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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
王志红, 王超, 彭锐*
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DOI:10.11984/j.issn.1000-7083.20180133
作者单位: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
中文关键字:日本三角涡虫;Caspase-7基因;克隆;组织重塑
英文关键字:Dugesia japonica; Caspase-7 gene; cloning; tissue remodeling
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-3PCNA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.
Baguñá J, Romero R. 1981. Quantitative analysis of cell types during growth, degrowth and regeneration in the planarians Dugesia mediterranea and Dugesia tigrina[J]. Hydrobiologia, 84(1): 181-194.
Chai J, Wu Q, Shiozaki E, et al. 2001. Crystal structure of a procaspase-7 zymogen: mechanisms of activation and substrate binding[J]. Cell, 107(3): 399-407.
Chaudhary S, Madhukrishna B, Adhya AK, et al. 2016. Overexpression of caspase 7 is ERα dependent to affect proliferation and cell growth in breast cancer cells by targeting p21Cip[J]. Oncogenesis, 5(4): e219. DOI: 10.1038/oncsis.2016.12.
DeHaan RL, Ebert JD. 2015. Fundamentals of planarian regeneration[J]. Annual Review of Cell and Developmental Biology, 20(1): 725-757.
Hill ME, Macpherson DJ, Wu P, et al. 2016. Reprogramming Caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition[J]. ACS Chemical Biology, 11(6): 1603-1612.
Hwang JS, Kobayashi C, Agata K, et al. 2004. Detection of apoptosis during planarian regeneration by the expression of apoptosis-related genes and TUNEL assay[J]. Gene, 333(3): 15-25.
Jang M, Park BC, Lee AY, et al. 2007. Caspase-7 mediated cleavage of proteasome subunits during apoptosis[J]. Biochemical and Biophysical Research Communications, 363(2): 388-394.
Kuranaga E. 2012. Beyond apoptosis: caspase regulatory mechanisms and functions in vivo[J]. Genes to Cells, 17(2): 83-97.
Liu PF, Hu YC, Kang BH, et al. 2017. Expression levels of cleaved caspase-3 and caspase-3 in tumorigenesis and prognosis of oral tongue squamous cell carcinoma[J]. PLoS ONE, 12(7): e0180620. DOI: org/10.1371/journal.pone.0180620.
Mangel M, Bonsall MB, Aboobaker A. 2016. Feedback control in planarian stem cell systems[J]. BMC Systems Biology, 10(1): 1-18.
Martinon F, Tschopp J. 2007. Inflammatory caspases and inflammasomes: master switches of inflammation[J]. Cell Death & Differentiation, 14(1): 10-22.
Mead RW, Christman J. 1998. Proportion regulation in the planarian, Dugesia tigrina following regeneration of structures[J]. Hydrobiologia, 383(1-3): 105-109.
Park JK, Doseff AI, Schmittgen TD. 2018. MicroRNAs targeting Caspase-3 and -7 in PANC-1 cells[J]. International Journal of Molecular Sciences, 19(4): 1206.
Pellettieri J, Fitzgerald P, Watanabe S, et al. 2010. Cell death and tissue remodeling in planarian regeneration[J]. Developmental Biology, 338(1): 76-85.
Putt KS, Chen GW, Pearson JM, et al. 2006. Small-molecule activation of procaspase-3 to caspase-3 as a personalized anticancer strategy[J]. Nature Chemical Biology, 2(10): 543-550.
Rink JC. 2013. Stem cell systems and regeneration in planarian[J]. Development Genes & Evolution, 223(1-2): 67-84.
Rouhana L, Weiss JA, Forsthoefel DJ, et al. 2013. RNA interference by feeding in vitro synthesized double-stranded RNA to planarians: methodology and dynamics[J]. Developmental Dynamics, 242(6): 718-730.
Tan TCJ, Rahman R, Jaber-Hijazi F, et al. 2012. Telomere maintenance and telomerase activity are differentially regulated in asexual and sexual worms[J]. Proceedings of the National Academy of Sciences of the United States of America, 109(11): 4209-4214.
Thomsen ND, Koerber JT, Wells JA. 2013. Structural snapshots reveal distinct mechanisms of procaspase-3 and -7 activation[J]. Proceedings of the National Academy of Sciences of the United States of America, 110(21): 8477-8482.
Wagner DE, Wang IE,Reddien PW. 2011. Clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regeneration[J]. Science, 332(6031): 811-816.
Weissman IL. 2000. Stem cells: units of development, units of regeneration, and units in evolution[J]. Cell, 100(1): 157-168.
Wolan DW, Zorn JA, Gray DC, et al. 2009. Small molecule activators of a proenzyme[J]. Science, 326(5954): 853-858.
Lei B, Zhou XM, Lv DJ, et al. 2017. Apoptotic and nonapoptotic function of caspase 7 in spermatogenesis[J]. Asian Journal of Andrology, 19(1): 47-51.
Yosefzon Y, Soteriou D, Feldman A, et al. 2018. Caspase-3 regulates YAP-dependent cell proliferation and organ size[J]. Molecular Cell, 70(4): 573-587.
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