Molecular Characterization Of Bursicon And Laccase1 Genes In Helicoverpa Armigera

Insect cuticle sclerotization is a complex biological process,during which a serial of hormones and associated enzymes take part in.As one of hormone,bursicon consist of two subuits bursicon-?(BURS)and bursicon-?(PBURS).Since it was found in 1962,bursicon was found to regulate cuticle tanning and wing expansion in insect.Beyond this,bursicon was found to form homodimer and corresponding homodimer was identified to take part in insect immunity.Most importantly,? homodimer exert more strengthen effect on immune reaction than that of ? homodimer.Even so,the detail manchanism of ? homodimer role in insect immunity keep still elusive.Laccase are key enzyme during insect cuticle tanning,which include laccase1(LAC1 or MCO1)and laccase2(LAC1 or MCO1).However the fuctions of two enzymes are different,MCO2 directly take part in the process of insect cuticle tanning,the function of MCO1 in insects seem diverse and the detailed role need to be explored.In the present study,the cotton bollworm Helicoverpa armigera was used as a model to study the molecular characteristics of bursicon and laccase1 by using molecular biology and biochemistry methods.The main results are as following:(1)Molecular mechanism of PBURS homodimer in regulating H.armigera immune reactionJAK/STAT signaling pathway is one of major signaling pathways that regulate insect immune reaction.Firstly,JAK2 and STAT5 gene sequences were obtained from transcriptome data of H.armigera.The immune related genes in fat body were found to be up-regulated significantly following PBURS homodimer challenge,which were expressed in mammalian cell.Further study found that the inhibition of JAK/STAT sigaling pathway could weaken the effect of PBURS homodimer on the expression of immune related genes.The results above indicated that PBURS homodimer regulate immune reaction via JAK/STAT signaling pathway.(2)Function of bursicon ? in H.armigera midgutBursicon genes were expressed in the whole tissues detected,which include midgut,malpighian tubules,fat body,epidermis,salivary gland and central nervous system(CNS).Results reveled that two subuit of bursicon have highest expression in CNS compared with that in other tissues.Moreover the expression of PBURS was significantly abundance than that of BURS in CNS,and results are reversed in other tissues.Gene function analysis manifested that RNAi-mediated knochdown of BURS lead to significant increase in cell proliferation related genes in midgut.These results demonstrated that bursicon probably take part in regulating cell proliferation process beyond regulating cuticle tanning,wing expansion and immune reaction.(3)Functional analysis of MCO1 gene in H.armigeraMCO1 gene sequence was obtained from transcriptome data of H.armigera.Sequence analysis revealed that MCO1 contains an open reading frame(ORF)of 2,430 bp,which encodes a putative protein of 810 amino acid residues,with a molecular weight with 91.998 k Da.The developmental expression pattern of MCO1 indicated that MCO1 was much more abundant in the molting stages of fourth-and fifth-instar larvae than in other stages.Hormone treatment suggested 20 E significantly inhibited MCO1 expression.Further knockdown of the recptor genes of 20 E,ECR and USP by RNAi and subsequent 20 E treatment confirmed that 20 E inhibited MCO1 expression via its heterodimer receptor.MCO1 tissue distribution revealed that MCO1 are most abundantly expressed in malpighian tubules in all the tissues tested.Further study of RNAi koockdown of MCO1 showed that MCO1 regulate iron metabolism in H.armigera.