Proteomics Of Malpighian Tubules And The Functional Analysis Of Important Enzymes,Bomyx Mori

Silkworm(Bombyx mori) belongs to the lepidopteran insect. Its malpighian tubules, the important organs for metabolism in the body, have not only the distinctive characteristics but also great relevance to the renal tube of other insects and even the kidney tube of mammals. The discovery and research, for significant functional proteins of the silkworm(Bombyx mori), will be favor of comprehending the function of malpighian tubules in insects. Moreover, it is helpful to find out the similar and different evolution results between the insects and the mammals, because of the different evolution requirements during long evolutionary process. What's more, so far, the economic value of Bombyx mori for silk has been paid long attention to. Because Metabolism plays the necessary role after the process of digestion, and it has the regulatory impact on the growth and development of silkworm. Hence, the research on malpighian tubules of silkworm will make contributions to enhancing the silkworm's physical health and impoving the the efficiency of silk production. Furthermore, fortunately, the malpighian tubules of silkworm possess the typical structure with various obvious sections,which provids better opportunity to research the functions of the different sections in the malpighian tubules.In this study, we analysised the proteins of malpighian tubules on day-5fifth larve with acrylamide gel electrophoresis (SDS-PAGE)-LC MS/MS and2D-MALDI-TOF/MS technology. And compared the different expression proteins between day-5fifth larve day-1pupa as well as each section of malpighian tublues. Also the main protein present in the moth of malpighian tubules metabolites were identified; Use of the silkworm genome sequence, expression sequence tags (ESTs) data as well as gene chip data, with significant periods and the section of the distribution characteristics of protein BmAGXT2, BmHYI, Bmhibadh the encoding gene Bmagxt2, Bmhyi, Bmhibadh carried out the cloning and analysis. RT-PCR, prokaryotic expression, Western blotting, and other technology is used to study the function of BmAGXT2, Bmhyi. Finally, we measured the activity of BmAGXT2in malpighian tublues from day-3fifth instars larvae to day-7pupa and in each section on day-5fifth instars larvae.1. Proteomics analysis of malpighian tubules of day-5fifth instars larvae using LC MS/MS combined with2D-MALDI-TOF MSLC MS/MS is a method of identifying proteins in complex mixtures using a combination of high performance liquid chromatography combined with mass spectrometry. The proteins of Day-5fifth instar larvae were identified LTQ VELOS mass spectrometer. To control the confidence, the identified peptides from SEQUEST were further validated by TPP analysis. Moreover, the FDR of the identifications estimated by searching MS/MS spectra against a target-decoy database. Finally, a total of1367proteins were identified. Approximately80.54%of these proteins mostly ranged between10-100kDa, and98.90%of these proteins had pis of4-12. Thus, the proteins were identified from9×silkworm genome that82.48%of proteins had the molecular weights mostly ranged between10-100kDa, and97.82%had pIs of4-12. Forty one proteins were simultaneously identified by2-DE. These proteins are indeed known to be highly abundant of the APEX results. Through the analysis of family classification, Gene Ontology and KEGG pathway of these identified proteins, we found the malpighian tubules of the Bombyx mori have completed the responsibility regulating the balance of water and salt in body. In addition to the similarity of function and multiple metabolic pathways, they may have more association. As a specific insect eating mulberry leaves, the Bombyx mori choses the malpighian tubules as a place to complete the metabolism of volatile components of mulberry leave. With the help of GSTs, P450s, alcohol dehydrogenases and other protein families in the malpighian tubules to deal with exogenously and endogenously harmful substances, the purpose of avoiding injuries can be achieved.2.2D electrophoresis of malpighian tubules from the Day-5fifth instars larvae and its metabolite product from the Day-2moth and protein identification by mass spectrometry.Through the observation of the color of Malpighian tubules and the previous literatures, we found that at least three sections of the silkworm Malpighian tubules can be divided. The DNA staining of these three sections by DAPI for30min showed that the chromatin of the first part mainly concentrated in the inside of lumen, and