Studies On Cloning And Expression Of Superoxide Dismutase Gene From Aleuroglyphus Ovatus(Troupeau) Exposure To Lead

Aleuroglyphus ovatus(Troupeau),Aacari and Acaridae,which is worldwide distribution stored-product pest mite and endagers human health.Heavy metal pollution is becoming increasingly serious,which has caused serious harm to the health and survival of organisms.The response mechanism of the superoxide dismutase SOD from A.ovatus to the Pb2+ stress is still unclear.In this study,we cloned the full length cDNA sequence of the three SOD genes from A.ovatus,and analyzed total SOD enzyme activity and mRNA expression at all developmental stages of A.ovatus under different concentrations of Pb2+stress.The main results are as follows:1.Response of the Superoxide Dismutase Enzyme Activity of A.ovatus from Different Developmental Stages and exposure of Different Concentrations of Pb2+The total SOD activity of A.ovatus eggs,larvae,protonymphs,tritonymphs and adult mites were also determined under five different concentrations(0,12.5,25,50,and 100 mg/kg while 0 mg/kg as the control group)Pb2+ stress.Our results found that,under the treatment of 0-100 mg/kg Pb2+,the SOD activity of A.ovatus showed a trend of rising first then decreasing with the development period.The activity of SOD in the egg stage was the lowest and the first nymph was the highest.As the concentration of Pb2+ increased,the SOD activity of A.ovatus at various developmental stages showed a trend of increasing first and then decreasing,but it was significantly higher than the control group.The SOD activity of each developmental stages at the concentration of 25 mg/kg Pb2+ was significantly higher than other treatment group.After that,the SOD activity decreases with increasing concentration.2.Characteristic analysis of the superoxide dismutase gene sequence of A.ovatusThe full length of the cDNA sequences of the three SOD genes from A.ovatus were cloned and named AoSOD1,AoSOD2 and AoSOD3,respectively.AoSOD1 gene is extracellular Cu/ZnSOD.The full length is 1100 bp.Open Reading Frame(ORF)length is 546 bp,encoding 181 aa.Molecular mass is about 18.999 kDa and theoretical protein isoelectric point(pI)is 9.10.Cu2+ binding sites are His73,His75,His90,His147,while Zn2+binding sites are His90,His98,His107,Asp 110.AoSOD1 gene contains two signature motifs,165-176(GNAGGRVGCGII)and 71-81(GFHIHQYGDTR),and a N-terminus signal peptide.AoSOD2 is mitochondrial MnSOD,with a full length of 1042 bp,693 bp ORF,encoding 230aa,molecular mass about 25.065 kDa,pI 9.10.Mn2+ binding sites are His57,His105,Asp189,His193,including a signature motif:189-196(DVWEHAYY).The AoSOD3 gene is also mitochondrial MnSOD,with full length of 1003 bp,an ORF of 786bp,encoding 261aa,molecular mass of 28.765 kDa,pI of 6.54,and Mn2+ binding site of His80,His 127,Asp211,His215,with a signature motif:211-218(DVWEHAYY).AoSOD2 and AoSOD3 are both glycoproteins.The phylogenetic analysis of the A.ovatus SOD gene showed that:AoSOD1 clustered into extracellular Cu/ZnSOD,AoSOD2 and AoSOD3 clustered with MnSOD.3.Analysis of the expression of the superoxide dismutase gene of A.ovatus in different developmental stages and under different concentrations of Pb2+ stressReal-time fluorescence quantitative Polymerase Chain Reaction was used to detect three SOD genes relative expression levels of A.ovatus under the stress of 0 mg/kg Pb2+ and different developmental stages,eggs,larvae,protonymphs,tritonymphs and adult mites with eggs as control group.The results showed that compared with the egg stage,the three SOD genes were significantly up-regulated during the nymph stages,and the expression levels of larvae and adult mites decreased,with no significant difference.AoSOD1 mRNA expression level was significantly up-regulated during the tritonymph stage,which was 2.63 times that of the control group;AoSOD3 was significantly up-regulated during the protonymph stage,which was 7.83 times that of the control group,and AoSOD2 was significantly up-regulated during protonymph and tritonymph stage,respectively 2.7 and 3.0 times that of control group.After 12.5,25,50 and 100 mg/kg Pb2+ treatment,qRT-PCR method was used to detect the three SOD gene from N3 stage of A.ovatus relative expression level with ?-Actin and ?-Tub gene as reference gene,0 mg/kg Pb2+ treatment as control,The results show that the expression of AoSODs gene could be detected in the range of 0-100 mg/kg Pb2+ treatment.Relative expression level of AoSODs gene under 12.5 and 25 mg/kgPb2+ stress decreased,but no significant difference.Gene expression levels of AoSOD1 and AoSOD2 significantly increased at 50 and 100 mg/kgPb2+stress(P<0.05),AoSOD3 only markedly unregulated under 100 mg/kg Pb2+ treatment.AoSOD1,AoSOD2,AoSOD3 gene relative expression levels peak vaule were at 100 mg/kg Pb2+treatment,respectively,3.1,4.1 and 2.3 times of the control group.In all,the total SOD activity of A.ovatus was development stage-specific,and Pb2+promoted its activity.The three AoSODs genes were all stage-specific expressions and high concentration of Pb2+ promoted its mRNA expression.The mRNA expression of mitochondrial MnSOD from A.ovatus had a stronger and broader response than extracellular Cu/ZnSOD.In addition,our study found that the enzyme activity of the SOD gene of A.ovatus was not synchronized with its mRNA expression level.