Cloning And Characterization Of Glucose Oxidase And Peroxidase Genes From The Oriental Armyworm,Mythimna Separata Walker

There are many kinds of biochemical reactions to maintain normal life activities of organisms.Oxidoreductase is the most basic and important reaction.The enzymes that catalyze the efficient reaction of this reaction are collectively called oxidoreductases.Oxidoreductases participate in redox reactions in organisms to generate energy as well as detoxify and have important functions in the process of forming physiologically active substances.Oxidoreductases can be classified into many types depending on the amino acid structure and the different activities involved.Among them,glucose oxidase and peroxidase are oxidoreductases which are widely present in plants,animals and microorganisms and have antioxidant functions.Glucose oxidase(GOX)is an aerobic dehydrogenase that has the specific recognition of ?-D-glucopyranose,its function is to reduce the concentration of oxygen in the body as an antioxidant,and to maintain the normal physiological activities of life;create a beneficial environment for anaerobic bacteria,inhibit the growth of harmful bacte ria.Peroxidase(POD)as a member of the antioxidant enzyme system.Its function is to remove excess reactive oxygen species in the living body and prevent a series of oxidative damages such as DNA damage,enzyme inactivation,lipid peroxidation and the like due to excessive active oxygen in the living body.Mythimna separata is one of the major agricultural pests of Chinese food crops.It has the characteristics of gluttony and seasonal long-distance migration,which seriously threatens the safety of food production.Its occurrence and harm have the following characteristics: a wide range of occurrence;many generations of damage;a large number of types and quantities of harmful crops;outbreak obvious can produce serious yield losses;long history of occurrence of hazards and so on.At present,the research on the GOX and POD genes of the M.separata is still in its infancy.According to the redox mechanism involved in GOX and POD and the related research on the related insect GOX and POD,it is suspected that the antioxidant enzyme system involved in these two enzymes is related to the digestion and bacteriostasis of insects,and thus can be silenced by RNAi technology.The gene affects its function and inhibits the physiological metabolic activities such as digestion of insects to achieve biological control.In this experiment,the molecular sequences of two new genes,glucose oxidase gene named MsGOX and peroxidase gene named Ms POD were cloned by using the M.separata as the test material;the nucleotide sequence and amino acid sequence were analyzed respectively;to study the differences of temporal and spatial expression levels of two genes in different tissues and different development stages;investigating the role of MsGOX gene in insect feeding by different concentrations of glucose induced treatment and starvation and refeeding treatment on M.separata;the treatment of the M.separata at different periods and different temperatures to explore the insect protective effect of the Ms POD gene induced by extreme temperature;using RNAi technology to explore the effects of MsGOX gene silencing on the growth and development of M.separata;to investigate the effect of MsGOX gene silencing on the bacteriostasis of this gene and its effect on the infection of Bacillus thuringiensis,to lay a theoretical foundation for the molecular design of this gene to control sticky insects.The main test results are summarized as follows:1.Cloning a new cDNA sequence of the GOX gene from M.separata.The GOX gene of M.separata named MsGOX,and was registered in Gen Bank with the accession number KY348779.It was 2187 bp in length and contained an open reading frame of 1821 bp.It encoded a polypeptide containing 606 amino acids.The molecular weight was 66.4 ku and the isoelectric point was 4.79.Amino acid sequence analysis showed that the gene was found it belonged to the glucose-methanol-choline oxidoreductase(GMC)family,included two conserved protein domains,GMC_oxred_N and GMC_oxred_C.The amino acid sequence was closely related to other insects of Lepidoptera,and it had a high consistency with Helicoverpa armigera,Spodoptera exigua,Helicoverpa zea and Heliothis viriplaca,which is more than 85%.Cloning a new cDNA sequence of the POD gene of the M.separata.The POD gene of M.separata named Ms POD,and was registered in Gen Bank with the accession number MH606240.The full length of POD gene was 2433 bp,which contained an open reading frame of 2061 bp,encoded 686 amino acids with isoelectric point of 5.68 and molecular weight of 76.1 ku.Amino acid sequence analysis showed that the gene possessed an heme peroxidase cell adhesion protein domain,belonging to the Peroxidase—Cyclooxygenase Superfarnily.Amino acid sequence analysis showed that the amino acid sequence of Ms POD was close to that of other Lepidoptera Noctuidae insects,and the homology with POD amino acid sequence of Helicoverpa armigera was the highest(92%).2.To explore the temporal and spatial expression differences between MsGOX gene and Ms POD gene,and to analyze the changes of expression levels of different genes in different developmental stages and different tissues.The MsGOX gene was expressed in different tissues and different developmental stages by experiments and analysis.Among them,the expression level of MsGOX gene was highest in the 4th instar and salivary glands,and lowest in the 1st instar and in the foregut.The Ms POD gene was also expressed in different tissues and different developmental stages,with the highest expression in the pupal stage and salivary glands,and the lowest in the 1st instar and in the foregut.3.Explore the relationship between MsGOX gene and insect feeding.4 different concentrations of glucose solution were treated on the M.separata.The results showed that 4 concentrations of glucose solution could induce the expression level of MsGOX gene,and the higher the concentration of glucose solution,the greater the effect on the expression level of MsGOX gene,the expression level of this gene was highest at a glucose concentration of 10%.The starvation treatment of the M.separata showed that the expression level of MsGOX gene increased first and then decreased with the increase of starvation time,and the expression level reached the highest at 24 h.The starvation treatment of M.separata was refeeding,and the results showed that the expression level of MsGOX gradually increased.To explore the insect protective effect of Ms POD gene induced by extreme temperature,different temperature gradients and different time treatments were carried out on the M.separata.The results showed that the expression level of Ms POD gene was significantly different at different time points after temperature gradient induction.The expression level reached the highest after treatment at 35 ? for 12 h.4.To explore the effect of MsGOX gene on the physiological indexes of M.separata after being interfered,the results showed that the body weight,body length and digestibility of the M.separata were affected,and it had obvious inhibitory effect after 12 hours of treatment.To investigate the effect of MsGOX gene on the bacteriostasis of this gene.The results showed that the number of colonies on the plate made by the treatment group injected with ds GOX was larger than that made by the control group injected with ds EGFP.To explore the synergistic effect of MsGOX gene on the infection of the M.separata by Bacillus thuringiensis.The results showed that the mortality of the treatment group(injected with ds GOX)was higher than the control group(injected with ds EGFP),indicating that the resistance to B.thuringiensis was reduced after RNA interference.The above results demonstrated that the expression of MsGOX gene was successfully inhibited by the RNAi method,and it was found that the MsGOX gene may play a role in the midgut digestion of the M.separata.