ABCA3转运蛋白在泥蚶镉富集中的表达特征及调控机制

ABCA3 is one of the ATP-binding cassette transporters which transports substrates from inside to outside of the cells. ABCA3 is an active transporter and plays critical roles in maintaining cellular homeostasis. The blood clam, Tegillarca granosa, is able to accumulate high levels of cadmium, and shows variationsin accumulation of Cd among individuals. Previous studies showed that the expression of ABCA3 is negative association with the content of accumulated Cd in T. granosa. Understanding of the expression pattern and regulation mechanisms of Cd accumulation in T. granosa will help us to unravel molecular mechanism for maintenance of cellular homeostasis and adaptation to Cd stress. In this study, we propose to characterize the expression patterns of ABCA3 in different tissues and during the Cd accumualtion of T. granosa, and then to identify the influence to Cd accumulation after theABCA3 mRNA interference, and to explore the transcriptional regulation of c-Myc in expression of ABCA3 gene. This work will reveal the relationship between dynamic alterations of ABCA3 expression and various phenotype of Cd accumulation, to reveal the molecular mechanism of the c-Myc mediating ABCA3 expression during Cd accumulation, and provide basis for studying depuration of heavy metalsand breeding strains of low Cd accumulation in T. granosa.

ABCA3转运蛋白通过主动运输将胞内物质转运至胞外，对维持细胞内稳态具有重要的调节作用。泥蚶对镉具有超富集现象，并且个体间存在非常大的差异，ABCA3基因转录水平与泥蚶个体镉含量呈反向相关。解析ABCA3在泥蚶镉富集中的表达特征和调控机制，有助于深入阐述泥蚶维持内环境平衡，适应镉逆境的分子机制。本项目以我国重要滩涂经济贝类泥蚶为研究对象，采用荧光定量PCR、免疫组化和免疫印迹等方法，分析ABCA3在泥蚶不同组织和镉富集过程中的表达规律；利用RNAi技术，解析ABCA3对泥蚶镉富集的调控作用；采用染色质免疫共沉淀技术，分析ABCA3基因与转录因子c-Myc的相互作用。通过本项目的实施，旨在明确泥蚶ABCA3基因的动态表达规律及其与镉富集差异表型的关系，阐明c-Myc介导的ABCA3表达影响镉富集的分子机制，为泥蚶重金属净化技术的研究和镉低富集品系的选育提供基础资料。

泥蚶是中国的重要的滩涂养殖贝类，泥蚶对镉的超富集现象越来越被人们所关注，转运蛋白ABCA3的表达水平与泥蚶镉富集能力呈反向相关。本项目对Tgc-Myc介导下的TgABCA3基因的表达特征及其对泥蚶镉富集的分子调控作用进行了深入研究。结果发现：(1)qRT-PCR和Westernblot技术检测发现泥蚶TgABCA3基因在鳃、外套膜、足、闭壳肌、内脏团等组织中均有表达，其中，鳃组织的表达量最高。(2)在不同浓度镉离子胁迫下，TgABCA3基因的表达呈上调趋势，并且呈现时间和剂量效应。(3)ABCA3基因敲降后，泥蚶鳃组织中的镉含量增加；泥蚶的耗氧率和排氨率上升，摄食率下降；抗氧化相关基因(MT、CAT)、免疫相关因子TRAF3，凋亡相关基因(JNK，BAX)的表达上调。(4)凝胶迁移电泳(EMSA)实验结果表明，Tgc-Myc核蛋白能够识别TgABCA3基因的启动子序列的CACGTG核心序列（E-box）并与之结合；染色体免疫共沉淀(CHIP)实验进一步验证了ABCA3基因DNA片段和c-Myc结合反应的序列位于ABCA3基因-40bp~-35bp的核心启动子区域。(5) 对Tgc-Myc进行RNAi沉默，结果显示Tgc-Myc在48 h时的表达量显著下调，TgABCA3基因的表达也相应下调50 %。综上，ABCA3基因作为转录因子Tgc-Myc的靶基因，其转录受到Tgc-Myc的正向调控作用；ABCA3基因参与泥蚶体内镉离子的转运，对维持泥蚶内环境的稳定，保证机体生理功能正常运行具有重要作用。研究结果不仅丰富了贝类重金属富集的分子机制的知识，也为泥蚶健康养殖及选育低镉富集品系奠定理论基础。