蚤状溞caspase-3基因在不同环境条件下的表达分析
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摘要
以蚤状溞(Daphnia pulex)为研究对象,采用Real-Time PCR技术分析了蚤状溞caspase-3 mRNA表达水平随着不同培养温度、种群密度和食物种类变化;用Caspase-3的特异性底物Ac-DEVD-AMC测定了Caspase-3酶活在不同培养温度、种群密度和食物种类条件下的变化,并通过整体原位杂交检测了caspase-3基因在蚤状溞不同生殖状态下的定位表达。研究结果:蚤状溞caspase-3 mRNA表达量随着温度和种群密度的增加而增加;以Banta培养液培养的蚤状溞caspase-3 mRNA表达量与小球藻培养的表达量无显著差异(P> 0. 05);且caspase-3 mRNA的表达量与Caspase-3酶活的变化规律相一致;整体原位杂交结果显示,caspase-3基因在蚤状溞活体细胞几乎都有表达,在两性溞中表达量较高,孤雌成溞中表达量较低。caspase-3基因的表达与环境有关,而蚤状溞的衰老和caspase-3 mRNA的表达及Caspase-3的酶活有关,但环境因子如何影响caspase-3基因的表达进而调控枝角类生殖转换还需今后进一步的研究。
Using Daphnia pulex as the researching object,q PCR was used to analyze the changes of expression level of caspase-3 mRNA in different culture temperatures,population densities and food types; then the enzyme activities of Caspase-3 under three different culture conditions were measured using the specific substrate Ac-DEVD-AMC; in situ hybridization was used to detect the localizations and changes of caspase-3 mRNA in different reproductive states of D. pulex. The main results were as follows: expression levels of caspase-3 mRNA were up-regulated with the increase of temperature and population density,but there was no obvious changes of caspase-3 mRNA expression in the food type group. In addition,q PCR results were in good agreement with the result of enzyme activity assays. Whole-mount in situ hybridization showed that caspase-3 was expressed in living cells of D. pulex at different growth stages and was highly expressed in adult individuals,which was essentially consistent with the result of q PCR. The expression of caspase-3 gene was relevant to changes of environmental factors,while aging of D. pulex was related to the expression of caspase-3 mRNA and the enzyme activity of Caspase-3. Thus,further research is needed to explore how environmental factors affecting the expression of caspase-3mRNA,which subsequently regulates cyclical parthenogenesis in Daphnia.
Using Daphnia pulex as the researching object,q PCR was used to analyze the changes of expression level of caspase-3 mRNA in different culture temperatures,population densities and food types; then the enzyme activities of Caspase-3 under three different culture conditions were measured using the specific substrate Ac-DEVD-AMC; in situ hybridization was used to detect the localizations and changes of caspase-3 mRNA in different reproductive states of D. pulex. The main results were as follows: expression levels of caspase-3 mRNA were up-regulated with the increase of temperature and population density,but there was no obvious changes of caspase-3 mRNA expression in the food type group. In addition,q PCR results were in good agreement with the result of enzyme activity assays. Whole-mount in situ hybridization showed that caspase-3 was expressed in living cells of D. pulex at different growth stages and was highly expressed in adult individuals,which was essentially consistent with the result of q PCR. The expression of caspase-3 gene was relevant to changes of environmental factors,while aging of D. pulex was related to the expression of caspase-3 mRNA and the enzyme activity of Caspase-3. Thus,further research is needed to explore how environmental factors affecting the expression of caspase-3mRNA,which subsequently regulates cyclical parthenogenesis in Daphnia.
引文
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[22]PENG X,CHEN K,CHEN J,et al. Aflatoxin B1 affects apoptosis and expression of Ba-x,Bcl-2,and caspase-3 in thymus and bursa of fabricius in broiler chickens[J] Environmental Toxicology,2016,31(9):1113-1120.
[23]SHINOURA N,MURAMATSU Y,YOSHIDA Y,et al. Adenovirus-mediated transfer of caspase-3 with Fas ligand induces drastic apoptosis in U-373MG glioma cells.[J]. Experimental Cell Research, 2000,256(2):423-433.
[24]FLANAGAN L,MEYER M,FAY J,et al. Low levels of caspase-3 predict favourable response to 5FU-based chemotherapy in advanced colorectal cancer:caspase-3 inhibition as a therapeutic approach[J]. Cell Death&Disease,2016,7(2):e2087. x
[25]陆开宏.淡水枝角类的培养技术[J].水产养殖,1992(1):2-4.
[26]赵满意,王伟洪,张健兵,等.种群密度和食物密度变化对蒙古裸腹溞产雄的诱导效应[J].浙江海洋学院学报(自然科学版),2014(1):59-64.
[27]王丹丽,徐善良,苏忠川.盐度和食物对蚤状溞(Daphnia pulex)生殖与生存的影响[J].宁波大学学报:理工版,1998(3):18-22.
[28]TONG Q,ZHANG M,CAO X,et al. Expression and activation of Daphnia pulex caspase-3 are involved in regulation of aging[J]. Gene,2017,634:37-46.
[29]ZHIVOTOVSKY B,SAMALI A,GAHM A,et al. Caspases:their intracellular localization and translocation during apoptosis.[J]. Cell Death&Differentiation,1999,6(7):644-651.
[30]张萌萌.蚤状溞衰老相关功能基因caspase-3和Sir2的研究[D].宁波:宁波大学,2017.
[2]CARVALHO G R,HUGHES R N. The effect of food availability,female culture-density and photoperiod on ephippia production in Daphnia magna Straus(Crustacea:Cladocera)[J]. Freshwater Biology,2006,13(1):37-46.
