The Function Of BmSTAT Transcription Factor In Antibacterial Immune Response Of Silkworm,Bombyx Mori

Insects are the most diverse and widely distributed animal groups on earth.They live in a complex and changeable environment and cope with the invasion of exogenous diseases such as bacteria,fungi and viruses through innate immunity.Insect innate immunity includes cellular immunity and humoral immunity.Studies have shown that JAK/STAT signaling pathway can participate in humoral immunity of insects by up-regulating the expression of immune effector molecules.JAK/STAT signaling pathway is one of the major intracellular signal transduction pathways,which is involved in many important biological processes,including cell proliferation,differentiation,apoptosis,and immune regulation.In mammals,loss of activity in this pathway leads to increased susceptibility to bacteria.The JAK/STAT signaling pathway has been shown to be involved in the antimicrobial immune response of crustaceans by regulating the expression of antimicrobial peptide genes.In insects,the JAK/STAT pathway has also been shown to be involved in immune responses against bacterial infections in Drosophila melanogaster,Anopheles gambiae,Cotton bollworm,and Acyrthosiphon pisum.Bombyx mori is an important economic insect,but it is vulnerable to diseases caused by pathogens such as bacteria and fungi in the process of large-scale breeding,thus causing serious economic losses to the silkworm industry.It has been found that Escherichia coli,Staphylococcus aureus and Beauveria bassiana infection can activate the JAK/STAT signaling pathway of silkworm,but whether and how the downstream key transcription factor Bm STAT is involved in the antibacterial immune response of silkworm remains to be further explored.In this study,we first investigated the response of key genes of JAK/STAT signaling pathway to bacterial and fungal infections in silkworm in different immune tissues,and then explored the function of transcription factor Bm STAT in antibacterial immune response at cellular and individual levels.The main research results are as follows:1.Immune response of silkworm JAK/STAT signaling pathway to bacteria and fungiThere are two shear forms of Bm STAT in silkworm: Bm STAT-S and Bm STAT-L.The expression of key genes of JAK/STAT signaling pathway in fat bodies,hemocytes and midguts was detected by q RT-PCR at 3,6,12,and 24 h after the injection of Escherichia coli(G-),Staphylococcus aureus(G+)and Saccharomyces cerevisiae(fungus)into the 3 days after 5th instar.The results showed that the expressions of Bm DOME,Bm HOP,Bm STAT-S and Bm STAT-L in the three tissues were significantly up-regulated after bacterial and fungal infection.These results indicated that the JAK/STAT signaling pathway could respond to the immune response process after bacterial and fungal stimulation,in other words,the signaling pathway might be involved in the antibacterial immune response of Bombyx mori.2.Bm STAT was involved in regulating the expression of antibacterial peptides in Bm E cellsAfter stimulation of lipopolysaccharide(LPS)and peptidoglycan(PGN),Bm STAT-S and Bm STAT-L gene transcription levels were detected at 0,6,12,and 24 h.The results showed that Bm STAT-S and Bm STAT-L gene expression levels increased significantly from 6 h.These results indicated that Bm STAT gene was responsive to LPS and PGN.Subcellular localization analysis showed that Bm STAT-S and Bm STAT-L,which were normally overexpressed in Bm E cells,were mainly localized in the cytoplasm.After LPS and PGN stimulation,Bm STAT-S and Bm STAT-L contents in nucleus increased significantly,indicating that LPS and PGN stimulation can promote the translocation of Bm STAT-S and Bm STAT-L from cytoplasm to nucleus,thus activating the Bm STAT transcription factor to regulate the expression of downstream target genes.To further investigate the effect of Bm STAT on antimicrobial peptide genes expression,we overexpressed and interfered with Bm STAT gene expression in cells.The results showed that after the overexpression of Bm STAT-S and Bm STAT-L,the expression levels of antibacterial peptide genes Gloverin 1,Gloverin 3,Gloverin 4,Moricin,Cecropin A,Cecropin B,and Lebcoin were significantly increased.In contrast,the expression of these antimicrobial peptide genes was significantly decreased after Bm STAT interference in cells.The results indicated that transcription factor Bm STAT could directly or indirectly regulate the expression of antimicrobial peptide genes.3.Bm STAT was involved in regulating the expression of antibacterial peptides in silkwormOE-Bm STAT-S and OE-Bm STAT-L Bombyx mori overexpressing Bm STAT were constructed by transgenic technology.The expression levels of Gloverin 1,Gloverin 3,Gloverin 4,Moricin,Cecropin A,Cecropin B,and Lebcoin in fat bodies of OE-Bm STAT-S and OE-Bm STAT-L Bombyx mori were significantly increased.On the contrary,the expression levels of these antibacterial peptide genes were significantly decreased in fat bodies of Bm STAT whole-body transgenic interference Bombyx mori A4-ds Bm STAT constructed by our team.This further proved that the transcription factor Bm STAT could regulate the expression of antibacterial peptide genes.The obtained transgenic silkworm was challenged by bacteria and fungi.After S.aureus infection,survival rate of OE-Bm STAT-S was 83% higher than that of control group;and there was no difference in survival rate of OE-Bm STAT-L,but the death rate was slower;on the contrary,the death rate of A4-ds Bm STAT silkworm was significantly accelerated.After infection with B.bassiana,compared to the control group,the survival rate of OE-Bm STAT-S and OE-Bm STAT-L silkworms increased by 47% and20%,respectively,whereas the survival rate of A4-ds Bm STAT silkworms decreased by17%.These results indicated that the transcription factor Bm STAT could affect the resistance of silkworm to S.aureus and B.bassiana.In summary,it was found that the JAK/STAT signaling pathway could be activated after bacterial and fungal infection.Overexpression of Bm STAT in silkworm cells and individuals could promote the expression of antimicrobial peptide genes and enhance the resistance of silkworm to S.aureus and B.bassiana.On the contrary,interference with Bm STAT could inhibite the expression of antimicrobial peptide genes and weaken the resistance of silkworm to S.aureus and B.bassiana.This study not only clarified the function of transcription factor Bm STAT gene in immune regulation of silkworm,but also obtained silkworm material with strong antibacterial ability.