贵州草海鲫鱼Spo11基因的克隆、生物信息学和表达分析
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摘要
【目的】探索环境内分泌干扰物壬基酚(Nonylphenol,NP)对贵州草海鲫鱼(Carassius auratus)Spo11基因表达的影响。【方法】通过PCR方法克隆实验鱼Spo11基因,利用生物信息学方法分析SPO11蛋白的氨基酸序列、理化性质和结构特点;并设置1个对照组和低、中、高共3个不同剂量NP处理组,用NP质量浓度分别为0,25,50和100μg·L~(-1)的水体浸浴实验鱼28d后,检测各组实验鱼Spo11基因的表达情况。【结果】Spo11基因cDNA序列长度为1 471bp(GenBank登录号:MF66470),包括66bp的5′非翻译区、编码383个氨基酸的1 152bp的开放阅读框和253bp的3′非翻译区。预测SPO11蛋白质相对分子质量为43.23kDa,等电点IP为6.89,主要由亮氨酸、丝氨酸和缬氨酸组成。该蛋白是含有信号肽的不稳性亲水蛋白,二级结构以α-螺旋和无规则卷曲为主。与对照组实验鱼的Spo11基因相对表达量相比,中剂量和高剂量的NP处理组实验鱼Spo11基因相对表达量均有所升高,与前者的差异均具有统计学意义(p<0.05)。【结论】环境内分泌干扰物NP可以影响贵州草海鲫鱼Spo11基因的表达。
[Purposes]To explore the effect of environmental hormone Nonylphenol(NP)on the Spo11 gene expression of the crucian carp(Carassius auratus)in Caohai lake of Guizhou Province.[Methods]The cDNA of Spo11 gene was cloned by PCR in crucian carp.And its physico-chemical properties and structure characteristics were analyzed by bioinformatics.Spo11 gene expression were detected after the fish were treated with NP mass concentrations of 0,25,50,and 100μg·L~(-1) for 28 days.[Findings]It was shown that the sequence of Spo11 gene was 1 471 bp(GenBank ID:MF66470)in length,containing 66 bp 5′UTR,1 152 bp ORF,and 253 bp 3′UTR.The predicted molecular weight was 43.23 kDa,and the value of PI was 6.89.Proteins are mainly made up of leucine,serine and valine.And the protein is an unstable hydrophilic protein containing signal peptide.Its secondary structures were shown to be mainlyα-helix and random coil.Compared with the control group,the relative expression of Spo11 gene were significantly higher in medium and high concentration groups after NP treatment(p<0.05).[Conclusions]It is shown that the environmental endocrine disruptor nonylphenol can affect the Spo11 gene expression of crucian carp in Caohai lake of Guizhou Province.
[Purposes]To explore the effect of environmental hormone Nonylphenol(NP)on the Spo11 gene expression of the crucian carp(Carassius auratus)in Caohai lake of Guizhou Province.[Methods]The cDNA of Spo11 gene was cloned by PCR in crucian carp.And its physico-chemical properties and structure characteristics were analyzed by bioinformatics.Spo11 gene expression were detected after the fish were treated with NP mass concentrations of 0,25,50,and 100μg·L~(-1) for 28 days.[Findings]It was shown that the sequence of Spo11 gene was 1 471 bp(GenBank ID:MF66470)in length,containing 66 bp 5′UTR,1 152 bp ORF,and 253 bp 3′UTR.The predicted molecular weight was 43.23 kDa,and the value of PI was 6.89.Proteins are mainly made up of leucine,serine and valine.And the protein is an unstable hydrophilic protein containing signal peptide.Its secondary structures were shown to be mainlyα-helix and random coil.Compared with the control group,the relative expression of Spo11 gene were significantly higher in medium and high concentration groups after NP treatment(p<0.05).[Conclusions]It is shown that the environmental endocrine disruptor nonylphenol can affect the Spo11 gene expression of crucian carp in Caohai lake of Guizhou Province.
引文
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[20]YEH H Y,LIN S W,WU Y C,et al.Functional characterization of the meiosis-specific DNA double-strand break inducing factor SPO-11fromC.elegans[J].Scientific Reports,2017,7(1):2370.
[21]SHARMA M,CHADHA P.4-Nonylphenol induced DNA damage and repair in fish,Channa punctatus after subchronic exposure[J].Drug and Chemical Toxicology,2016,40(3):320-325.
[22]ZHAO Y,WANG C,XIA S,et al.Biosensor medaka for monitoring intersex caused by estrogenic chemicals[J].Environmental Science and Technology,2014,48(4):2413-2420.
[23]MIURA C,HIGASHINO T,MIURA T.A progestin and an estrogen regulate early stages of oogenesis in fish1[J].Biology of Reproduction,2007,77(5):822-828.
[2]OZAKI Y,MIURA C,MIURA T.Molecular cloning and gene expression of Spo11 during spermatogenesis in the Japanese eel,Anguilla japonica[J].Comparative Biochemistry&Physiology Part B,2006,143(3):309-314.
[3]PASQUIER J,CABAU C,NGUYEN T,et al.Gene evolution and gene expression after whole genome duplication in fish:the Phylo Fish database[J].Bmc Genomics,2016,17(1):368.
