engleski

Gene expression in sugar beet cell lines

Introduction: As direct gene products, proteins and isoenzymes clearly reflect changes in gene expression patterns. We compared four sugar beet (Beta vulgaris L.) cell lines with regard to their protein patterns obtained by one- or 2-D electrophoresis. A normal (N) tissue is hormone dependent, and unable to produce any organised structures. Two habituated cell lines (unorganogenic (HNO) and organogenic (HO) one) are hormone independent. The tumorous line (T) was obtained by Agrobacterium tumefaciens mediated transformation of leaf fragments. We analyzed patterns of soluble intracellular proteins by means of one and 2-D electrophoresis, extracellular proteins and extracellular glycoproteins by electroblotting. Materials and methods: For protein extraction tissues were homogenised in the extraction buffer and centrifuged. Protein content of supernatants was determined according to Bradford. Crude extracts were analysed by SDS-PAGE or the proteins were precipitated with cold aceton for 2-D electrophoresis. After centrifugation the pellet was vacuum dried and solubilised in sample buffer. IEF was performend for the first dimension and SDS-PAGE for the second. Extracellular proteins were obtained from a liquid medium of 4-day old suspension cultures. Proteins were concentrated with Sephadex G-25, analysed by SDS-PAGE and visualised by silver staining. They were electroblotted onto a nitrocellulose membrane. Glycoproteins with D-manose in their glycan component were detected by reaction with concanavalin A and peroxidase staining. Results and discussion: Only a few specific cellular proteins were detected. The bands of 18 and 19 kDa were constantly expressed in the HNO line. The polypeptide of 24 kDa was the most prominent in the HO line. N and T line had some bands in common. Extracellular protein patterns showed significant differences between callus lines. HNO cells produced a lot of proteins in a molecular weight range from 25 to 66 kDa. This line was characterised by deficient cellulose and lignin deposition in cell walls and by a reduced cell to cell adhesion. The T tissue did not excrete any specific proteins. Some proteins were common to the N and T lines. Con A detection of extracellular protein blots revealed that the glicoprotein of 45 kDa was characteristic for both habituated lines and the one of 43 kDa for the HNO only. The protein of 34 kDa was present in all lines except the HNO. Their characterisation should contribute to better understanding of the habituated phenotype in tissue culture. To improve the cellular protein separation 2-D electrophoresis was performed. The majority of total soluble proteins were detected in the pH range between 5 and 8. One of the further steps should be the identification of specific spots on 2-D gels of cellular proteins as well.

nije evidentirano

nije evidentirano