Interrelation Between Hemolin And Phenoloxidase In The Immune Process Of Bombyx Mori

Two important processes in insect development and innate immunity have been shown to use many of the same or similar molecular and signaling pathways,and Hemolin protein and phenoloxidase(PO)of silkworm are bifunctional molecules belong to this class during immunization and development.Both of which already have many reports,butresearch revealed that the relationship between this two proteins is minimal.Both proteins can be stimulated by pathogens,induced by ecdysone,adhered to the cell surface and play a role,and are closely related to liposome particles.After silencing the hemolin gene by RNA interference,the activity of phenoloxidase is reduced,suggesting that Hemolin may play an important role in triggering the activation of phenoloxidase.So what's kind of the relationship between the Hemolin protein and the phenoloxidase?We first prepared antibodies of the two proteins,and the antibodies prepared were used to study the tissue localization and expression of the silkworm after induced by pathogen.And then according to the gene sequence we design fluorescent qRT-PCR primers to study the transcription of these genes after induced by pathogen.In addition,we also preliminarily studied the polymorphism of phenoloxidase 1(PO1)sequence and the protein modification of Hemolin protein in different cases.The main results are as follows:1.Recombinant plasmids were constructed using the two cloned subtype genes po1 and po2 of po,and the two proteins were expressed respectively,then immunized New Zealand white rabbits to prepared polyclonal antibodies of PO1 and PO2.2.The co-localization of PO and Hemolin was studied by double immunohistochemical method using paraffin section of silkworm midgutand hemolymph smear after infection.PO2 is distributed throughout the midgut tissue,and Hemolin binds to the blood cells in the haemolymph tissue,and PO1 and PO2 are distributed throughout the haemolymph tissue.3.The transcription of three genes under different conditions was studied byfluorescence qRT-PCR using the cDNA of each tissue in different infection periods as templates.The results showed that the transcription level of po1,po2 and hemolin increased whether it is by feeding infection or injection infection,but the degree of up-regulation and the time of up-regulation were different,and the whole showed a trend of increase first and then decrease.4.The protein expression of three proteins in different conditions was studied by Western blot.The results showed that PO1 was mainly expressed in hemolymph tissue,and there was almost no expression in the midgut,and less expression in the fat body.Expression of hemolymph increased mainly after 32-48 h of infection,and then gradually decreased.PO2 was less expressed in the midgut and expressed in the fat body.Hemolin protein was expressed in all three tissues.The injection group had a longer duration than the feeding group.5.The diversity of po1 gene in different strains of silkworm was studied.It was found that the sequence of po1 gene was different in different strains of silkworm,and the conservability was not as good as po2 gene.We also received Hemolin protein in different states to study its glycosylation and phosphorylation modification.From the above studies we concluded that PO protein and Hemolin protein in the immune process to work together to resist the infection of pathogens.First of all,PO1,PO2 and Hemolin protein are located in the surrounding of aggregated blood cells after bacterial infection.Secondly,the time of high expression of Hemolin after infection was earlier than that of PO.These conclusions and our proposed assumptions are consistent.