Dynamic Changes Of Global Proteins During Ovarian Development In Haemaphysalis Longicornis And Premilinary Study Of Key Regulatory Proteins

Ticks are kinds of extracorporeal parasites which are mainly blood sucking,belonging to Arthropoda,Arachnidae,Acarina,Parasitiformes.A total of 907 species of 20 genera and 3 families have been found worldwide,and 125 species of 10 genera and 2 families have been found and reported in China.The subject of this study is Haemaphysalis longicornis,belonging to Ixodidae and Haemaphysalis,widely distributed in eastern Asia and Australia.This speciesis one of the main tick species in China.It can spread many diseases and do great harm to human beings,livestock and wildlife.It is of great significance for its research.Female ticks have huge blood sucking capacity and strong reproductive capacity,causing great harm to the host.Ovary,as an important part of the reproductive system of female ticks,plays an important role in the reproductive process,so the research on ovarian physiology and development has always been a hot spot.It is very important to study the regulation mechanism of tick ovary development for effectively controlling tick population and reducing tick harm.In this study,female ticks of H.longicornis after engorged were taken as the research object.Quantitative proteomics and phosphorylated proteomics of ovaries of H.longicornis at different stages after engorged were studied in order to find out the key factors controlling ovarian development and lay a foundation for further understanding its molecular regulation mechanism.The experiment is divided into two parts: The first part is to study the dynamic changes of global proteins at five time points during ovarian development of H.longicornis after engored by using data-independent quantitative proteomics technology DIA.Four repetitive experiments were carried out in this study.A total of 1170 proteins were identified.Bioinformatics analysis of 555 proteins with significant differences was carried out.A large number of proteins possibly involved in the degradation of yolk protein and cell proliferation were found.RNA interference experiments were carried out on some proteins.It was found that the two proteins,leucine aminopeptidase and protein kinase B,were interfered and the female was found to be involved in the degradation of yolk protein and cell proliferation.The development of tick ovaries slowed down obviously.Leucine aminopeptidase is one of the key proteins in the process of growth and development,which can regulate the metabolism of amino acids in cells,and it also participates in the synthesis of yolk protein.Because vitellin is synthesized during vitellogenesis,in order to understand the role of leucine aminopeptidase in vitellin synthesis,RNAi was applied to female ticks with engored.When leucine aminopeptidase was knocked down,the ovarian development rate of female ticks slowed down and the number of eggs laid decreased by 30%.Protein kinase B can regulate cell survival,growth,proliferation and other biological processes.In order to understand the role of protein kinase B in the whole ovarian development period of ticks,RNA interference experiments were carried out on unfed female ticks.When the gene was knocked down,the engored weight of female ticks decreased by 60%,ovarian development slowed down significantly and oviposition decreased by 78%.In the second part,phosphorylated proteomics was studied at different developmental stages of ovaries of female ticks with full blood.840 phosphorylated proteins were identified by DIA,involving 454 phosphorylated proteins.Bioinformatics analysis of 404 phosphorylated proteins corresponding to 782 phosphorylated proteins with significant difference was carried out.A variety of proteins regulating growth and development were found.Through a large number of RNA interference experiments,it was found that the protein Yorkie was interfered in unfed female ticks.Engored weight decreased by 70%,ovarian development was significantly inhibited,and spawning decreased sharply.Yorkie is a transcriptional co-activator,which can bind to DNA-binding transcription factors and activate the transcription of downstream target genes.These regulated downstream target genes are mainly involved in cell proliferation,growth and inhibition of apoptosis,thus mediating Yki-induced tissue growth.In conclusion,the dynamic changes of protein and phosphorylation modification during ovarian development of female ticks with engored were systematically studied by using DIA.Through the analysis of quantitative proteomics results,key proteins that can control the development of ticks’ ovaries and the growth and proliferation of ticks’ cells were finally found.The research results fill the gap in this field and provide a theoretical basis for tick control,which has important theoretical and practical significance.