Cloning And Function Exploration Of Superoxide Dismutase And Its 5' Flanking Sequence From The Plutella Xylostell

Plutella xylostella is an important pest of cruciferous vegetables.Due to the large amount of chemical pesticides have been used in agricultural production activities for a long time,not only the natural enemies of P.xylostella are killed,but also their resistance is increasing.Therefore,it is an important way to find effective biological control method for the prevention and control of P.xylostella.Previous studies have shown that external environmental stress induces excessive ROS in the body of P.xylostella,which causes damage to the host,SOD,as the first line of defense of the antioxidant system in cells,plays a key role in the scavenging ROS.Therefore,it is of great significance to study the function of SOD for studying the interaction between P.xylostella and the environment,in order to find a more effective control method for P.xylostella.In this study,three PxSOD genes were cloned from the genome of P.xylostella,and its enzymatic properties,physiological functions and expression regulation patterns were studied.The main results are as follows:1.Based on the genomic information,three SOD genes of P.xylostella?PxSOD1,PxSOD2,PxSOD3?were cloned by RACE-PCR and analyzed by bioinformatics.The results showed that the protein PxSOD1 encoded by PxSOD1 belonged to intracellular Cu/ZnSOD,which contains six conserved active sites of Cu/ZnSOD and two conserved motifs of RHAGDLGN and TGN.The protein PxSOD2 encoded by PxSOD2 belonged to extracellular Cu/ZnSOD,which contains six conserved active sites of Cu/ZnSOD and one conserved motif of RHAGDLGN.The protein PxSOD3 encoded by PxSOD3 belonged to mitochondrial MnSOD,which contains four MnSOD-conserved active sites and two conserved motifs of QGSGW and DVWEHAYY.2.The results of gene expression of different PxSOD in response to external factor stress by Quantitative Real-time PCR showed that In response to high temperature stress,the expression of PxSOD2 increased by 115%,which may play an important role in host resistance to high temperature,and while the expression of PxSOD3 did not change significantly.The expression of PxSOD3 increased by 20%and 60%respectively,while the expression of PxSOD2 decreased significantly under Mn²⁺and Cu²⁺stress.When Salmonella infects P.xylostella,the expression of PxSOD3 is increased by 40%,and it is hypothesized that PxSOD3 may be involved in the defense against Salmonella infection;The expression levels of PxSOD2 and PxSOD3 decreased to different degrees under the stress of three different bactericidal mechanisms?Avermectin,Bacillus thuringiensis preparation,Cypermethrin?and different times of UV radiation,The specific mechanism of the stress response to pesticides and UV radiation remains unclear,which needs further research.3.To study the enzymatic properties of PxSOD,prokaryotic expression vector pET28a-PxSODs was constructed and the protein PxSOD3 was successfully purified by Ni-NTA resin affinity chromatography.The analysis of enzymatic characteristics showed that the enzyme activity of PxSOD3 is 871.23 U/mg;the maximum reaction enzyme activity(V_max)was 1002 U/mg,and the affinity coefficient K_m was 1.027 mmol/L xanthine.The optimum temperature is in the range of 15°C to 25°C;the optimum pH is 6;the half-life of enzyme activity is about 13 days;Mn²⁺can be used as an activator of PxSOD3.In addition,in the CHP oxidation resistance experiment,after adding purified PxSOD3 to the strain BL21?DE3?-pET28a as the experimental group or the strain BL21?DE3?-pET28a-PxSOD3 was used instead of BL21?DE3?-pET28a as the experimental group,The results showed a significant decrease in the diameter of the inhibition zone,which indicating that PxSOD3 had the physiological function of resisting CHP oxidative damage,and PxSOD3may play an important role in the process of antioxidation.4.In order to study the expression regulation of PxSOD3,the 5'flanking sequence of PxSOD3 was cloned,5'flanking sequence vector pMD19-T-PxSOD3-aP_-1137-+184 of PxSOD3 was constructed.The results of bioinformatics prediction showed that the sequence contains four promoter regions:aP_-957-_-908?aP_-691-_-642?aP_-561-_-512?aP_-476-_-427,and several basic elements of promoters,such as CAAT-box and TATA-box,with the TATA box is located at-947 bp to-944 bp;the transcription factor binding sites was located at-917 bp.The deletion cloning of the 5'flanking sequence of PxSOD3,the results showed that aP_-1137-_-712?aP_-1137-_-712?aP_-398-_-212212 and aP_-605-_-1 all have promoter function,but because aP_-1137-_-712 contains aP_-1041-_-702702 which belongs to the coding region of solute vector superfamily members of P.xylostella,so it is suspected that although aP_-1137-_-712 can initiate the expression of green fluorescent protein in the vector with high fluorescence intensity,but it is not the promoter of PxSOD3.The fluorescence intensity of aP_-211-_-1 was significantly higher than that of aP_-398-_-212?aP_-605-_-1,suggesting that aP_-211-_-1 was probably the promoter region of PxSOD3.In summary,Three PxSODs were cloned,their physicochemical properties and expression patterns in response to various environmental factors were analyzed,and the prokaryotic expression of PxSOD3 was carried out to verify the related properties of enzymes and antioxidants.At the same time,the promoter of PxSOD3 was preliminarily studied,which laid a foundation for further study on the function of SOD in the stress resistance of P.xylostella.It provides a new idea for finding more effective genetic control method for P.xylostella.