Role Of Noduler Gene From Spodoptera Litura

Noduler gene was first discovered and named in Antheraea mylitta.A Study show that Noduler gene control the formation of the nodules.A study about the Bombyx mori also found that the gene involved in silkworm phenol oxidase activation reaction.In this study we cloned and identified Noduler gene sequences from the Spodoptera litura.In vitro we prokaryotic express and purify Noduler recombinant protein and obtain the multi-clonal antibody against Noduler,then do a series of immune related experiments.RNA interference assays were used to study the immune function in vivo about the Noduler gene.The results are as follows1.cloning and multiple sequence alignment of Noduler:the length of Noduler cDNA sequence is 501 bp,contained an ORF of 501 bp.It encoded a protein with a 166-amino acid.Software prediction show that the protein isoelectric point is 9.33,and the molecular weight is 17.35 kDa.After multiple sequence alignment analysis,the result shows that the amino acid sequence of Noduler had striking similarities with the Noduler sequences from other lepidoptera insects.2.Tissue specificity analysis:Noduler was expressed in hemocytes,fat bodies,midguts,epidermis and malpighian tubule.from the picture we can conclude that Noduler was expressed at a higher level in midguts.The highest Quantity expressed is in the hemocytes.3.The result of qRT-PCR and western blot show that:After stimulation with Escherichia coli and Staphylococcus aureus,The expression of Noduler Gene increases significantly in hemocytes and fat bodies at different time points.The results of western blot test show that the amount of Noduler protein expression in the plasma were significantly raised.These results show that Noduler gene is likely plays an important role in innate immune response of the Spodoptera litura..4.The function experiments of Noduler gene in vitro show that:(1)rNoduler can be combined with Escherichia coli and Staphylococcus aureus.(2)rNoduler can enhance the prophenoloxidase activation function.5.The results of RNAi assays show that:When the Noduler gene was knocked down off in the Spodoptera litura after injected double-stranded RNA:(1)phenoloxidase activity in plasma was significantly decreased,proved that the genes involved in cascade phenol oxidase reaction;(2)After injection of Staphylococcus aureus,the number of the formed nodules in the control group are significantly higher than the experimental group,proved that the genes involved in nodulation;(3)After injection of Staphylococcus aureus,in the experimental group,the number of staphylococcus aureus is higher than the control group,proved that after interference the haemolymph's abilityto remove bacteria is decreased.The results of function experiments in vitro and RNA interference experiment show that Noduler gene involved in innate immune response in the Spodoptera litura.When the pathogen invasion of host,Noduler gene can enhance cascade phenol oxidase reaction and participate in nodulation to achieve the purpose of clearing pathogenic content.