Study On The Expression And Function Of Phenolic Oxidase Gene In Locust Migratory Migratoria Asiatica

Phenoloxidase(PO),an enzyme that synthesizes melanin and plays an important role in insect defense,has been found to be a proenzyme(Prop O),which is activated by proteolysis in hemolymph and cuticle.Active intermediates produced during the PO-catalyzed melanism process have been shown to directly kill bacteria,fungi,viruses and parasitic wasps.Phenolic oxidase plays a key role in insect development and metamorphosis.PO is uniquely associated with a number of important physiological and biochemical processes,such as corneous hardening,melanosis,and wound healing.Corneous hardening and blackening are important for insect coating,defensive coating and blackening of exotic organisms.In this study,the expression characteristics and function of phenol-oxidase gene of Locusta migratoria were studied.The main research contents of this paper are as follows:1.Establishment of Real-time PCR assay for phenol oxidase genereal-time PCR is the preferred method for quantitative analysis of gene expression in many laboratories.In this study,a real-time PCR method for the detection of phenol oxidase gene was established,and the correlation coefficient R~2 value of PPO1 and PPO2plasmids was 0.9979 and 0.9951,respectively,the slope was-3.794 and-2.332,respectively,and the amplification efficiency was 99%and 90%,respectively.Therefore,it is proved that the method is successfully established.2.Expression of phenolic oxidase gene in locust migratory migratory speciesReal-time PCR was used to analyze the transcription level of phenol oxidase gene.The results were as follows:The expression of PPO1 gene existed in every developmental age of locust,and the highest expression was on the third day of the third instar,while the lowest expression was on the third day of the fifth instar.The expression level of PPO2 was the highest on the third day of the first instar,and the lowest on the third day of the third instar.PPO1 is mainly expressed in the midgut,while PPO2 is expressed in the hindgut.3.Study on the biological function of phenol oxidase geneRNA interference(RNAi)technique was used to study the function of phenoloxidase gene PPO2 and its effect on insect growth and development.The results showed that the expression level of PPO1 gene was relatively high after 48h silencing,but there was no significant difference in the expression level of other time periods.The silencing efficiency of PPO2 gene was basically unchanged within 24h after injection,but the silencing efficiency of PPO2 gene decreased significantly at 48h and 96h after injection.In different time periods after gene silencing,the feeding situation of treatment group was significantly different from that of control group.Although there was no significant change in body weight,compared with the control group,the weight gain of the treatment group was significantly different.