The Study Of The Transcript Level Of Six Genes Of Bombyx Mori Related To Anti-Virus After Bombyx Mori Infected With BmNPV And BmBDV

Silkworm in China has a long history of cultivation, it not only has high economic value, also can be used as a good model organism and has high research value. But in the process of breeding of Bombyx mori, Bombyx mori are easily affected by many factors that cause the loss. In our country, the viral disease caused by Bombyx mori Nuclear Polyhedrosis Virus(Bm NPV) accounts for a large part of the whole virus diseases and can cause great harm to Bombyx mori. Bombyx mori bidensovirus(BmBDV) is the first to be found in the binary DNA virus family members and the typical virus in this family.In this study, we reported the changes of transcription level of several immune genes, including bmi, argo, dicer, cap1, cap3 and car, in Bombyx mori midgut after exposure to BmBDV or BmNPV. Silkworm strains 798(anti-BmBDV) and 306(susceptible to BmBDV) were subjected to BmBDV infection, and NB(anti-BmNPV)and HUABA(35)(susceptible to BmNPV) were subjected to BmNPV infection. The results showed that the transcript levels differ largely among different silkworm strains, and that the extent to which the gene transcriptions were affected by the viruses was different. However, both BmNPV and BmBDV viruses can reverse the transcription patterns of these genes when the silkworms were administered with the viruses compared with those control groups. The transcript levels of bmi and dicer were decreased in 798 and 306 strains that were inoculated with BmBDV compared with their respective controls, but were increased in NB and HUABA(35) inoculated with BmNPV. The transcript levels of argo and cap3 were risen in 798, 306 and NB strains when inoculated with their respective viruses, but were decreased in HUABA(35) strain. The transcript levels of cap1 were risen in all silkworm strains, while the levels of car were decreased in 798, 306 and HUABA(35) strains, and increased in NB strain when inoculated with their respective viruses. These findings may contribute to more in-depth understanding on functions of these genes in virus infection and proliferation.The further research of the car gene has been carried on in this paper, this gene belongs to carboxylesterase family. Using PCR technology to clone the car gene with the template of the cDNA of Bombyx mori. The fragments of car gene were connected to the cloning plasmids for amplification. The fragment of car gene after amplification were connected to the expression plasmids, and then transfected them into BL21. Using IPTG to induce the expression of protein. Main target proteins existed in inclusion body. Using Ni-NTA to purify target proterins after them had been denatured.The purification proteins can be divided into two parts, one part was used for preparation of polyclonal antibody, another part was used for the determination of activity units of this proteins after renaturation of the denatured proteins. After 4weeks when the mice were immunized by target proteins, the serum including polyclonal antibody was obtained successfully. After determination, it was found that the enzyme activity was highest in 39 ?.Fluorescence quantitative PCR method was used to study the car gene transcript level of different growth stage and the organization in the silkworm with the template of the gene of p50 silkworm. It is found that the transcript level of car gene was higher in fat body than any other organizations and lowest in the head; the transcript level of car gene in pupal stage quantity is higher than other growth stages.