Selection Of Reference Genes For Rt-qPCR,Genomic And Proteomic Analysis Of Heliothis Virescens Ascovirus

Ascoviruses are double-stranded DNA viruses that mainly infect Lepidoptera larvae of Spodoptera exigua,Spodoptera litura and Helicoverpa armigera.A chronic but ultimately fatal disease was the obvious symptom infected by ascoviruses.In nature,ascoviruses are transmitted mainly through the oviposition behavior of parasitic wasps.Ascoviruses have good potential application value in the field of biological control of important pests,such as S.exigua larvae,but there were relative lack of basic research in the molecular mechanism of the pathogenesis of ascoviruses.This study focuses on Heliothis virescens ascovirus 3h?HvAV-3h?,a case for the investigation into the selection of reference gene in the cassettes of the“Ascovirus–Microplitis similis Lyle–Spodoptera exigua”dissemination system,and Heliothis virescens ascovirus 3i?HvAV-3i?,a case for the comprehensive investigation into the genome and proteomic analysis with other reported.The major results are shown as follows.1.Selection of reference genes for expression analysis using RT-qPCR in the dissemination system of ascovirusSeries of 10-fold dilution(10⁰-,10²-,10⁴-,10⁶-,10⁸-and 10¹⁰-fold)of HvAV-3h-containing hemolymph?at a titer of 5.6×10⁸ pfu/ml?were used as inoculations applied to S.exigua larvae,and were harvested at points-in-time of experimental treatments.And 11candidates reference genes were evaluated using the geNorm,NormFinder,BestKeeper,RefFidner and Delta Ct methods,including?-actin1?ACT1?,?-actin2?ACT2?,elongation factor 1?EF1?,elongation factor 2?EF2?,ribosomal protein L10?L10?,ribosomal protein L17A?L17A?,superoxide dismutase?SOD?,28S ribosome?28S?,Tubulin?TUB?,18S ribosome?18S?,peptidyl prolyl isomerase A?PPI?,TATA-binding protein?TBP?,glyceraldehyde-3-phosphate dehydrogenase?GAPDH?and ribosomal protein L35?L35?.The results showed that EF1 was the most stable reference gene in S.exigua and M.similis infected-ascovirus;The elongation factor 2?EF2?and 28S ribosomal RNA?28S?was the the most stable reference gene in the IOZCAS-Spex-II-A cell line and Sf9 cell line,respectively.And the stability of reference genes were confirmed via the expression levels of two inhibitor of apoptosis-like?iap-like?genes from HvAV-3h.2.Genome analysis of ascovirusAs a novel ascovirus isolate of the species Heliothis virescens ascovirus?HvAV?,HvAV-3i genome size is 185,650 bp with a G+C content of 45.4%and predicted 181hypothetical open reading frames?ORFs?,which include 4 unique genes,25 baculovirus repeat ORFs?bros?,5 microsatellites and no homologous repeat regions?hrs?.Here,the definition of ascovirus repeated ORFs?aros?with 3 aro genes detected in the HvAV-3i genome were proposed for the first time.Further,the aros based on the unrooted phylogenetic trees were classified,in which 29 aros from the all sequenced genomes of Ascoviridae were clearly divided into 6 groups.Additionally based on the conjoint analysis of common genes in ascoviruses,the phylogenetic tree clarified that the phylogenetic relationship within the series isolates of the HvAV variants is?HvAV-3f,?HvAV-3h,?HvAV-3e,?HvAV-3g,HvAV-3i????with well supported by Bayesian posterior probability of 1.00 and Bootstrap value of 100%.3.Proteomic analysis of ascovirusIn this chapter,the virions of a sequenced HvAV-3i strain was used to perform proteomic analysis based on both in-gel and in-solution digestion.A total of 81 viral proteins were identified in HvAV-3i virions.Among the proteins,35 viral proteins with annotated function were identified to be involved in the DNA/RNA metabolism/transcription,virion assembly,sugar and lipid metabolism,signaling,cellular homoeostasis and cell lysis.20viral proteins were identified to contain transmembrane domains.This study would help us to understand the virions structure of ascovirus,and provide the predicted functional domains of viral and host proteins for further investigation the function in the ascovirus infection cycles.