Fluorescent differential display analysis of gene expression for NPV resistance in Bombyx mori L.

Abstract

Using the fluorescent differential display (FDD) technique, we analysed the differential expression of genes related to BmNPV (Bombyx mori nuclear polyhedrosis virus) resistance. Silkworm strains used included a highly resistant strain named NB, a highly susceptible strain named 306 and a near-isogenic line named 306NNZZ. Two differential bands, G12 532 and G13 313 , were found linked to BmNPV resistance and were further identified. Band G12 532 was found in the midguts of the eighth generation of backcross (BC 8 ) of strain 306NNZZ but was not present in those of strain 306. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirmed that the band existed in BC 8 of strains 306NNZZ and NB while not in strain 306. Northern blot analysis showed that G12 532 expressed actively in BC 8 of strains 306NNZZ and NB while expressed inactively in strain 306. These results suggest that the G12 532 is a fragment of the gene related to silkworm's resistance to BmNPV disease. The sequence of G12 532 has an 85% similarity to sequences with GenBank accession numbers of AV398077 and AV398034 that are relative to BmNPV infection demonstrated by previous studies. Its coding region (37 amino acids) has 54% similarity with B. mori endonuclease and reverse transcriptase-like protein. Band G13 313 was appeared in the hemolymph of highly resistant strain NB and near isogenic line 306NNZZ in FDD, but was not found in highly susceptible strain 306, all of which were confirmed by RT-PCR and Northern blot analyses. All the experimental results obtained suggest that G13 313 is also a fragment of the gene related to BmNPV resistance. The sequence of band G13 313 does not have a high similarity to the above relative sequences in GenBank. It might be a new RNA fragment related to NPV resistance. Our study also showed that the FDD technique could be a promising approach in identifying novel genes while appropriate silkworm strains are used.