Molecular Tagging And Mapping In Bombyx Mori Against BmNPV And The Differential Protein Expression Profiling In The Midgut Tissue Of Silkworm Infected By BmNPV

Bombyx mori nuclear polyhedrosis virus(BmNPV),the insect virus first discovered,causes nuclear polyhedrosis disease in Bombyx mori, which is the most serious of three viral diseases in sericulture.The disease often breaks out in sericultural countries,and due to its high infectivity,it is difficult to control,thus resulting in enormous economic loss.Currently, cocoon loss caused by BmNPV accounts for 70%-80%of the total loss caused by silkworm diseases in major sericultural areas,such as Sichuan, Zhejiang,Jiangsu and Guangdong.For years,scientists in sericulture have been striving to search for resistant genes and elucidate the resistance mechanism as well as to breed resistant races.So far,great progress has been made in many fields.However,many problems remain unsolved.Using backcross population BC1,established with strain NB(resistant to BmNPV) and strain 306(susceptible to BmNPV),and the constructed linkage map of SSR molecular marker,this study aimed to screen molecular markers of the genes associated with NPV resistance and make linkage mapping analysis as well as to establish new germplasms employing molecular marker-assisted breeding technique.Furthermore, near isogenic line(NIL) was established,a combination of 2-dimensional electrophoresis and mass spectrum was adopted to study the changes in expression patterns of BmNPV-infection related proteins in midgut tissue at different time points post inoculation and to reveal the response pattern of Bombyx mori to BmNPV at the proteomic level.Through comparison with the expression patterns of the control,a number of infection-related protein markers and resistance-related proteins were discovered,providing theoretical basis for the early diagnosis of BmNPV disease and developing new ways of preventing the disease as well as for breeding resistant races. The major results achieved are as follows:1.Eight molecular markers linked with resistant genes were screened out on the two chromosomes and then located.Based on the fact that,in the meiosis of Bombyx mori,while there is no crossover between genes on female chromosome there will be crossover between those on male chromosome,we used the following strains as research materials:strain NB, which is highly resistant to BmNPV;strain 306,which is highly susceptible to BmNPV;and two types of backcross populations,namely, (306×NB)♀×306♂and 306♀×(306×NB)♂,designated as BC1F and BC1M,respectively.103 molecular marker specific primers were designed according to the published SSR molecular linkage map for screening molecular markers associated with BmNPV resistance.By using 22 individuals of BC1F population,5 SSR markers linked with resistant gene KN1 were screened out on the third linkage group,and 3 SSR markers linked with resistant gene KN2 were screened out on Chromosome 25,and then 192 individuals of BC1M population were used to locate these markers and resistant genes,revealing that the resistant gene on Chromosome 25 was located at 10.8 cM,with the nearest primer being LFL0442 and arrangement order being LFL0442,KN2,LFL1125 and LFL0771,and that the resistant gene on the third linkage group was located at 45.3 cM,with the nearest primer being LFL0454 and the arrangement order being LFL0542,LFL0773,LFL1148,KN1,LFL0454 and LFL0326.2.New germplasm was created by constructing near isogenic line. According to the principle of molecular marker-assisted breeding technique, genes associated with BmNPV resistance were transferred to hypersilkgenous race C03 with good characters through successive backcrossing.Using a routine inoculation method and molecular marker-assisted breeding technique,a new silkworm race called "Xinyue" was obtained after backcrossing for 6 generations,selecting after virus inoculation and self-crossing for 3 generations.This race is resistant to BmNPV and possesses similar economic characters with C03.The result showed that races bred with molecular marker-assisted breeding technique and those bred with conventional breeding techniques have little difference in economic characters.However,molecular marker-assisted breeding technique needs less time and smaller scale,and enables higher possibility of fixing resistant genes due to its ability to detect SSR marker in the progenies.3.Using 2-dimersional electrophoresis and mass spectrometry,36 known proteins were screened out by comparing the expression patterns of proteins in midgut tissue of newly fourth-exuviated larva of different strains at different time points post inoculation with NPV with those of the control newly fourth-exuviated larva,and functional analyses were performed on part of the proteins.With strain NB,which is highly resistant to BmNPV,strain 306,which is highly susceptible to BmNPV,and strain BC8,a near isogenic line,which was established through cross and backcross between NB and 306 as research models,and using a combination of 2-dimensional electrophoresis and mass spectrometry, proteins were extracted from the midgut tissue of silkworms infected with BmNPV first feeding of the fifth instar at 24 h,48 h,72 h post inoculation and from that of the control silkworms,and then analyzed,and differential expression patterns of proteins of the three strains at the three time points post inoculation with BmNPV were obtained.Expression patterns of proteins in the midgut tissue of silkworms infected with BmNPV were discussed among the same strain at the same time point or at different time points,and among different strains at the same time point.After being in-gel digested,the differential protein spots were identified by mass spectrometry,and peptide mass fingerprints(PMF) were used to search NCBI protein database.A total of 256 differential protein spots were cut from 18 gels,and analyzed by mass spectrometry,and 64 of them had satisfactory results(Scores are higher than 79.Or the alignment shows high homology with Bombyx mori proteins with a few of them being identical). That is,the reliability was higher than 99.5%.MASCOT software was used to analyze these results,and 36 known proteins were identified:(1) proteins that may relate to NPV resistance:arginine kinase,thiol peroxiredoxin, serine proteinase inhibitor(Serpin),aspartame aminotransferase(AST); (2)proteins that may relate to immuifaction:Enoyl-CoA Hydratase(ECH), triose phosphate isomerase(TIM),heat shock proteins;(3) the correlated proteins involved in vital movement:lipoamide dehydrogenase, fumary-lacetoacetate hydrolase,phosphatidylethanol-aminesn(PE); H-ATPase;(4) other differential proteins:IDGF- like proteins,DNA helicase,nonmuscl or smooth muscle protein,Translation elongation factor, Actin-depolymerizing factorand so on.