| Insect lack acquired immune system processed by higher animal, so they developed a complicated innate immune system. The special system is different from vertebrate in the physiologcal and pathologcal processes against the invaded pathogenic microorganism. Therefore, the molecular study of innate immunity not only useful in understanding the physiologcal and pathologcai process, as well as beneficial to comprehend the host and pathogen interaction. Locust is the representative of Orthoptera and major agricultural pest with a strong denfense system, so the study of locust innate immunity also profitable to optimize the strategy of pest control.Fungi are major pathogenic microorganism of insects, play an important role in the regulation of insect populations. Insect pathogenic fungi as insecticides has broad application prospects, for example, Metarhizium acridum has been widely used for locust control. But the immune response is an important factor affecting the insecticidal rate. Consequently,the studies about the immune system of locust are increased, which made us understanding the locust immune system and the differences of locust different immune tissues against the invading pathogens. However, it lack the characteristic study about the immune response in different stages of infection process that the insect pathogenic fungi infecting the locust. Therefore, it is essential to build the immune response differentially expressed genes library of different immune tissues, which is in the different infection stages of pathogenic fungi invading the locust. It can help us understand the dynamic changes in locust immune process and comprehended the mechanism of pest against pathogenic fungi. Besides, it have important meanings in highly virulent strains bred.In this study, the deep sequencing and transcriptome analysis technology has been applied to generate the differentially expressed genes and obtained the immunity-related genes of fat body and hemocyte in different infection stage. Validate the up-regulated genes with immunity at prepenetration stage via RT-q PCR. Through the knockdown of RNAi, we confirmed the mechanism of locust in sensing the pathogenic fungi at prepenetration stage.The main results were as follow:(1) Through the Illumina Hiseq2000, we establish the high quality representative database of locust immune tissues. 128988 Unigenes were obtained with an average length 920 bp, among them, 33396 Unigenes were longer than 1000 bp.(2) The differentially expressed genes of two immune tissues during locust defense Metarhizium acridum were identified. After Metarhizium acridum challenge, at the different time point of infection process, the up-regulated genes of fat body were 722?1103?901?1476?5123 and 1251, the down-regulated genes were 4519?5077?9020?10299?4879 and 8870. Correspondingly, the up-regulated gene of hemocyte were 193?1616?1031?1593?1406 and 2234, the down-regulated gene were 134?155?7248?4207?5420 and 4457. At prepenetration(4 h and 12 h) stage, many gene were differentially expressed in fat body and hemocyte; At the penetrating stage(24 h and 36 h), the numbers of DEGs in fat body and hemocyte were biggest, so the immune responses was strongest; At the colonization stage(48 h and 72 h), the total numbers of DEGs in two immune tissues were stable, but the down-regulated genes were increased.At the early stage of infection process, the DEGs of two immune tissues main refer to energy metabolism, biosynthesis, sensing, signal transduction, environmental adaptation and some immunity-related protein. At the penetrating stage, the primary genes of early stage were increased. Besides, some other GEGs involved in cell migration and aggregation, cell phagocytes and cell cycle. At the colonization, there is no big change for those genes that differentially expressed at penetrating, but the genes of cell apoptosis and anti-microbial protein were increased. Certainly, many unknown annotation genes or novel proteins not previously linked to immune response were obtained.(3) Through tblastn with the immune related protein database, the immunityrelated genes of two immune tissues have been identified. At the different time points of infection process, the immunity-related genes of fat body were 61?98,153?171?106 and 115, and the hemocyte immunity-related genes were 13?40?80?81?95 and 120. For fat body, at the early stage, the major immunity-related genes refer to immune recognition and immune effectors. At the penetrating stage, these two types genes were increased and many signal transduction genes begin differentially expressed. At the colonization, the immune recognition and signal transduction gene were decreased and the effector genes were not change. Specially, the gene related to the anti-microbial protein have been differentially expressed. For hemocyte, only few immunity-related genes differentially expressed at the early stage. These genes main included immune recognition and immune effectors. At the penetrating stage, the immune related genes were dramatic increased, the main types were immune recognition, signal transduction and immune effectors. At the colonization, the immune recognition and transduction genes have not significant change, but the effector gene were increased, especially related to the anti-microbial protein.(4) Through the RT-q PCR, we validate the expression level of 57 immune related genes(include the serine protease genes and PPO genes) in fat body and hemocyte, only two genes were found to be inconsistent with the respect to expression between the transcriptomic and q-RT-PCR data, it certificate the reliability of the transcriptomic data.(5) Through RNAi knockdown of the serine protease related to the prophenolo xidase system, at the early stage, the expression level of PPO were significant decreased. That illustrate the locust already initiated the immune response indeed, Potentially, the PPO system play an important role in early response against the invaded pathogen fungi.Through this study, the gene expression characteristics of locust against the Metarhizium acridum were revealed. The defense strategy of locust was changed over time. The fat body and hemocyte not only have synergy, and have different functions respectively. For locust sensing the pathogenic fungi at the prepenetration stage, the PPO system play an crucial role in this process. |
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