 117 annotated sequences,the most abundant upregulated transcripts identified in the forward SSH libraries were associated with cell cycle regulation, sex steroid biosynthesis, DNA repairing and respiratory processes, while genes encoding proteins of DNA replication, transcription and translation regulation, cytoskeleton were found in reverse SSH libraries. Altogether, the results showed that different pathways (reproduction control, respiratory processes and stress adaptation) of mussel metabolism were changed in both forward and reverse directions after exposed to triazophos. Several reproduction-related transcripts (cyclin, cyclin-dependent kinase, zygotic DNA replication licensing factor, sperm-specific protein, estrogen sulfotransferase and vitelline envelope zona pellucida domain) identified in forward library might be the key genes involved in reproductive disturbance of triazophos, and provided the basis for the study of reproductive toxicity of organophosphorus pesticide.