背角无齿蚌钙调蛋白基因的克隆及Ca2+和Cd2+对其表达的影响
Characterization of AwCaM1 from Freshwater Clam Anodonta woodiana and Effect of Ca2+ and Cd2+ on Its Expressions
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摘要: 钙调蛋白（CaM）是Ca2+结合蛋白，参与程序性细胞死亡、自噬、肌肉收缩、炎症和免疫应答等多种生物学过程。为探讨Ca2+和Cd2+对背角无齿蚌（Anodonta woodiana）的生理效应，克隆出AwCaM1全基因序列，分析Ca2+和Cd2+对AwCaM1表达的影响。结果显示，背角无齿蚌AwCAM1 cDNA全长由692个碱基组成，包含一个516 bp开放阅读框编码的172个氨基酸的多肽链，AwCaM1氨基酸序列包含有4个Ca2+结合EF-手形结构域。Ca2+处理后，肝胰脏中AwCaM1表达水平在0.01、0.02、0.04和0.08 mg·L-1处理组呈时间和剂量依赖性方式上调；Cd2+处理后，肝胰脏中AwCaM1表达水平在8 mg·L-1和16 mg·L-1处理组增加了65.04%（P<0.01）以上。与对照组相比，Ca2+处理后鳃中AwCaM1表达水平增加了79.41%（P<0.01）以上，Cd2+处理后AwCaM1表达水平增加了88.23%（P<0.01）以上。与对照组相比，外套膜中AwCaM1表达水平在0.16 mg·L-1的Ca2+处理组增加了1.69倍以上（P<0.01），AwCaM1表达水平在8 mg·L-1和16 mg·L-1的Cd2+处理组增加了1.65倍（P<0.01）以上。以上结果表明，Ca2+和Cd2+处理对背角无齿蚌AwCaM1表达水平具有显著的诱导作用，其原因与钙的吸收和胁迫效应有关。Abstract: Calmodulin (CaM), a Ca2+-binding protein, is involved in programmed cell death, autophagy, muscle contraction, inflammation and the immune response. In order to explore effect of Ca2+ and Cd2+ on the CaM, one complete cDNA sequence (AwCaM1) was cloned and characterized from the freshwater mussel Anodonta woodiana and its expressions were analyzed. The complete cDNA sequence of AwCaM1 was comprised of 692 bp contained a 516 bp open reading frame which was encoded 172 amino acids. AwCaM1 contained four putative Ca2+-binding EF-hand motifs. The AwCaM1 was cloned from the A. woodiana using the rapid amplification of cDNA ends methods and its expression was determined by real-time PCR. In the hepatopancreas, AwCaM1 expression was up-regulated with a time and dose dependent pattern in the Ca2+ treated groups (0.01, 0.02, 0.04 and 0.08 mg·L-1). AwCaM1 mRNA level increased more 65.04% (P<0.01) in the Cd2+ treated groups (8 mg·L-1 and 16 mg·L-1). In the gill, AwCaM1 expression increased more than 79.41% (P<0.01) compared with that of control group in all the Ca2+ treated groups, and more than 88.23% (P<0.01) in all the Cd2+ treated groups. In the mantle, AwCaM1 mRNA levels increased more than 1.69 times (P<0.01) in the Ca2+ treated group (0.16 mg·L-1) in contrasted with that of control group, 1.65 times (P<0.01) in Cd2+ treated groups (8 mg·L-1 and 16 mg·L-1). These results indicated that up-regulations of AwCaM1 expression in bivalve A. woodiana are associated with Ca2+ absorption and environmental adaption derived from Ca2+ and Cd2+ treatment.
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Key words:
- Cd2+ /
- Anodonta woodiana /
- cytotoxicity /
- AwCaM1 /
- Ca2+
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