the second part formed a network structure, whereas the third part was in a particle pattern, indicating that the significant differences are present in these three sections of Malpighian tubules. To further understand the protein distribution from the different sections, we performed2D electrophoresis and found that the expression of BmAGXT2, BmHYI, Bmhibadh, and BmActin3were varied. For example, Bmagxt2was mainly expressed in the first section (away from the rectum side), but with slight expression in the second and third sections. BmHYI and Bmhibadh were also enriched in the first section. However, the high expression of BmActin was in the third section. In the study, we identified proteins form the contents of malpighian tubules of Day-2silkworm moth using two-dimensional electrophoresis and mass spectrometry. There were three proteins successfully identified which was typical antimicrobial peptides-Hemolin. These results suggest that they maight be involved in the silkworm defense system of pupal and moth stages.3. Proteomic comparison on malpighian tublues between larvae stage and pupal stage of silkworm, Bombyx moriTo Get more imformation of malpighian tublues on the proteome changes between larvae stage and pupal stage and provide new evidences on the protein level at different developmental stages of malpighian tublues of silkworm, Bombyx mori. In this study, the matrix-assisted laser desorption ionization time of flight mass spectrometry were applied for identifying the different spots which had more expression of the2D map, and all the protein sequences from P50/Dazao on NCBI combined silkworm proteins database, were used to build local-database by the software GPMAW8.00. GPMAW also used to analyze peptide fingerprint masses. There were about360-430spots were obtained by silver staining from the malpighian tublues after2D-PAGE. Most of them were distributed in the area from15to80kD with pI4-9.5. We had successfully identified17markedly different proteins between larvae stage and pupal stage. They included not only the proteins in association with energy metabolism, but also heatshock proteins, Vacuolar-type H+-transporting ATPase,30K lipoprotein, and alanine-glyoxylate aminotransferase2,3-hydroxyisobutyrate dehydrogenase which have important functions in mammal kidney. The discovery and identification of these proteins can offer a valuable insights into how the malpighian tublues of silkworm adaptting the great changes on the protein level, from the larvae stage transporting the metabolic products of mulberry leaves digestion to the pupal stage transporting the metabolic products of fat body and storage proteins digestion.4. Cloning, sequence analysis and mRNA expression of Bmagxt2, Bmhyi and BmhibadhThree pairs of primers were synthesized based on the Bmagxt2, Bmhyi and Bmhibadh. Three positive clones including three genes were obtained. The CDS of Bmagxt2is2080bp, which codes260aa. Its putative molecular weight is50.38KD and pI is7.70. Seven exons and six introns are contained and only1copy exists which locates on nscaf3031of the11th chromosome on the genome. SignalP predicts that it has no signal peptide and it is secreted protein. TMHMM predicts no transmembrane structure. The CDS of Bmhyi is783bp, the proteins of which code260aa. Its putative molecular weight is29.17kD and pI is6.10. It contains4exons and3introns. Only one copy exists on the genome and locates on nscaf2360, but the condition of location chromosome is unknown. The CDS of Bmhibadh is969bp, the proteins of which code322aa. Its putative molecular weight is34.01kD and pI is9.24. It contains6exons and5introns. Only one copy exists on the genome and locates on nscaf2360of the17th chromosome. SignalP predicts that signal peptide is the1st to21th amino acid residues (MAARTILSTQCLYTAARRAYS). TMHMM predicts that the preceding60aa are transmembrane structure.In order to research the various periods of space-time expression profile of the three genes in Bombyx mori, we performed an expression analysis by using ESTs data, microarray data and RT-PCR. The ESTs analysis result shows that Bmagxt2, Bmhyi and Bmhibadh all have the EST evidences. In summary, ESTs evidences of Bmagxt2were found in silkworm malpighian tubules and in midgut. ESTs evidences of Bmhyi were found in silkworm ovary, blood cells, malpighian tubules, midgut, fat body and silkgland. ESTs evidences of Bmhibadh were found in silkworm midgut, wing primordium, malpighian tubules, embryo, blood cells, fat body and silkgland.The genome-wide microarray data indicates that the expressions level of Bmagxt2in each