Cloning And Function Exploration Of Superoxide Dismutase Gene In Panonychus Citri

The accumulation of reactive oxygen species(ROS) which was induced by environmental stresses, which causes oxidative damage to macromolecular structure, including proteins, nucleic acids, and lipids. Superoxide dismutases(SODs) are one of the most important antioxidant enzyme of organism, and they play a key role in maintaining the balance of intracellular ROS. Panonychus citri(P. citri) is a worldwide pest mites with wide host range and continuously long damage, and the major control method of P. citri is chemical control, and the mite has evolved serious resistance to long-term unscientific use of acaricide, hence, control stratergy of the mite have necessitated rethink with sustainability and green. Hereby, to insight into the P. citri adaptation mechanisms for environmental stresses, the SODs of P. citri were systematically studying in this dissertation, and the main contents are as follows:1. Sequence and phylogenetic analysis of three SOD genes of P. citriAccording to the transcriptome sequencing data of P. citri from our research group, the ORF of three SOD genes were cloned by screening of subject sequences and designing of primers, and named Pc SOD1(KJ522695), Pc SOD2(KJ700412) and Pc SOD3(KJ700411), respectively. This three isoforms of SOD genes were identified to belong to intracellular Cu Zn SOD, extracellular Cu Zn SOD and mitochondrial Mn SOD, respectively, based on the analysis of the molecular weight, signature sequence and signal peptide. The results of multiple protein alignments showed that high similarity(about 70%) existed among species for SOD1 and SOD3, whereas low similarity(about 35%) was found for SOD2. The phylogenetic relationship analysis showed that SODs were divided into three groups, it is indicated that there are differences of three SOD genes of P. citri in their binding sites, signature sequence and molecular weight, which may lead to differently antioxidant function.2. The expression patterns under environmental stresses of SOD genesThe accumulation of ROS was induced by environmental stress, thereby induced the SOD gene overexpression. The current study focuses on four types of environmental stress, including heavy metals, acaricides, UV-B and extreme temperatures, the expression patterns of Pc SOD1, Pc SOD2 and Pc SOD3 were analysised under oxidative stress by q RT-PCR. As the results, the m RNA expression levels of three isoforms of SOD genes were all significantly overexpressed with high levels under ultraviolet radiation and extreme temperature stresses(0°C and 41°C), it is indicated that the UV-B and extreme temperature were potential environmental inducers of ROS in P. citri. Furthermore, the fold changes of Pc SOD3 were significantly higher than that of the other two Cu-Zn SOD genes in various treatments, including heavy metal stresses(Mn2+, Cu2+ and Cd2+), 0°C and UV-B, these results indicated the Mn SOD was a typical inducible gene, and the Pc SOD3 provided more broad-spectrum protection against oxidative damage induced by environmental stress than Pc SOD1 and Pc SOD2.3. Prokaryotic expression of Pc SOD3The p ET28α-Pc SOD3 recombinant protein was expressed by E. coli BL21(DE3), and the purification of protein was related to the uniting of His tag and Ni column. The recombinant protein was comfirmed by Western Blot. The activity of Pc SOD3 recombinant protein is 276.61 U/mg, it is indicated that this recombinant protein has antioxidant function. Analsis of recombinant protein characterization showed that Pc SOD3 had maximum activity at 35℃ and p H 7.0 by enzymatic reaction.4. Analysis of antioxidant function between Mn SOD and Cu Zn SOD in P. citriThe Pc SOD1, Pc SOD2 and Pc SOD3 were overexpressed in E. coli cells, and the empty p ET28α vector was as control, the diameters of the death zones were measured by disc diffusion assays under three types of oxidative stress, therefore we analysis the antioxidant function of three SOD recombinant proteins. The results showed that diameters of the death zones of Pc SOD3 were smaller than that of Pc SOD1 and Pc SOD2 in all the three oxidative stress, it is indicated that the antioxidant function of Mn SOD gene(Pc SOD3) is stronger than that of Cu Zn SOD gene(Pc SOD1 and Pc SOD2) in P. citri.In conclusion, in this study of P. citri against to environmental stress with SOD gene, we found three isoforms of SOD gene sequences from transcriptome sequencing data, namely Pc SOD1, Pc SOD2 and Pc SOD3 after validation, and analysis of their sequence characteristics, phylogenetic relationships and the expression patterns under environmental stress. Preliminary analysis of the antioxidant function of this three genes through prokaryotic expression and disc diffusion assay. The research results provided basic data for deeply investigation of SOD physiological function under oxidative stresses in P. citri and helped us to make new control stratergy of pest mites.