Comparison of proteomics between acute myeloid leukemia and acute lymphoid leukemia / 中国实验血液学杂志

The aim of this study was to explore the distinct protein profiles of different subtype of acute myeloid leukemia (AML), including M(1), M(2), M(3) and acute lymphoid leukemia (ALL) by differential proteomic expression analysis. The proteins of bone marrow leukemia cells from AML and ALL patients were separated by two-dimensional electrophoresis (2-DE). 2-DE patterns were analyzed by PDQuest 7.4 software and the differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and bioinformatics. The results indicated that 21 differentially expressed proteins were found by 2-DE and 15 were identified by MS to be significantly differentially expressed. In AML, seven proteins were highly expressed such as MPO, PRDX3, CALR and ECH1 and so on, and eight proteins were highly expressed in ALL, including ARHGDIB, PFN1 and ACTG1 and so on. It is concluded that the distinct protein profiles between AML and ALL have been proved. It may be helpful for the identification of new targets for specific treatment approaches and the molecular markers for the early diagnosis of leukemia.

Establishment and optimization of two-dimensional gel electrophoresis technique of proteome from bone marrow leukemia cells / 中国实验血液学杂志

The aim of this study was to establish and optimize two-dimensional electrophoresis method for human bone marrow leukemia cells in order to obtain the profiles with high resolution and reproducibility. The total protein was extracted and separated by isoelectric focusing (IEF) and SDS polyacrylamide gel electrophoresis (SDS-PAGE). The gels were stained with silver nitrate or Coomassie brilliant blue, and then scanned and analyzed with PDQuest 7.4 analysis software. The effects of different protein preparation methods and electrophoresis conditions on the profiles were compared. The results indicated that by optimizing preparation of protein sample and electrophoresis protocols, clear profiles with 780 +/- 73 well separated protein spots on an average were obtained and the match rate was 82 +/- 5% between reproducible gels from leukemia cells of different sub-type. It is concluded that the two-dimensional electrophoresis method of proteome from human bone marrow leukemia cells is established successfully and is suitable for the further comparative proteomic research between leukemia of different types.

Construction of a vector conferring herbicide and pest resistance in tobacco plant / 生物工程学报

A binary plant expression vector, pCM12-slm, carrying the aroAM12 mutant gene encoding bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and the Bts1m recombinant gene consisting of 331 N-terminal amino acids of CryIAc and 284 C-terminal amino acids of CryIAb has been constructed. The truncated Bts1 gene was fused with the PR1b signal peptide sequence and expressed in tobacco plants under the control of 2E-CaMV35S promoter and the omega (omega) translation enhancer sequence from tobacco mosaic virus. The mutant aroAM12 was fused with the transit sequence of tobacco EPSPS and expressed in tobacco plants under the control of the CaMV35S promoter. Tobacco leaves were transformed with Agrobacterium tumefaciens LBA4404 harboring the pCM12-slm plasmid, and the transgenic plants were selected directly on medium containing the herbicide. Forty glyphosate resistant plants were regenerated, with a transformation frequency of 27%. Transgenic plants were initially assessed for glyphosate resistance by placing leaf discs on shoot induction media containing the herbicide. Rooted plantlets, propagated from selected transgenic tobacco, were transferred to soil in a greenhouse and tested for glyphosate resistance by spraying them with Roundup at a commercial recommended dose. The glyphosate resistance assay indicated that all the transgenic plants showed highly resistant to the herbicide. The PCR assay showed that the aroAM12 gene was present in all of the 40 T0 transfer plants, and Bts1m genes present in 28 of 40 of the transgenic plants. Southern blot analysis further confirmed that the copy number of the transgenes varied from one to three copies in different transgenic plants. Northern blot and immunodot blot showed that the aroAM12 and Bts1m genes were expressed at the transcription and translation levels. Transgenic plants containing both the aroA M12 and Bts1m genes were further assessed for insect resistance. Tobacco leaves of T0 transgenic plants were infested with tobacco bollworm H. assulta larvae for 6 days. The result (table 1) showed that the survival rate of insect larvae was between 0-10%, and the growth of insect larvae was seriously inhibited, suggesting pCM12-slm as a dual functional vector with potential application in breeding of glyphosate and insect resistance transgenic plants.