| Silkworm is the typically mode insect in Lepidotera.It plays an important role in exploring the sex-related regulation mechanism and its application in insects to study development- related proteome in Bombyx mori.Exploring for the specific proteins which related to development,hemolymph and fat body proteins from male and female silkworm during different stages were studied based on 1DE-LC-MS,2DE and MALDI-TOF-MS. The conclusions are following:1.Studied the feasibility on 1DE LC-ESI Q-TOF MS(1DE-LC-MS) technology for high-throughput analysis of silkworm's proteome by using the newly hatched larvae as materials.The results showed that 1DE-LC-MS displayed a unique using value in high-throughput analysis,mixed sample separation,specific proteins' detection,as well as differential proteome.Furthermore,constructed the proteome database based on 1DE-LC-MS in silkworm,Bombyx mori,171 protein compositions were identified and defined as 121 kinds of proteins or protein subunits respectively,and 36 kinds of those have not been deposited in the DNA or protein database of silkworm as well as other insects.In which more than 64 components were relevant to cell structure and protein metabolism among 171 identified proteins.2.Exploring for the molecular mechanism of sexual development and regulation in silkworm,hemolymph proteins of male and female silkworms were extracted respectively from 5th instar larvae and pupae.After treatment,the proteins were isolated and identified based on 1DE-LC-MS.The results showed that differential expression of hemolymph proteins on contents and types were detected which is relevant to sex and development. Ninty-six matchable protein candidates were searched from database.By eliminating the redundant protein candidates,a total of 27 protein compositions were identified and defined as 13 kinds of proteins or protein subunits.The different protein compositions between male and female silkworms are mainly on 70kD to 100kD in which Arylphorin, Vitellogenin and Antichymotrypsin were female-specific expressing components.In addition,30K protein family was highly expressed composition in silkworm hemolymph. The identification and functional analysis of these different proteins provide information for molecular mechanism of sexual development and regulation in silkworm.3.The technology of IPG-IEF / SDS-PAGE was optimized to be suitable for fat body proteome using fat body from male and female silkworm during development stages.After 2DE,the maps were compared and analyzed with Image Master software.The result showed that 56 protein components which differential expression was more than twice were detected in fat body between male and female silkworm.In which 28 proteins were in 5th instar larvae,12 proteins were in unpupated larvae of silking end,16 proteins were in pupa.In addition,11 protein components which between male and female silkworm were detected,among which 5 proteins were in 5th instar larvae,4 proteins were in unpupated larvae of silking end and 2 proteins were in pupa.4.Fourteen spots which volume ratio was more than three times and 11 spots which specific expressed were in-gel digested and analyzed on MS.After that 12 proteins were identified,in which 8 proteins were related to sexual development(Actin and calponin were up-regulated expression in male,cytoplasmic dynein light chain,75kDa subunit NADH and RACK were female-specific expression,IMP cyclohydrolase and tropomyosin 1 were male-specific expression).4 proteins were related to metamorphosis development (30K protein and 30K lipoprotein were up-regulated expression in pupa,antichymotrypsin was pupa-specific expression).5.Receptor for activated protein kinase C(RACK) was an important protein which was differential expressed between male and female silkworm during developmental stages. So Bmrack gene was in silico cloned based on Bioinformatics and a conting of 1224bp was obtained.The coding region was 960bp and coding 319aa.Its molecular weight and pI was 36 kD,7.64 respectively.Moreover the amino acid sequences were highly conserved in Lepidotera.After that Bmrack gene was cloned from silkworm genome by PCR and a sequence of 2122bp was obtained which contained 4 introns and 5 exons.Blasting in silkworm wgs database 5 sequences were found and the identities with Bmrack gene reached to 99%and 8 GAP were repaired.
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