[3]FONTANA L,PARTRIDGE L,LONGO V D. Extending healthy life span-from yeast to humans[J]. Science,2010,328(5976):321.
[4]李超,伏圣博,刘华玲,等.细胞凋亡研究进展[J].世界科技研究与发展,2007,29(3):45-53.
[5]赵瑞杰,李引乾,王会,等. Caspase家族与细胞凋亡的关系[J].中国畜牧杂志,2010,46(17):73-78.
[6]梁先敏,杨克敌. Caspase和JNK/SAPK、p38 MAPK与细胞凋亡[J].环境卫生学杂志,2008,35(1):5-10.
[7]李艳萍,王琳,伍思琪,等.凋亡调节蛋白Bcl-2、p53和caspase-3在乳腺癌组织中的表达及其相互关系[J].解剖科学进展,2010,16(2):167-170.
[8]毛德文,陈月桥,王丽,等. Caspase-8及caspase-3与细胞凋亡[J].辽宁中医药大学学报,2008,10(10):148-150.
[9]李洪波. XIAP及CASPASE-3在年龄相关性听力减退C57BL/6J小鼠初级听皮层中的表达及其意义[D].重庆:第三军医大学,2009.
[10]胡艳丽,李守民,傅希玥,等. caspase-3在D-半乳糖致衰老SD大鼠海马的表达[J].昆明医学院学报,2009,30(6):9-13.
[11]ZHAO M,SU J,HEAD E,et al. Accumulation of caspase cleaved amyloid precursor protein represents an early neurodegenerative event in aging and in Alzheimer's disease.[J]. Neurobiology of Disease,2003,14(3):391.
[12]SU J H,ZHAO M,ANDERSON A J,et al. Activated caspase-3 expression in Alzheimer's and aged control brain:correlation with Alzheimer pathology[J]. Brain Research,2001,898(2):350-357.
[13]杨启莲.大黄鱼HSP及Caspase家族部分基因对温度胁迫的响应[D].厦门:集美大学,2011.
[14]贾旭颖,张丹,路允良,等.温度突变和非离子氨胁迫对淡水养殖凡纳滨对虾cyt-C和caspase-3的影响[J].中国水产科学,2014(4):700-710.
[15]LIVAK K J,SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method.[J]. Methods,2001,25(4):402-408.
[16]曹双俊,金国平,赵新国.蚤状蚤的生长和生殖[J].四川动物,1996(3):100-104.
[17]DU Z,LI S,LIU L,et al. NADPH oxidase 3 associated oxidative stress and caspase 3 dependent apoptosis in the cochleae of Dgalactose induced aged rats. Corrigendum-in/10. 3892/mmr. 2015. 4590[J]. Molecular Medicine Reports,2015,12(6):7883-7890.
[18]LIU J,MAO J,JIANG Y,et al. AGEs induce apoptosis in rat osteoblast cells by activat-ing the caspase-3 signaling pathway under a high-glucose environment in vitro[J]. Applied Biochemistry and Biotechnology,2016,178(5):1015-1027.
[19]NICHOLSON D W,THORNBERRY N A. Caspases:killer proteases.[J]. Trends in Biochemical Sciences,1997,22(8):299-306.
[20]DIRKS A J,LEEUWENBURGH C. Aging and lifelong calorie restriction result in adaptations of skeletal muscle apoptosis repressor,apoptosis-inducing factor,X-linked inhibitor of apoptosis,caspase-3,and caspase-12.[J]. Free Radical Biology&Medicine,2004,36(36):27-39.
[21]ROTSCHAFER S E,ALLENSHARPLEY M R,CRAMER K S. Axonal cleaved caspase-3 regulates axon targeting and morphogenesis in the developing auditory brainstem[J]. Frontiers in Neural Circuits,2016,10:84.
[22]PENG X,CHEN K,CHEN J,et al. Aflatoxin B1 affects apoptosis and expression of Ba-x,Bcl-2,and caspase-3 in thymus and bursa of fabricius in broiler chickens[J] Environmental Toxicology,2016,31(9):1113-1120.
[23]SHINOURA N,MURAMATSU Y,YOSHIDA Y,et al. Adenovirus-mediated transfer of caspase-3 with Fas ligand induces drastic apoptosis in U-373MG glioma cells.[J]. Experimental Cell Research, 2000,256(2):423-433.
[24]FLANAGAN L,MEYER M,FAY J,et al. Low levels of caspase-3 predict favourable response to 5FU-based chemotherapy in advanced colorectal cancer:caspase-3 inhibition as a therapeutic approach[J]. Cell Death&Disease,2016,7(2):e2087. x
[25]陆开宏.淡水枝角类的培养技术[J].水产养殖,1992(1):2-4.
[26]赵满意,王伟洪,张健兵,等.种群密度和食物密度变化对蒙古裸腹溞产雄的诱导效应[J].浙江海洋学院学报(自然科学版),2014(1):59-64.
[27]王丹丽,徐善良,苏忠川.盐度和食物对蚤状溞(Daphnia pulex)生殖与生存的影响[J].宁波大学学报:理工版,1998(3):18-22.
[28]TONG Q,ZHANG M,CAO X,et al. Expression and activation of Daphnia pulex caspase-3 are involved in regulation of aging[J]. Gene,2017,634:37-46.
[29]ZHIVOTOVSKY B,SAMALI A,GAHM A,et al. Caspases:their intracellular localization and translocation during apoptosis.[J]. Cell Death&Differentiation,1999,6(7):644-651.
[30]张萌萌.蚤状溞衰老相关功能基因caspase-3和Sir2的研究[D].宁波:宁波大学,2017.