日本三角涡虫 Caspase-7基因的克隆及功能研究
Cloning and Functional Analysis of Caspase-7 Gene from Dugesia japonica

中文摘要：本文以日本三角涡虫 Dugesia japonica为研究对象，通过cDNA末端快速扩增技术首次克隆得到了涡虫 Caspase-7基因，全长935 bp，编码251个氨基酸，5'-UTR和3'-UTR长度分别为86 bp和93 bp。涡虫Caspase-7具有Caspase家族保守而关键的LSHG与QAC（Q/R）G两大结构域。此外，基于SWISS-MODEL同源建模对涡虫Caspase-7蛋白三维结构的预测和分析，发现其包含2个催化单位，且潜在催化位点由4个表面环构成。进化分析也显示，涡虫 Caspase-7基因未与扁形动物门Platyhelminthes物种聚为一支，而与刺胞动物门Cnidaria水螅 Hydra vulgaris有较近的亲缘关系，说明 Caspase-7基因可能发生了趋同进化。本文通过实时荧光定量PCR检测了 Caspase-7基因在涡虫再生不同时间段的表达变化，发现在涡虫切后再生6 h和3 d， Caspase-7基因表达量升高，这2个时间点是涡虫切后凋亡发生的2个高峰期。此外，通过喂食dsRNA干扰涡虫体内 Caspase-7基因的表达，结果发现，干扰 Caspase-7基因并不影响涡虫的再生，但再生完成后或未切割的成体涡虫在饥饿环境下，受 Caspase-7基因干扰，虫体逐渐溶解直至死亡，而 Djclg-3及 PCNA基因的表达量也显著降低，说明干扰 Caspase-7基因表达可能引起涡虫细胞凋亡与增殖减少，涡虫组织重塑失衡，导致虫体溶解并死亡。该结果揭示 Caspase-7基因可能在涡虫组织重塑中起关键作用。
英文摘要：In this study, the full length of the Caspase-7 gene of Dugesia japonica was first cloned using the rapid amplification of cDNA ends technique. The Caspase-7 gene of D. japonica is 935 bp in length, encoding 251 amino acids, and containing 86 bp and 93 bp of the 5'-UTR and 3'-UTR regions, respectively. It also contains 2 key conserved domains of Caspase family, LSHG and QAC(Q/R)G. The three-dimensional structure of Caspase-7 protein was predicted using SWISS-MODEL workspace. The result showed that Caspase-7 protein contains 2 catalytic units and the potential catalytic sites consist of 4 surface rings. The result of phylogenetic analysis reveals that planarian Caspase-7 gene has closer relationship with that of Hydra vulgaris than other species of Platyhelminthes. Quantitative real-time PCR technology was used to evaluate the expression of Caspase-7 gene at different times of planarian regeneration, and the result suggested that Caspase-7 gene might be involved in the regulation of apoptosis in planarian. In addition, knocking down the in vivo expression of Caspase-7 gene by feeding dsRNA interference in the planarian did not influence the regeneration of planarian, but led to a significantly decreased expression of Djclg-3 and PCNA gene, indicating a significantly reduced apoptosis and proliferation. Finally, the regenerative or untreated planarians were dissolving under the starvation condition, and this should be due to the down-regulated expression of Caspase-7, thereby implied that Caspase-7 gene plays a key role in planarian tissue remodeling. 2019,38(1): 11-19 收稿日期：2018-04-26 分类号：Q959.151+.3;Q78 作者简介：王志红(1992—),硕士,主要从事基因功能研究,E-mail:zhihong1992@foxmail.com *通信作者：彭锐,E-mail:pengrui@scu.edu.cn 参考文献： 郭素英, 舒瑞阳, 彭建新, 等. 2009. 昆虫中分离与鉴定的caspase及其作用[J]. 应用昆虫学报, 46(5): 678-683. Bagu？á J, Romero R. 1981. Quantitative analysis of cell types during growth, degrowth and