Preliminary Research On Non-susceptible Factor Of Silkworm To B.mori Parvo-like Virus (China Isolate)

Bombyx mori parvo-like Virus China isolate(BmPLV-Z) which belongs to BmDNV-â…¡before,is one of the most disastrous viruses in silk cocoon production.Non-susceptibility to DNV-Z was found under the control of a recessive gene(nsd-Z) that was located on linkage 15.The silkworm non-susceptibility to DNV-2 was found under the control of a recessive gene(nsd-2).The nsd-2 gene was well mapped with flanking DNA markers and is located on linkage 17.However,its mechanism is still unknown.In 2008,a gene of silkworm,which is susceptible to the parvo-like virus BmDNV-2,has been certificated by gene +^nsd-2 encoding a 12-pass transmembrane protein with positional cloning.The researchers have determined that the candidate gene nsd-2 has a great significance for the invasion of the virus by transgenes.BmPLV-Z,isolated in Zhenjiang of China,has a high homology with BmDNV-2 in serological characteristics and genome structure.Thus,it is tempting to speculate that silkworm may employ similar strategy with nsd-2 and +^nsd-2 gene to defend themselves against BmPLV-Z.In present study,we performed PCR and RT-PCR analysis detected the nsd-2 and +^nsd-2 gene in most of the tested silkworm strains'genomes and cDNAs.Meanwhile,the candidate gene nsd-2 and +^nsd-2 was expressed by E. coli Rosetta and the baculovirus expression system.1.To detect nsd-2 and +^nsd-2 in the genome and cDNAsWe use three pairs of primers D1,D2,D3,to detect the different varieties of nsd-2 in silkworm genomes and cDNAs.The +^nsd-2 gene could be detected from the genome and the cDNA pool from BmDNV-Z susceptible silkworm strains JingSong,HuaBa35 and 306,as well as the C terminus truncated nsd-2 gene from the BmDNV-Z non-susceptible silkworm strains QiuFeng and BC8.Interestingly,all of our PCR products are smaller than those described by Kidokoro,being about 300 bp less in size,indicating a 300 bp deletion in the thirteenth intron between D3-F and D3-R.There is no different nucleotide in cDNAs.The transcription pattern in different tissues and different developmental stages of silkworm HuaBa35 were analysised by RT-PCR. Our results revealed that +^nsd-2 gene was only expressed in larval midgut.It is not expressed in the stage of egg and pupa,but only expressed in the larva stage.2.nsd-2'and +^nsd2' gene cloning and functional domain analysisThe nsd-2'and +^nsd-2' gene were cloned and sequenced from silkworm strain which is resistant or susceptible to BmPLV-Z.The results showed that there is no difference in the nucleotide sequence between two fragments in cDNAs.So we suspect that the two viruses have the same resistance mechanism.Bioinformatic study showed that +^nsd-2 is a putative amino acid transporter with three glycosylation sites and located on chromosome 11.N-glycosylated and O-glycosylated residues were also predicted,which is at 155,178,520 site of the protein respectively.3.Prokaryotic expression of nsd-2The nsd-2 gene was cloned from silkworm strain Qiufeng according the sequence of nsd-2 in NCBI.The protein NSD-2 was expressed in pET-30a vector with expected molecular mass of 27 kDa,which was further confirmed by Western blot.4.+^nsd-2 gene expressed by eukaryotic system+^nsd-2 fragment was inserted into pFastBacHTb,within E.coli DH10Bac,the +^nsd-2 gene was transposed into the Bacmid.The recombinant Bacmid DNA was isolated,purified and transfected into Sf9 cells to yield the recombinant AcNPV carrying the +^nsd-2 gene.Cells were harvested 72 h post-infection,then scraped into the medium.The His-+^nsd-2 fusion protein examed by Western blot.