| Silkworms are another genome-sequenced insect after Drosophila and Anopheles,and are an important economic model inscet.China's cocoon and silk production rank first in the world,but there is one of the most difficult diseases in silkworm industry,namely Bombyx mori nuclear polyhedrosis virus(BmNPV),which caused great losses in silk industry in annual.Our group have identified 344 silkworm variety resources in China and found a high resistance to BmNPV local species,which the semi-lethal dose is 1000 times more than susceptible strains of silkworm.After 10 years of cultivation to screen a high antihomozygous line NB,and the near isogenic line BC8 of susceptible strain 306 was constructed by hybridization and backcross.Genetic analysis showed that the resistance of NB to Bm NPV showed the inheritance of dominant single gene control.Using the high-resistance NB and the second generation of high-throughput sequencing technology to screen the molecular markers related to the resistance gene,and the linkage of polymorphic molecular markers and resistance genes was analyzed by genetic population.The resistance gene was located on the chromosome 23 of silkworm.The region's potential 22 genes were sequenced,it was found that compared the susceptible strains of silkworms with resistant strains were some mutations.Carboxylesterases are multifunctional superfamily,widely exist in animals,plants,insects and microorganisms,with catalytic hydrolysis of ester-containing compounds.It plays an important role in pheromone degradation,exogenous compounds detoxification,lipid transport,neurogenesis and developmental regulation.The insectic carboxylesterases were divided into 8 subfamilies according to substrate specificity and sequence similarity,contain ?-esterase,?-esterase,acetylcholinesterase,juvenile hormone esterase,neurotactins,neuroligins,glutactin and gliotactin.In previous studies,Drosophila and Anopheles were used as model organisms,and the study of carboxylesterase was mainly focused on its resistance to insecticides.Recently,it has been reported that human carboxylesterase 1(CES1)is a very important host enzyme in the replication process of hepatitis C virus,suggesting that carboxylate esterase may have resistance to viruses.Therefore,this paper uses the Bmae42 gene of silkworm to analyze the Bmae42 gene on BmNPV resistance by using a variety of molecular biology methods.The main results are as follows:(1)Using bioinformatics to analyze the Bmae42 gene,it was found that there were unique triad catalysis center Gly-X-Ser-X-Gly.(2)The gene of Bmae42 of silkworm 306,NB and BC8 were obtained by cloning.Comparison of different varieties of carboxylesterase Bmae42 gene found that the mutations of the five loci occurred in the resistant varieties of silkworm.Two of these sites are glycosylation sites that may be associated with resistance to viruses.(3)Quantitative real-time polymerase chain reaction(RT-PCR)analysis showed that the transcription level of Bmae42 gene was the highest in the fifth instar silkworm.In addition,higher levels of transcription were detected in the fat body and midgut.(4)Constructed the Bmae42 recombinant plasmid containing His-tag,and the prokaryotic expression was expressed to obtain soluble protein.The activity of the protein was analyzed and compared of the activity differences of different strains of silkworm carboxylesterase Bmae42.Simultaneously,recombinant proteins were used to immunize mice,and polyclonal antibodies were obtained,which provided the basis for the follow-up protein research.(5)Construction of Bmae42 transient expression plasmid,to explore its impact on the proliferation of the virus.The results of the above studies show that silkworm carboxylesterases Bmae42 is a hydrolyzed protease and the difference of Bmae42 activity was not obvious,but it is closely related to Bm NPV virus invasion and value. |
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