The Regulative Functions Of Serine Protease Gene On Trade-offs Between Reproduction And Immune Responses In Locusta Migratoria Manilensis

The locusta migratoria manilensis is an important agricultural pest in China,which is distributed widely and brings too much damage.In this study,Locusta migratoria manilensis(Meyen)was selected as research object.Four pieces of differential expressed serine protease genes in L.migratoria after parasitized by flesh fly Blaesoxipha lapidosa were screened using the deep sequencing and transcriptome analysis technology and biological information was further analyzed.In different development stages of female adult L.migratoria,immune challenges were simulated by injecting Sephadex G-50 or infecting Micrococcus luteus at different concentration.Relative expressional levels of serine protease genes mentioned above were determined by fluorescence quantitative PCR.Combining the expressional level of genes related to reproduction such as vitellogenins as well as immune-related factors such as phenoloxidase activity,the roles of serine protease genes in the reproduction and immunization of L.migratoria manilensis were revealed.The main conclusions of this research are showed as followed.I)The differentially expressed genes during locust defense the parasite of Blaesoxipha lapidosa were identified.There were four differentially expressed serine protease genes,CL7471,CL12613,Unigene20743 and Unigene24864 which involved in immune defense.The expression of CL7471,CL12613,Unigene20743 and Unigene24864 were screened by EDG.The full-length cDNA sequences of CL7471,CL12613,Unigene20743 and Unigene24864 genes respectively were 2209,1861,692 and 1627bp.The open reading frames were 1374,1065,363 and 1206bp,which encoded 468,354,120 and 401 amino acids respectively.The molecular weights were 48.89kDa,37.22kDa,12.98kDa and 42.03 kDa,respectively.The CL7471,CL12613 and Unigene 24864 genes have a trypsin domain,which belongs to the family of trypsin.Unigene20743 may be associated with an insulin-growing factor that contains an insulin-growing factor domain.2)Real-time quantitative PCR was used to determine the expression of four serine protease genes in female adults of L.migratoria invade by parasite(simulated using injection of Sephadex G-50)at different developmental stages.The results showed that the expression of serine protease CL7471 was up-regulated at the maturation stage,pre-mating stage and mating stage,and the higher the injection concentration,the effect is more significant.The expression of Unigene20743 gene appeared down-regulated after invaded by the dextran beads.The expression of two genes,CL12613 and Unigene24864,was affected slightly by invaded dextran beads.The expression of Vg and its receptor gene VgR,which is involved in reproduction,was down-regulated by Sephadex G-50 at the time of maturation and pre-mating stage,but just has little effect at mating stage.The results showed that the two serine protease genes of CL7471 and Unigene20743 could enhance the immunity by down-regulating the synthesis and transport of yolk protein in order to response to the invasion of dextran microbeads,which means the two serine protease genes of CL7471 and Unigene20743 could involve in trade-off between reproductive and immunity in female adults of L.migratoria manilensis.3)The expression of serine protease gene CL7471 was up-regulated at the maturation stage and pre-mating stage,but was inhibited during the mating stage when the female adults infected with pathogens(infection of micrococcus luteus).The expression of Unigene20743 gene in the fat body was down-regulated at the maturation stage,whereas down-regulated injected with low concentration and up-and down-regulated injected with higher concentration,when the infection was happened at the pre-mating and mating stage.The expression of Unigene20743 gene in the ovary was down-regulated at all three stages when infected by micrococcus luteus.There was no obvious effect of infection of micrococcus luteus on the expression of CL12613 and Unigene24864 gene.The expression of Vg and VgR were down-regulated when infected by micrococcus luteus,and the higher the concentration was,the stronger the effect.Infected by the micrococcus luteus can also induce the immune activity(activity of lysozyme and phenoloxidase)of locust.These results also show that CL7471 and Unigene20743 may be involved in the regulation of reproduction and immunity in female adults of L.migratoria manilensis.