A Preliminary Study Of Destruxins A-binding Proteins In Bombyx Mori Bm12 Cell Line

Destruxin A(DA), a cyclodepsipeptidic mycotoxin isolated from the entomopathogenic fungus, Metarhizium anisopliae, has bioactivities against insects and virus, etc., but the molecular mechanism of DA affecting target insects is not clear yet. It is necessary for illustrating the molecular mechanism to discovery the DA-binding proteins and to define their molecular compositions, structures and the interactions with other proteins. In the current experiment, taking silkworm cell line Bm12 as the material,protease digestion after DA treatment, gel electrophoresis and mass spectrum technologies were used to isolate and identify DA-binding proteins. Then, homology modeling and melecular docking were employed to evaluate the interaction relations between DA and the proteins. Finally, by means of fluorescence quantitative PCR(q PCR), the silkworm cell line Bm12 was used to survey the expression level of antimicrobial peptides cecropin B and gloverin 4 genes and Bm Rel/Bm Relish genes after the cells treated with DA or silence of transcription factors in TOLL and IMD signal pathways.The main result were shown as follows:(1) Isolation and identification of destruxins A-binding proteins After Bm12 cells were treated with 200 μg/m L DA at 6 h, the total proteins were extracted; then, the proterins were degradated by pronase and proteinase K for 1, 3 and 5min. After treated by proteinase K, the distinct three electrophoretic bands of protein(A,B1, B2) were found in DA treatment, while no difference were observed in pronase treatments and control. Furthermore, the protein bands were identified by means of mass spectrum technology. The results indicated that there were 112 same proteins with the molecular mass of 50-100 KDa in the three distinct protein bands.From the above 112 proteins, 26 different proteins of relatively high score were selected. They were subjected to homology modeling and molecular docking with MOE2014.09 software. The results showed DA had a good binding with the proteins because their combination free energies of were between-9.93 kcal/mol and-5.46 kcal/mol,especially, 7 proteins of them had the free energies <-8.00 kcal/mol. The 26 proteins involved in different functions including regulation of gene expression, molecular chaperone, protein degradation enzymes, protein synthesis, cellular structural proteins, etc.Moreover, 6 kinds of proteins related to gene expression; 5 kinds of proteins related to molecular chaperone; 4 kinds of proteins related to protein degradation enzymes; 3 kinds of proteins related to cellular structural; 2 kinds of proteins related to energy metabolism; 1kind of protein related to immune; 1 kind of protein related to transmembrane transport;and 1 kind of protein was unknown function.(2) Homology modeling and molecular docking of DA-binding proteins(3) Effect of destruxin A on expression of antimicrobial peptide cecropin B and gloverin 4 genes The cecropin B gene was up-regulated and gloverin 4 gene was down-regulated after the DA treatment. When the transcription factors Bm Relish1, Bm Relish2, and Bm Rel were respectively silenced specific si RNA, it was found that cecropin B and gloverin 4 genes were all down-regulated in the Bm Relish2 gene silence treatment but not in Bm Relish1, and Bm Rel treatments. This illustrated that the two antimicrobial peptides were biosynthesized through IMD signal pathway. Furthermore, when the silence of transcription factors Bm Relish1 or Bm Rel genes and DA treatment were combined, cecropin B gene was significantly down-regulated, the results suggested that there were some synergism between DA and transcription factors Bm Relish1 or Bm Rel to inhibit the biosynthesis of cecropin B. Similarly, there were close synergistic relations between DA and transcription factors Bm Relish2 to promote the biosynthesis of gloverin 4.(4) Destruxin A influences gene expression of regulation of silkworm’s Bm Rel/Bm Relish gene The similar phenomena were observed in gene expressions of Bm045 and Bm066, i.e.,down-regulation with the individual Bm Relish1 gene silence or DA treatment, and up-regulation with joint Bm Relish1 gene silence plus DA treatment; while up-regulation with the individual Bm Relish2 gene silence and down-regulation with joint Bm Relish2 gene silence plus DA treatment, but no changes in the Bm Rel gene silence and joint DA treatment. For the Bm133 gene, the down-regulated expressions were found in individual Bm Relish2 gene silence or DA treatment, and up-regulation in the combination treatment of Bm Relish2 gene silence plus DA and the indiviual treatment of Bm Rel or Bm Relish1 silence.It is suggested that expressions of the Bm045, Bm066 and Bm133 genes are closely related to the IMD signal pathway. Meanwhile, there are some synergism between DA and transcription factors Bm Relish1/2 to expressions of the three genes, which imply DA may promote splicing Bm Relish into the Bm Relish2 other than Bm Relish1.