tissue of fifth-instar day3larvae are very low, besides a gleam expression in ovary, testis and fat body.The gene of Bmhyi has different expression level in silkgland, ovary, testis, malpighian tubules, body wall, head, blood, fat body and midgut. Among them, the expression level in silkgland is low, but is high in malpighian tubules, blood, fat body and midgut, especially in malpighian tubules. The gene of Bmhibadh has hardly expressed in these tissues of day-3in fifth-instar of larvae. The expression data from early embryo, the mature stage to the moths stage indicates that there exists no datum in the developmental stage and early embryo. The expressions level of Bmagxt2is low in early embryo and on the day-8to9of mounting stage but hardly appear in other stages. In contrast, the gene of Bmhyi has no expression in the whole embryonic period.For the purpose of the further addition and verification of the expression profiling, we made use of Semi-quantitative RT-PCR before. Bmagxt2is widely expressed in gonad, body walland fat body in fifth-instar day3larvae, while the expression level of the head and malpighian tubules is low, though there has expression from1st molting stage to day-75th stage of larve. The expression level of Bmhyi is low in fat body, epidermis and blood cell, but, On the contrary, the data of malpighian tubules is high. There has the expression during the period of ant silkworm and the day-10of mounting stage. The whole process almost shows that the expression level of larval stage is higher than that of pupal stage, but the level after36-72h of mounting stageis lower than that after96h to the day-10of mounting stage. Bmhibadh is expressed in all the tissues during fifth-instar day3larvae, but the expression level in the malpighian tubules and silkgland is a little higher than that in other tissues. However, the larval stage expresses a certain amount of data, and the overall trend is that the expression level of sleep period is lower than that of wandering stage. It hardly has no expression after on mounting stage, until the day-8of mounting stage to moths stages.5. Prokaryotic expression and antibody preparation of Bmagxt2, Bmhyi and wester blotting examinationFor the further research regarding gene function, the cDNA of Bmagxt2and Bmhyi were respectively connected with p29and p28to construct expression vector, which named as Bmagxt2/p29and Bmhyi/p28, and transformed into expression strain BL21(DE3). After induced by IPTQa51kD and a30kD recombinant protein were obtained. However, the target protein molecular weights are about50.38kD and29.17kD. In addition of a6×His-tag and a MBP-tag, the molecular weights are about51kD and30kD, which is consistent with the forecasted molecular weights.We obtained high concentration Bmagxt2and Bmhyi with the methods of affinity chromatograph and electroelution. With these two proteins, immunize male rabbits, get the serum, and purify polyclonal antibodies. Then, examin the expression level of the Bmagxt2and Bmhyi in tissues with the antibodies on day-5of larve. The research comes out that Bmagxt2protein has expression in blood and especially high in malpighian tubules, which is consistent with the phenomenon in the experiment of two-dimensional electrophoresis. In contrast, Bmhyi protein only expressed in malpighian tubules. However, the further western blotting results shows that, compared with the condition on day-5of larve, Bmagxt2and Bmhyi in malpighian tubules reduced in a huge degree on the first day in pupal stage, which is consistent with the results of two-dimensional electrophoresis.6. Detecting the activity of alanine-glyoxylate aminotransferase2(BmAGXT2)In order to study BmAGXT2protein activity in the malpighian tubules its expression of the correlation, we refer to the homologous proteins in human and mouse activity was measured, and measured the activity of BmAGXT2in malpighian tublues from day-3fifth instars larvae to day-7pupa and in each section on day-5fifth instars larvae. The results indicate that:1.The activity of BmAGXT2increased from day-3to day-7fifth instars larvae, then reduced throughout the pupal stage and stable in a relatively low level.2. In larval malpighian tublues, the activity of BmAGXT2the highest in the first section, and the lowest in the third sectin. Suggesting that BmAGXT2may be associated with metabolic intensity and silkworms eat mulberry leaves have a certain degree of correlation. BmAGXT2play its role mainly in the first section of the the silkworm, suggesting that BmAGXT2may be play its role mainly in the first section of malpighian tublues..