| In recent years,it has gradually become a hot topic in the international environmental science and water ecology to use aquatic animals as subjects to evaluate the toxic effects of drugs.Rotifers are the main groups of zooplankton and often used in ecotoxicology studies because of its propagation speed,short life cycle and high sensitivity to toxicants.Paraquat is a type of organic heterocyclic herbicide,as for juglone,a kind of naphthalene quinones,it is extracted from J.mandshurica Maxim or J.regia L.It is generally believed that both the two drugs can induce the formation of excess ROS(reactive oxygen species,ROS),and thus cause oxidative damages to organisms.This study selected two different types of rotifers as experimental materials,B.calyciflorus together with A.brightwelli,to investigate the sensitivity of both the two for poisen,then further explored the mechanism of action of both of the drugs.We cloned the full length of A.brightwelli hsp70 gene by RACE technology.Based on this,we used Real-time quantitative PCR to determine the hsp70 mRNA expression of B.calyciflorus and A.brightwelli treated by paraquat and juglone.In traditional ecology and molecular ecology,these two kinds of rotifers were compared to determine their sensitivity of poison and then explore the feasibility of A.brightwelli to be a potential model of environmental toxicological and thus to investigate the role of HSP70 against oxidative damage.This research mainly involved four aspects as follows:1.The acute toxicology effect of paraquat and juglone on B.calyciflorus and A.brightwelliWe conducted acute toxicology experiments on B.calyciflorus and A.brightwelli.Exposed in different concentration of paraquat after 24h,B.calyciflorus mortality rate increased along with the concentration increase of paraquat.Then we worked out that the B.calyciflorus's 24h-LC50 under paraquat treatment was 49.1588 mg/L,the linear regression equation was Y = 7.7928X-8.1824(R2=0.9739),and the 95%confidence limits was 43.3978?55.6846 mg/L.Experiments of A.brightwelli exposed in paraquat showed that A.brightwelli is very sensitive to paraquat as well as B.calyciflorus.We have obtained that A.brightwelli's 24h-LC50 under paraquat treatment was 22.0874 mg/L,the linear regression equation is Y=4.9927X-1.7108(R2=0.9263),and the 95%confidence limits was 16.3690?29.8034 mg/L.It is showed in the acute toxicology experiments that juglone has strong toxic effects on B.calyciflorus.Exposed in different concentrations of juglone after 24h,B.calyciflorus's mortality rate increased alone with the concentration increase of juglone.The B.calyciflorus's 24h-LC50 under treatment of juglone we've got was 196.3306?g/L,the linear regression equation was Y=7.7157X-12.692(R2=0.9647),and the 95%confidence limits was 171.1099?225.2688?g/L.Similarly,the acute toxicity experiments showed that juglone's toxic effects on A.brightwelli was quite strong,dozens or hundreds ?g of juglone showed a significant lethal effects on A.brightwelli.We have obtained A.brightwelli of juglone 24h-LC50 was 92.5658?g/L,the linear regression equation was Y=8.1195X-10.967(R2=0.9739),and 95%confidence limits was 82.3666?104.0279?g/L.2.Duration of developmental stages and mean lifespan of B.calyciflorus and A.brightwelli at different paraquat and juglone concentrationsDifferent paraquat and juglone concentration has significantly effects on duration of development stages and mean lifespan of B.calyciflorus and A.brightwelli.With the increase of paraquat concentration,B.calyciflorus and A.brightwelli's duration of development stages and mean lifespan showed similar trend:a substantially continuous extension was showed in pre-reproductive period,while the reproductive period,the average life expectancy and fecundity reduced gradually.The trend of B.calyciflorus's postreproductive initially decreased and then increased,while there is no certain trend in that of A.brightwelli.Among all data of B.calyciflorus,pre-reproductive period extended clearly in the high concentration of paraquat(5 and 10 mg/L)(P<0.05),the reproductive period was shorten in all concentrations of paraquat(P<0.05),the average lifespan and fecundity have reduced obviously in high concentrations(2.5,5 and 10 mg/L)of paraquat(P<0.05),however,there is no significant change in postreproductive.As for the rotifer of A.brightwelli,the pre-reproductive period significantly extended(P<0.05).A.brightwelli's reproductive period,average life expectancy and fecundity decreased in all the concentrations of paraquat(P<0.05).The postreproductive shortened after exposed in 1.25 and 10mg/L of paraquat.Duration of development