Screening for Bombyx mori nonsusceptibility to DNV-2(nsd-2) gene through cDNA subtraction.

抄録

In Bombyx mori nonsusceptibility to B. mori densonucleosis virus type 2 (BmDNV-2) is controlled by a recessive nonsusceptibility gene, nsd-2. In susceptible insects, BmDNV-2 specifically colonizes the columnar cells of the larval midgut epithelium. We have mapped nds-2 on linkage group 17 by RFLP. Differential expression of genes within the midgut of susceptible (C124; +^nsd-2/+^nsd-2) and nonsusceptible (J124; nsd-2/nsd-2) strains to DNV-2 was evaluated through cDNA subtraction using the strains C124 and J124 as tester and driver (control), respectively. Since nsd-2 is a recessive gene, the dominant allele, +^nsd-2 will cause susceptibility and contain specific transcripts. cDNA was synthesized from mRNA of the midgut of these strains and the subtracted cDNA library was constructed in pTAdv vector (Clontech). Two percent of the clones which were differentially expressed hybridized to both the subtracted and unsubtracted tester probes. On the other hand, 12% of the clones hybridized only to the subtracted probes indicating differentially expressed transcripts of low abundance. Inserts from the two categories of clones were amplified by PCR and used to screen the midgut cDNA library of C124 to get the full-length clones. Positive clones obtained were used as probes in Southern blot analysis of genomic DNA digests derived from BF₁ larvae inoculated with DNV-2. Linkage analysis of these clones and possible facilitation of map based cloning of nsd-2 gene will be discussed.