| Comparative proteome of the midguts of different developmental period of 5th instar was investigated by high resolution two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF-MS techniques. The proteome of the midgut in the 3rd of 5 instar was investigated by Multi-Dimensional Liquid Chromatography and LTQ-Orbitrap mass spectrometry. The proteome of BmN cells infected and uninfected by BmNPV were investigated by SDS-PAGE, Multi-Dimensional Liquid Chromatography and LTQ-Orbitrap mass spectrometry. The molecular characters and antiviral activity of 2 antiviral proteins were investigated by bioinformatics and molecular biology technology.1. High resolution two-dimensional polyacrylamide gel electrophoresis and and ImageMaster 2D platinum techniques to investigate the total midgut proteins of silkworm, Bombyx mori in the 2nd and 5th and 7th day of the fifth instar stage. The resulted showed that the characteristic of midgut proteomic is remarkably different from other reported silkworm tissues. Most protein spots were unevenly distributed in the area from 20 to 70 kDa with pH 4-8. Among them most spots located in acidic area, and this characteristic was particularly observed in the map of midgut in 7th day. There were 869 spots in the 2D map of midgut in 2th day, and 966 in 5th day. Within this duration,97 new sopts were increased, and mainly located in the area of PH 6-9, size 20-40 KDa. Along with larval maturation, the spots in the map were found to be decreased obviously, with 420 in 7th day, less than that of 5th day by 56.5%。Some spots expressed differently were excised and identified by using MALDI-TOF MS, and the proteins abundantly expressed at the early, middle 5th stage include actin, cytoplasmic actin A3a1, TBA8_RAT, keratin 1 which constitute muscle tissue and epithelial tissue, include HSP7C_DROME, VATA_MANSE, thiol peroxiredoxin, AGL165Wp, arginine kinase, which are related with digestion and absorption of the rnidgut, include hemolymph 30K protein and major plasma protein 30K which are related to restaining cell apoptosis. The expression volume of these proteins except for VATA_MANSE decreased, some proteins are completely degraded at the late 5th stage. The expression volume of VATA_MANSE increased because a mass of energy is need to degrade tissue protein of the midgut of the silkworm. This study will help us to understand the silkworm midgut well.2. In order to learn more about the constitution of the midgut, its proteome was studied by using nanoLC electrospray ionization MS/MS. Mass spectrometry data were analyzed by X! Tandem searching software using different parameters and validated by Poisson model.Total'90 proteins were identified and 79 proteins were described for the first time. Among the new proteins,22 proteins were closely related to the digestive function of the midgut, including 11 proteins of digestive enzymes secreted by the epithelium,8 proteins of intestine wall muscle and mechanical digestion,3 proteins of peritrophic membrane that could prevent the epithelium from being mechanically rubbed; 44 proteins were involved in metabolism of substance and energy; 11 proteins were associated with signal transduction, substance transport and cell skeleton.3. Cell line is an important experimental platform for biological sciences as it can basically reflect the biology of its original organism. We firstly characterized the proteome of cultured BmN cells, derived from Bombyx mori. Total 1478 proteins were identified with two or more peptides by using 1-D SDS PAGE and LTQ-Orbitrap. According to the gene ontology annotation, these proteins presented diverse isoelectric points and molecular weights, involved in various molecular functions, including catalytic activity, binding, molecular transducer activity, motor activity, transcription regulator activity, enzyme regulartor activity, as well as antioxidant activity. We used the same technique to investigate the proteome of cultured BmN cells infected by BmNPV and concluded that total 303 proteins are identified and 8 proteins are expressed only in cultured BmN cell infected, which are Lipase, SP-2, CLH-17, GBP-1, ICE5, microtubuleβ-1, Apaf-1 and unknown protein. In the protein, CLH-17 and microtubuleβ-1 are related to the BmNPV infecting path; Lipase, SP-2 and GBP-1 are related to antiviral activity; Apaf-1 and ICE5 are related to BmN cells apoptosis.4. In order to learn more about antiviral proteins, two cDNAs encoding two antiviral proteins, Lipase (GenBank accession AY945209 and AY945212) were cloned from Chinese domestic and wild silkworm Bombyx mandarina Moore based on the available information of silkworm. The size of the cDNA from the wild silkworm is 906 bp, encoding 301 amino acids and molecular weight is predicated as 28.9kD. Its full length of genomic sequence was also analyzed. It consists of 2147 bp, including 4 exons and 3 introns. The gene expression analysis showed that the lipase gene was only expressed in midgut, and its expression level was higher during the ingesting stage while almost no during molting and mature stage. In order to identify its bioactivity, large amount of recombinant lipase was obtained by efficient gene expression system in vitro, and activity assay showed that this protein has strong antiviral activity against BmNPV.5. A cDNA encoding a antiviral protein, SP-2 (GenBank accession:AY945210) was cloned from Chinese wild silkworm Bombyx mandarina Moore based on the available information of silkworm. The length, of its cDNA is 855 bp, encoding 284 amino acids and molecular weight is predicated as 29.6kDa. Sequence alignment analysis indicated that its amino acids is different from that of domesticated silkworm, specially at C-terminal with 8 various amino acids. In order to identify its bioactivity, large amount of recombinant SP-2 was obtained by efficier(?)t gene expression system in vitro, and activity assay showed that its antiviral activity against BmNPV is much stronger than that of silkworm Bombyx mori by 1.6 fold. The big difference of the antiviral activity between the wild and domesticated worm was suppositionally attributed to the variation at its C-terminal sequence.
|
PDF Full Text Request