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

作　　者：王志红 王超 彭锐[1] WANG Zhihong;WANG Chao;PENG Rui(Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education,College of Life Sciences,Sichuan University,Chengdu 610065,China)

机构地区：[1]四川大学生命科学学院生物资源与生态环境教育部重点实验室,成都610065

出　　处：《四川动物》2019年第1期11-19,共9页Sichuan Journal of Zoology

摘　　要：本文以日本三角涡虫Dugesia japonica为研究对象,通过c DNA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ds RNA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。In this study,the full length of the Caspase-7 gene of Dugesia japonica was first cloned using the rapid amplification of c DNA 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 ds RNA 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.

关 键 词：日本三角涡虫 Caspase-7基因 克隆 组织重塑 

分 类 号：Q959.151.3[生物学—动物学] Q78