Cloning And Expression Analysis Of Superoxide Dismutase Gene From Tetranychus Cinnabarinus(Boisduval)(Acari:Tetranychidae)

Tetranychus cinnabarinus?Boisduval?,belonging to the Arachnoidea,Acari,Tetranychidae,is a worldwide agricultural pest mite that harms the cash crops and ornamental plants such as beans,cotton,vegetables,and fruit trees and leads to serious economic losses to agricultural production.For a long time,the prevention and control of T.cinnabarinus has been mainly controlled by chemical agents.Under environmental stress,antioxidant enzymes undergo a series of changes in physiology and biochemistry levels,and changes in the molecular level of antioxidant enzyme genes are unclear.In this paper,the full-length cDNA of three superoxide dismutase?SOD?genes of T.cinnabarinus was cloned using RT-PCR and RACE technology and the bioinformatics analysis of the sequences was performed.The qRT-PCR technique was used to study the mRNA expression level of SOD genes under various stress conditions.The main results are as follows:?.Analysis of the characteristics of the superoxide dismutase gene sequence of T.cinnabarinusThe full-length cDNAs of cytoplasmic Cu/ZnSOD,extracellular Cu/ZnSOD and mitochondrial MnSOD of T.cinnabarinus were cloned and named as TcSOD1,TcSOD2 and TcSOD3.Among them,the GenBank accession number of TcSOD1 gene is KY348746,which is 827 bp in length.The open reading frame?ORF?is 459 bp and encodes 152 amino acid residues.Its protein molecular weight is approximately15.559 kDa,and the theoretical pI is 6.02.It includes two signature motifs,43-53?GFHIHEFGDNT?and 137-148?GNAGARSACGVI?.Cu²⁺binding sites are His₄₅,His₄₇,His₆₂ and His₁₁₉,and Zn²⁺binding sites are His₆₂,His₇₀,His₇₉ and Asp₈₂.The TcSOD2 gene has a GenBank accession number of KY348747 and a total length of1034 bp.The ORF is 699 bp,encodes 232 aa.Its protein has a molecular weight of25.154 kDa,and has a theoretical pI of 6.48.Its amino acid sequence contains a signature motif:109-119?GFHIHQFGSIL?,the Cu²⁺binding sites are His₁₁₁,His₁₁₃,His₁₂₈ and His₁₈₆,and the Zn²⁺binding sites are His₁₂₈,His₁₃₆,His₁₄₅ and Asp₁₄₈.The TcSOD2 amino acid sequence has a 17 aa signal peptide?MKISFCFLILLITKILS?at the N-terminus.The TcSOD3 gene has a GenBank accession number of KY348748and a total length of 1003 bp.The ORF is 678 bp,encodes 225 aa.Its protein has a molecular weight of approximately 24.657 kDa,and has a theoretical pI of 7.86.Its amino acid sequence contains a signature motif:186-193?DVWEHAYY?,and the Mn²⁺binding site is His₅₃,His₁₀₁,Asp₁₈₆,and His₁₉₀.The results of sequence multiple alignments showed that the cytoplasmic Cu/ZnSOD and mitochondrial MnSOD were relatively conserved,while the extracellular Cu/ZnSOD was more diverse.Phylogenetic analysis showed that SOD is divided into two major branches,one is Cu/ZnSOD,and the other is MnSOD,while Cu/ZnSOD can be divided into two branches,one is cytoplasmic Cu/ZnSOD,and the other is extracellular Cu/ZnSOD.?.Analysis of superoxide dismutase gene expression in T.cinnabarinus under temperature stressesThe relative expression levels of TcSOD1,TcSOD2,and TcSOD3 genes were measured by real-time fluorescence quantitative PCR after treatment at different temperatures?4°C,25°C,and 40°C?for 2 hours.The treatment at 25°C is as a control,and RPS18,?-TUB were as reference genes.It was found that the relative expression levels of TcSOD1,TcSOD2,and TcSOD3 genes in the 4°C treatment group were significantly down-regulated compared to the 25°C control group?P<0.05?,and were0.344,0.287 and 0.358-fold of 25°C control groups,respectively.The relative expression levels of TcSOD1 and TcSOD2 genes in the 40°C treatment group were significantly down-regulated?P<0.05?,being 0.481 and 0.291-fold that of the control group,respectively,while there was no significant difference in the relative expression level of TcSOD3 gene.?.Analysis of superoxide dismutase gene expression in T.cinnabarinus under acaricide stressesLeaf disc method was used to treat female adults of T.cinnabarinus with LC₅₀doses of abamectin,fenpropathrin,propargite and cyflumetofen,respectively,and clear water treatment was used as a control.The relative expression levels of TcSOD1,TcSOD2,and TcSOD3 genes treated after different acaricides were measured by real-time fluorescence quantitative PCR,and RPS18 and?-TUB were as reference genes.The results were as follows:compared with the control group,the relative expression levels of TcSOD1,TcSOD2,and TcSOD3 genes in the abamectin treated treatment group were significantly down-regulated?P<0.05?,being 0.450,0.147 and0.663-fold of the control group,respectively.The relative expression levels of TcSOD1 and TcSOD2 in the fenpropathrin treatment group were significantly down-regulated?P<0.05?,being 0.794 and 0.201-fold of the control group,respectively,while the relative expression of TcSOD3 was significantly increased?P<0.05?,being 2.774-fold of the control group.The relative expression of TcSOD2was significantly down-regulated both in the propargite and cyflumetofen treatment groups?P<0.05?,which was 0.655 and 0.397-fold of the control group,but TcSOD1and TcSOD3 had no significant changes.In summary,the three SOD genes of T.cinnabarinus were cloned for the first time in this study:TcSOD1,TcSOD2,and TcSOD3,and their expression and regulation at different temperatures and acaricides were studied,laying a foundation for further investigation of the antioxidant properties of SOD,and providing the theoretical data for the formulation of pest control strategies.