[4]尚婉婧,罗凤,罗廷文,等.尼罗罗非鱼Spo11基因的克隆表达及RU486处理对其表达的影响[J].水生生物学报,2016,40(2):403-407.SHANG W J,LUO F,LUO T W,et al.Molecular cloning and expression analysis of spo11 gene and expression change under RU486treatment in Nile tilapia[J].Acta HydrobiologicaSinica,2016,40(2):403-407.
[5]DACHS J,RY D A V,EISENREICH S J.Occurrence of estrogenic nonylphenols in the urban and coastal atmosphere of the lower Hudson river estuary[J].Environmental science and technology,1999,33(15):2676-2679.
[6]DONG C D,CHEN C W,CHEN C F.Seasonal and spatial distribution of 4-nonylphenol and 4-tert-octylphenol in the sediment of Kaohsiung Harbor,Taiwan[J].Chemosphere,2015,134:588-597.
[7]KANNAN K,KEITH T L,NAYLOR C G,et al.Nonylphenol and nonylphenol ethoxylates in fish,sediment,and water from the Kalamazoo river,Michigan[J].Archives of Environmental Contamination&Toxicology,2003,44(1):77-82.
[8]LIBER K,KNUTH M L,STAY F S.An integrated evaluation of the persistence and effects of 4-nonylphenol in an experimental littoral ecosystem[J].Environmental Toxicology and Chemistry,1999,18(3):357-362.
[9]KINNBERG K,KORSGAARD B,BJERREGAARD P.Concentration-dependent effects of nonylphenol on testis structure in adult platyfish Xiphophorus maculatus[J].Marine Environmental Research,2016,50(1):169-173.
[10]LEE C C,JIANG L Y,KUO Y L,et al.Characteristics of nonylphenol and bisphenol A accumulation by fish and implications for ecological and human health[J].Science of the Total Environment,2015,502:417-425.
[11]ASIFA K P,CHITRA K C.Effects of nonylphenol-induced oxidative stress in ovary of cichlid fish,Etroplus maculatus(Bloch,1795)[J].2016,58:11-15.
[12]CAREGHINI A,MASTORGIO A F,SAPONARO S,et al.Bisphenol A,nonylphenols,benzophenones,and benzotriazoles in soils,groundwater,surfacewater,sediments,andfood:a review[J].Environmental Science and Pollution Research International,2015,22(8):5711-574.
[13]SAYED A E,HAMED H S.Induction of apoptosis and DNA damage by 4-nonylphenol in African catfish(Clariasgariepinus)and the antioxidant role of Cydonia oblonga[J].Ecotoxicology and Environmental Safety,2017,139:97.
[14]SHARMA M,CHADHA P.Study on DNA damaging effects of 4-nonylphenol using erythrocytes from peripheral circulation,gill and kidney of fish Channa punctatus[J].Journal of Environmental Biology,2016,37(2):313-318.
[15]THOMAIDI V S,STASINAKIS A S,BOROVA V L,et al.Is there a risk for the aquatic environment due to the existence of emerging organic contaminants in treated domestic wastewater?Greece as a case-study[J].Journal of Hazardous Materials,2015,283:740-747.
[16]刘晓聪,赵元莙,张金叶.瓶囊碘泡虫HSP70基因的克隆、鉴定及功能预测[J].重庆师范大学学报(自然科学版),2016,33(2):37-42.LIU X C,ZHAO Y J,ZHANG J Y.Cloning,identification and functional prediction of heat shock protein 70gene in Myxobolus ampullicapsulatus[J].Journal of Chongqing Normal University(Natural Science),2016,33(2):37-42.
[17]ARNOLD K,BORDOLI L,SCHWEDE T.The SWISSMODEL workspace:a web-based environment for protein structure homology modelling[J].Bioinformatics,2006,22(2):195-201.
[18]GUEX N,PEITSCH M C.SWISS-MODEL and the SwissPdb Viewer:an environment for comparative protein modeling[J].Electrophoresis,1997,18(15):2714-2723.
[19]SCHWEDE T,KOPP J,GUEX N,et al.SWISS-MODEL:an automated protein homology-modeling server[J].Nucleic AcidsResearch,2003,31(13):3381-3385.
[20]YEH H Y,LIN S W,WU Y C,et al.Functional characterization of the meiosis-specific DNA double-strand break inducing factor SPO-11fromC.elegans[J].Scientific Reports,2017,7(1):2370.
[21]SHARMA M,CHADHA P.4-Nonylphenol induced DNA damage and repair in fish,Channa punctatus after subchronic exposure[J].Drug and Chemical Toxicology,2016,40(3):320-325.
[22]ZHAO Y,WANG C,XIA S,et al.Biosensor medaka for monitoring intersex caused by estrogenic chemicals[J].Environmental Science and Technology,2014,48(4):2413-2420.
[23]MIURA C,HIGASHINO T,MIURA T.A progestin and an estrogen regulate early stages of oogenesis in fish1[J].Biology of Reproduction,2007,77(5):822-828.