stages of B.calyciflorus and A.brightwelli at different juglone concentrations is similar in general.With the increasing of juglone concentration,the pre-reproductive period of B.calyciflorus and A.brightwelli initially decreased and then increased,and the reproductive period,the average life expectancy and fecundity reduced.The pre-reproductive period of B.calyciflorus exposed in 12.5 and 25?g/L of juglone occured to be significantly longer(P<0.05),while the reproductive period significantly shortened when exposed in 12.5,25,50?g/L concentration ofjuglone(P<0.05).The average life expectancy of B.calyciflorus greatly shortened when exposed in 25,50 ?g/L of juglone(P<0.05).B.calyciflous fecundity has been reduced obviously in all concentrations of juglone(P<0.05).There is no certain trend of B.calyciflorus postreproductive,only in the group of highest concentration of juglone(50?g/L),B.calyciflorus postreproductive was significantly shortened than the control group(P<0.05).A.brightwelli reproductive period visibly shortened in all concentrations of juglone(P<0.05),however,that of pre-reproductive period can be seen increased or decreased significantly in the 6.25?g/L and 50?g/L of juglone,respectively.The average life expectancy and fecundity of A.brightwelli reduced obviously in 12.5,25,50?g/L of juglone(P<0.05).The postreproductive of A.brightwelli exposed in 25?g/L of juglone showed a significant decline.3.A.brightwelli hsp70 gene cloning and research of its structure and functionAccording to hsp70 gene sequences from other species,we designed degenerate primers by homologous comparing and looking for their conserved regions.After NCB1 BLAST confirmation,the short fragment was amplified to full length using RACE technology.In this way,we finally got A.brightwelli hsp70 gene sequences.A.brightwelli hsp70 gene was 2134bp in length.There was non-coding regions in both 5' and 3' ends,which were 80bp and 125bp in length.The open reading frame(ORF)was 2069bp in length,encoding 642 amino acids and a stop codon TAA.There was a polyadenylation addition signal(Polyadenylation Signal Site AATAAA)and a poly A tail in its 3' non-coding region.The protein molecular it translated weighted 70.2337kDa and theoretical isoelectric point was 5.84.A.brightwelli HSP70 is an intracellular protein,because that we didn't find the presence of a signal peptide sequence.Homologous comparison showed that the full-length gene we got belongs to hsp70 gene family in the NCBI homologous comparison.Amino acid sequence alignment showed the highest similarity between Asphsp70 amino acid sequence and mouse M.musculus(BAE41082)amino acid sequence,which reached 87%.The N-terminus of HSP70 is more conservative than the C-terminus.4.Effects of paraquat and juglone on B.calyciflorus and A.brightwelli hsp70 gene expressionWe used real-time quantitative PCR to determine the effect of paraquat on the mRNA expression of B.calyciflorus hsp70.The results showed that most of the treatment groups significantly increased(P<0.05)the mRNA expression levels of B.calyciflorus hsp70,except for the group of 1.25mg/L-1h,2.5mg/L-6h and 5mg/L-12h.The RT-PCR results of A.brightwelli treated with paraquat showed that short-term treatment of paraquat caused the hsp70 mRNA expression greatly increased.After 1.25,2.5,5 and 10 mg/L of paraquat treated for 1h,the hsp70 mRNA expression increased significantly in A.brightwelli(P<0.05).In addition,A.brightwelli hsp70 mRNA expression also increased significantly(P<0.05)after 2.5mg/L of paraquat treated for 3h and 6h,or 10mg/L of paraquat treated for 3h.With the extension of treatment time,the hsp70 mRNA expression of A.brightwelli gradually declined.In the experiments of B.calyciflorus treated with juglone,after 6.25 and 12.5 ?g/L of juglone treated for 6h and 12h,the hsp70 mRNA expression greatly increased in B.calyciflorus(P<0.05).Similarly,after 25 and 50 ?g/L of juglone treated for 1h,3h and 6h,the hsp70 mRNA expression also greatly increased in B.calyciflorus(P<0.05).After 12.5,25 and 50 ?g/L of juglone treated 1h,the hsp70 mRNA expression greatly increased in A.brightwelli(P<0.05).With the extension of processing time,the trend of A.brightwelli hsp70 mRNA expression occurred to fall.Except for hsp70 mRNA expression in the group of 25?g/L of juglone treated for 6h greatly increased in A.brightwelli(P<0.05)when compared with the control group,the rest of the groups did not change significantly.In addition,in 6.25?g/L concentration group,the hsp70 mRNA expression greatly increased in A.brightwelli,which was not affected by the treat time. |
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