Cloning,Expression And Functional Analysis Of 102 Gene In Oriental Armyworm,Mythimna Separata

Mythimna separata?Walker??Lepidoptera:Noctuidae?is a major pest of grain crops in China.Its larval body color is blackened or altered as the density of larvae changes:such as high density and low density induction.The black type and light color type,and the middle?gray type?type between these two.Different types of larvae not only differ greatly in their growth and development characteristics,feeding and digestive physiology,but also their immune systems such as phenol oxidase and lysozyme activity as well as their ability to withstand natural enemy parasite or pathogen infection.In addition,the flight and reproductive ability of adults that emerge from different types of larvae are also different.However,whether these differences are related to genes or regulated by genes has not yet been reported yet.Therefore,based on the cloning of the 102 gene of the M.separata,we have studied the sequence of 102 gene and the spatio-temporal expression pattern,and revealed the effect of the larval density,parasitism of Exorista civilis Rondani and the infection of Metarhizium anisopliae on the expression of the 102 gene of the larva,and finally the role of the larvae in the immunization,flying and reproduction of the adult.The main results were as follows:The study on the relations between 102 gene sequences of M.separata and other Lepidopteran insects.According to the transcriptome database of M.separata,the open reading frame of the immune-related gene 102 of M.separata was obtained and named 102?MG844422?,which included perfect open reading frames of 1050 bp,encoding 349 amino acids.The molecular formula of the protein was C₁₇₆₃H₂₇₇₈N₄₆₈O₅₂₅S₁₅,and the predicted molecular weight was 39.411 kDa.The theoretical isoelectric point was 5.81.The sequence analysis results showed that the sequence similarity between the insects of the family Noctuidae:Helicoverpa armigera,Heliothis virescens,Spodoptera littoralis,and Spodoptera litura is over 90.0%.The insect similarity with other subjects is about 70%,and the phylogenetic tree analysis results also coincided with the status of M.separata in traditional taxonomy.The study showed the spatio-temporal expression pattern of 102 gene.The results of the relative expression of 102 gene by qPCR showed that the temporal and spatial expression of 102 gene was significantly different at the level of mRNA.As far as the developmental stage was concerned,although the 102 gene could be expressed in the egg,1-6th larvae,pre pupae and adult,the highest expression of the 6th larvae was followed by the adult,the pre-pupae,the 5th and 4th larvae.The tissues expression of the 5th larvae was significantly different,and significantly higher than that of the eggs and 1-3rd larvae.In the 4-6th larvae,pre-pupae and adult,the expression was the highest in the fat body,and significantly higher than the epidermis,head,digestive tract and hemolymph,and the expression of the latter four was not significant;among the adults.The expression of 102 gene in flight muscle was significantly lower than that in fat body,but significantly higher than that in other tissues.Therefore,102 gene had the highest expression level in the fat body of 6th instar larvae at high density.The study have confirmed the effects of larval density,E.civilis parasitism,and M.anisopliae infection on the expression of 102 gene.1)The results of determination of the relative expression levels of 102 gene of 4-6th larvae and pre-pupae in fat bodies and other tissues under 1,10,and 20 larvae/jar conditions indicated that in fat bodies,10 larvae/jar,the highest expression level was followed by 20 larvae/jar and 1 larva/jar,the difference between the three densities was significant.Expression levels in other tissues were also highest at 10larvae/jar,but they varied with stages or tissues;2)After being parasitized by E.civilis,the expression level of 102 gene in 5-6th larvae was significantly increased:The expression in the fat body and haemolymph of parasitic larvae increased more than twice as much as in the control;3)The expression level of gene 102 in larvae after infection by M.anisopliae changed significantly:compared with the expression levels in the body of control larvae in the fat,the expression levels were significantly higher in the 10 and 20 larvae/jar conditions than in the control,but 1 larva/jar was lower than the control.These results indicated that the 102gene plays an important role in adapting density change or resisting invasion of the parasite.The study identified the role of 102 gene in larvael immune defense,flight and reproduction of adult.After studying the silencing effect of the RNA interference technique on 102 gene of larva and early adult adult,it was found that the protein content of P102 in the treated larvae was significantly lower than that of the other control groups,and the protein content of P102 was still lower than that of the control group after the induction of M.separata.In addition,the activity of larvae resistant to M.separata was significantly reduce and the mortality of larvae after M.separata infection.2)The activity of phenol oxidase?PO?in the larvae decreased significantly,and the activity of PO was significantly lower than that of the control;3)The distance,time and speed of the treatment of adults were lower than those of the control.The proportion of individuals in the flight was also lower than that of the control.Moreover,RNAi interference had an effect on the early spawning stage of adults,but the effect on spawning duration and egg production was not significant.These results indicate that the 102 gene plays an important role in the maintenance of adult immune defense and flight ability.The results showed that the sequence and structure of 102 gene,as well as spatio-temporal expression,revealed the role of the 102 gene in the immune defense of M.separata,the change of adaptive density and the role of the flight,which laid a solid foundation for the continued study of the immune defense ability of the gene and the control of the migratory flight.In order to reveal M.separata outbreak mechanism and provides a new experimental basis to improve the early warning and prevention and control technology.