Studies On The Molecular Marker And The Gene Related To The Resistance To Bombyx Mori Densovirus-Z

Silkworm, Bombyx mori, is an important economic insect and the model insect of Lepidoptera. Bombyx mori densovirus is a kind of harmful virus that may lead to serious loss in sericicultural industry. Three densovirus have been isolated from Bombyx mori. BmDNV-Z is very similar to BmDNV-2, but is different in serological characteristics. A recessive gene nsd-1 and a dominant gene Nid-1 control the resistance to BmDNV-1 of Bombyx mori. The resistance to BmDNV-Z is controlled by another recessive gene nsd-z that is located on the 15^th chromosome. In our study, RAPD technique was used for screening molecular markers linked to the resistance gene. The molecular marker we obtained was converted to SCAR that may lay a foundation to molecular marker assisted breeding. Fluorescent differential display technique was also applied to identify genes linked to silkworm resistance to DNV disease on the isogenic line. The research process and the main conclusions are presented as the following:1. Two silkworm strains, namely QIUFENG that is resistant to DNV and HUABA35 that is highly susceptible to BmDNV-Z and their isogenic line BC₆,were used. 495 RAPD random primers were used to screen molecular markers in DNA mixtures of these strains respectively. One molecular marker named S366 was found linked to major gene resistant to DNV. We cloned and sequenced the molecular marker. According to the sequence, special primer was synthesized and tested in several silkworm strains that are highly susceptible or resistant to DNV. With bioinformatic analysis, we found S366 has 91% homology to xanthine dehydrogenase gene. Xanthine dehydrogenase playes an important role in the innate immune system, which means that xanthine dehydrogenase gene may have some relation to the resistance to BmDNV-Z.2. Near isogenic line BC₆ was set between the highly susceptible silkworm strain HUABA35 and the highly resistant silkworm strain QIUFENG. FDD RT-PCR was carried out and a special fragment was identified. The result of FDD RT-PCR was confirmed by real-time PCR. After 5'-RACE, a full-length of 1096bp cDNA was obtained and resorted to bioinformatic analysis. Bioinformatic analysis suggests that it was a protein kinase C inhibitor (PKCI) gene. This gene was the first case of the PKCI reported in Bombyx mori. PKCI can induce cell apoptosis through different pathways. A model is presented suggesting that PKCI may be involved in the resistance toBmDNV-Z in silkworm, Bombyx mori.3. In our FDD RT-PCR research we found a cDNA fragment from Bombyx mori which had high homology to Bombyx mori EST. Special primer was designed for 5'-RACE. An 814bp cDNA clone containing a 675bp open reading frame (ORF) was identified. This gene encodes 225 amino acids and contains a conserved motif from Drosophila and Anopheles gambiae. Bioinformatic analysis suggests that its deduced amino sequence has 82.6% homology to the Tpn I (Troponin I) gene of Anopheles gambiae and 84% to Drosophila, while the similarity to mammal such as Rattus norvegicus and Homo sapiens was only 23.5%.