Studies On Differentially Expressed Genes In The Silkworm Infected With Beauveria Bassian

Fungicidal disease is one of the major contagions in Bombyx mori. Beauveria bassian isone of the major fungi pathogens for the silkworm, causes enormous damages to thesericultural industry. In this research, the molecular mechanism of silkworm-fungiinteractions was studied. The results would be useful in understanding of the host responseto Bombyx mori B. bassian infection, the diagnosis of B. bassian-caused silkworm diseaseand the development of new methods for controlling the agricultural and forestry pests.For better understanding of the molecular regulation of immune response and theinteractive mechanism between the silkworm and B. bassiana, suppression subtractivehybridization was employed to identify differentially expressed genes in thepathogen-stimulated silkworm larvae. Complementary DNA libraries were constructed, inwhich240clones were sequenced. Some genes were found to be involved in the infectionprocess, among which56were known genes and32were novel. Expression profiling for theperiods post-infection of6h,8h,10h,12h,15h,18h,24h in6genes (B. mori heat shockprotein70, B. mori cyclophilin-like protein, B. mori translationally controlled tumor protein,B. mori Alcohol dehydrogenase, B. mori Catalase, B. mori ribosome-associated membraneprotein4) by real-time quantity PCR showed that they were induced by fungal challenge.This study establishes the first step to understanding the molecular mechanisms by whichsilkworm responds to fungal infection.Variations of gene expression in the silkworm following injection of conidia of B.bassiana were investigated by using the suppression subtractive hybridization method, andthe individuals which were injected with distilled water were used as control. Two cDNAlibraries were constructed and a total of140cDNA clones were isolated. Total55differentially expressed genes were identified, of which48(112clones) in the forwardlibrary and5(28clones) in the reverse library. The frequencies of genes for proteinsynthesis, tansportor, storage in this library were higher than those of dip-treated library.The genes which were coresponding with immunity were up-regulated in both libraries, butthere were some differences in category. There were heat shock proteins,Antibacterialpeptide B precursor and Catalases in both libraries, and Ubiquitin, Ribosomal protein L10A,Alcohol dehydrogenase, Cytochrome b only existed in the dipped-library, and Lysozyme，Moricin, Gloverin4, Rubisco activating enzyme only existed in the injected-library. Expression profiling for the periods post-infection of3h,6h,9h,12h,24h of the hemolymphin6genes (B. mori Chemosensory protein11, B. mori Muscle LIM protein isoform1, B. moriTransferrin, B. mori Sex-specific storage-protein SP1, B. mori arylphorin, B. mori Low molecularlipoprotein30K pBmHPC) by real-time quantity PCR showed that they may be induced byfungal challenge.In silkworm, the metabolism will be disordered after it was infected by B. bassiana.Some genes which function were concerned with protein synthesis, transportor and storagewould be up-regulated to adapt to the replication requirements of the fungi. In addition, thesilkworm would express not only the natural immune response genes to defense the invasionof B. bassiana, which caused the immune related gene expression level increased; but alsoincreased the expression level of apoptosis-related gene to initiate apoptosis mechanism toreduce infected cells.The full length gene of B. mori zinc finger protein1(BmZF1) and high mobility groupbox protein (BmHMBG) were cloned from the dipped-library by using RACE methods.The full length of BmZF1cDNA is1850bp, wherein the5’ untranslated region is195bp,and3’ untranslated region of464bp, open reading frame is1191bp, encoding396aminoacids. In the polypeptide chain, the ZnF-C2H2domains existed at the regions of4-28,66-90,172-195,223-250amino acids residues contain, which means the protein belongs to theC2H2type zinc finger protein family. The full length cDNA of BmHMBG gene is3015bp,and313bp is5’ untranslated region and3’ untranslated region is2114bp,588bp openreading frame, encoding195amino acids. This is a hydrophilic protein, there is aHMGB-UBF_HMG-box domain (107-173aa) in the polypeptide chain. It is a DNA bindingprotein, which belongs to HMG-Box superfamily.BmZF1and BmHMBG were both expressed in silkworm fat body, hemolymph,epidermis, middle silk gland, posterior silk gland, midgut, malpighian tubules, gonad et al.RT-qPCR results showed that BmZFP1were up-regulated obviously in the infected tissues.These results suggested that as a universal transcription factors, it caused the expressionlevel of other proteins in the infected tissue, so that the host cannot use the apoptosispathway to kill the infected cells. The infected cell number gradually increased, and thenumber of fungal spores also gradually increased. RT-qPCR results of BmHMGB showedthat its expression level in the infected tissues were significantly higher than those of normaltissues. For example, after the tissues were infected for24h, the expression quantity in postsilk glands was5.98times as controll, and7.31times in the middle silk gland,10.31times in midgut,4.56times,4.20times in malpighian tubules,6.11times in fat body,8.34times inepidermis,21.71times in haemolymph respectively. Suggesting that the host’s innateimmune response is activated by B. bassiana infection, the immune function cells secretedBmHMGB, the infected and apoptosis cells also passive released this protein. Finally,BmHMGB was up-regulated to a high level, coordinating with other factors led to thedysfunction of the silkworm, and ultimately died.This research would elucidate the initial molecular response for silkworm against B.bassiana infection, and provide a new perspective and new basis for the nosogenesismechanism for B. bassiana in silkworm. In the future, we will focus on the research of therelationship among B. bassiana and the immune proteins such as B. mori zinc finger proteinand BmHMGB.