| The Mythimna separata(Walker)is migratory pest,belonging to Insecta,Lepidoptera,Noctuidae.The armyworm is one member of the holometabolous class of insects,which possesses four distinguished developmental stages: egg,larva,pupa,and adult.The larvae have six instars.Guttony would outbreak after the fifth instar larve,feeding wheat,corn,rice and other crop,which causes significant damage to food production in China.The feeding behavior and migration of larvae are density-dependence.The larvae have more active feeding and greater intake at the density of 10 larvae per jar,which was significantly higher than 1 larvae per jar.And high density(10 per jar)feeding larvae emergence into adults with migratory characteristics.PKG is a cGMP dependent protein kinase,a family of serine / threonine protein kinases,widely distributed in eukaryotes.It has been reported that PKG gene regulates animal feeding behavior that can be a candidate gene for the foraging behavior of armyworm.Therefore,studying the effect of PKG gene on the feeding of two kinds of life type armyworm larvae(1 per jar and 10 per jar)provides the basic data for the molecular mechanism of armyworm migration.First,we measured activity and expression of PKG protein in the brain from different life forms of 5 instar larvae of armyworm.RNA interference and pharmacological methods were used to verify the function of PKG gene and detecting intake changes of 10 per jar.The results are as follows:1.Setting 6 groups of different feeding density(1per jar?5per jar?10per jar?15per jar?20per jar and 30 per jar)to detected six kinds of life form of the PKG protein in the larval brain.The results showed that the activity of armyworm PKG protein in 1 larvae per jar was highest.10 larvae per jarand15 larvae per jar significantly lower than 1 larvae per jar.No significant difference between 10 larvae per jarand15 larvae per jar in the activity of PKG protein.2.Detection expression level of two life forms larvae of armyworm by western blot.The result shows there was no electrophoretic bands.3.Use RNAi interference PKG gene,and four dsRNA were designed to interference.The result shows that only fragment 2 of dsRNA had effective interference in the case of high injection volume which decreased expression of PKG gene,but decrease is not significantly.4.Feeding 8-Br-cGMP(cGMP agonist)with a concentration of 1000?M to the larvae,the feed intake reduced but the activity of PKA and PKG increased significantly.But feeding 8-Br-cAMP(cAMP agonist)with a concentration of 1000?M to the larvae,the feed intake reduced and the activity of PKA significantly increased but the activity of PKG was not noticeable.In this study,we first measured PKG protein in the brain based on 6 different feeding density of armyworm larvae,finding that PKG protein activity was significantly different between 1 larvae per jar and 10 larvae per jar.Previous research found that the Mythimna separate in 10 larvae per jar has the strongest migration ability.Therefore,we determine 1 larvae per jar and 10 larvae per jar as research object.Western blot was used to make quantitative analysis of PKG protein and we also tried to interfere with PKG gene by RNA interference technology,although the results of Western blot and RNAi are not ideal,but also provide some suggestions for our follow-up experiments: Before the Western blot experiment,to ensure the good quality of protein;RNA interference in the process,continue to increase the amount of injection of fragment 2 dsRNA should be successful interference of PKG gene,the PKG gene expression was significantly reduced.Finally,by means of pharmacology treatment results showed that the food intake decreased of armyworm larvae,treated feed with cGMP agonist and cAMP agonist respectively.The larvae feeding were treated with cGMP decreased significantly,this show that PKG gene may be involved in regulation of feeding behavior of the larvae.These results are beneficial to further study on the molecular mechanism of feeding behavior of armyworm larvae. |
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