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Establishment of sorghum
cell suspension culture system for proteomics studies
Rudo Ngara, D. Jasper, G.
Rees and Bongani K. Ndimba*
Proteomics
Research Group, Department of Biotechnology, University of
the Western Cape, Private Bag X17, Bellville, 7535, Cape
Town, South Africa.
*Corresponding author. E-mail:
bndimba@uwc.ac.za.
Tel: +27 21 959 2468. Fax: +27 21 959 1551.
Accepted 22 January, 2008 |
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This study describes the establishment of sorghum cell
suspension culture system for use in proteomics studies.
Friable sorghum callus was initiated from young shoots under
completely dark conditions on MS medium supplemented with 3
mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 2.5 mg/L
1-naphthaleneacetic acid (NAA). Additionally, sorghum cell
suspension cultures have been initiated from the friable
callus masses in liquid medium with the same composition as
the callus initiation medium. Total soluble proteins (TSP)
and culture filtrate (CF) proteins were extracted from the
cell culture system and solubilised in urea buffer (9 M
urea, 2 M thiourea and 4% CHAPS). Both one-dimensional (1D)
and two-dimensional (2D) gel analysis of these two proteomes
show that the TSP and CF proteomes have different protein
expression profiles. The sorghum TSP proteome, which is
highly complex, is best resolved when separated on large
format, 18 cm, pH 4 - 7 isoelectric focusing (IEF)
immobilised pH gradient (IPG) strips. On the other hand, the
sorghum CF proteome (secretome) is less complex with most
proteins being resolved on mini format, 7 cm, pH 3 - 10 IPG
strips. Furthermore, narrowing down the pH range from 3 - 10
to 4 - 7 for the CF proteome resulted in improved protein
spot resolution.
Key
words:
Sorghum, proteomics, callus, cell suspension cultures, total
soluble protein, secretome. |
