Expression and properties of diacylglycerol acyltransferase from cell-suspension cultures of oilseed rape

2000 ◽  
Vol 28 (6) ◽  
pp. 684-686 ◽  
Author(s):  
R. J. Weselake ◽  
C. L. Nykiforuk ◽  
A. Laroche ◽  
N. A. Patterson ◽  
W. B. Wiehler ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Cell Suspension ◽  
Molecular Mass ◽  
Oilseed Rape ◽  
Cell Suspension Cultures ◽  
Transcript Level ◽  
Diacylglycerol Acyltransferase ◽  
Suspension Cultures ◽  
Dgat Activity ◽  
Brassica Napus L

The expression of diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) with predicted molecular mass of 56.9. kDa (BnDGAT1) was examined using microspore-derived cell suspension cultures of oilseed rape (Brassica napus L. cv Jet Neuf). As well, a recombinant histidine-tagged N-terminal fragment of BnDGAT1 [BnDGAT1(1–116)His6], which was relatively hydrophilic, was partially characterized. A temporal increase in DGAT activity occurred within a 24 h period following transfer of cells from 6% (w/v) sucrose to 14% (w/v) sucrose. Western blotting indicated that the abundance of BnDGAT1 protein was closely correlated with DGAT activity. BnDGAT1 mRNA also exhibited a temporal increase within the 24 h period following transfer of cells into higher sucrose concentrations, but the transcript level was not closely associated with DGAT activity as BnDGAT1 protein. The fragment BnDGAT1(1–116)His6, interacted with [1-14C] oleoyl-CoA, suggesting that the N-terminal region of BnDGAT1 may have a role in binding cellular acyl-CoA.

Microspore-derived cell suspension cultures of oilseed rape as a system for studying gene expression

2007 ◽  
Vol 92 (2) ◽  
pp. 131-139 ◽  
Author(s):  
Yongzhong Shi ◽  
Gangbiao Xu ◽  
Timothy B. Warrington ◽  
Gordon K. Murdoch ◽  
E. Chris Kazala ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Suspension ◽  
Oilseed Rape ◽  
Cell Suspension Cultures ◽  
Suspension Cultures

scholarly journals Increased BnaMFT-transcript level is associated with secondary dormancy in oilseed rape (Brassica napus L.)

2020 ◽  
Vol 19 (6) ◽  
pp. 1565-1576
Author(s):  
Lei LIU ◽  
Wen-qi FAN ◽  
Fu-xia LIU ◽  
Xin YI ◽  
Tang TANG ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Oilseed Rape ◽  
Transcript Level ◽  
Brassica Napus L ◽  
Secondary Dormancy

scholarly journals Solubilization and characterization of diacylglycerol acyltransferase from microspore-derived cultures of oilseed rape

1994 ◽  
Vol 304 (3) ◽  
pp. 951-958 ◽  
Author(s):  
D Little ◽  
R Weselake ◽  
K Pomeroy ◽  
T Furukawa-Stoffer ◽  
J Bagu
Keyword(s):  
Enzyme Activity ◽  
Suspension Culture ◽  
Cell Suspension ◽  
Oilseed Rape ◽  
Cell Suspension Culture ◽  
Solution Structure ◽  
Specific Activity ◽  
Diacylglycerol Acyltransferase ◽  
Dgat Activity ◽  
Protein Ratio

Particulate fractions prepared from microspore-derived (MD) embryos of oilseed rape (Brassica napus L. cv. Reston) and an embryogenic MD cell suspension culture of oilseed rape (B. napus L. cv. Jet Neuf) were used as a source of diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) for enzyme characterization and development of a solubilization procedure. DGAT activity in the 1500-100,000 g fraction from MD embryos was stimulated 4-5-fold by 3 to 4 mg of BSA/ml of reaction mixture. DGAT activity from MD embryos was stimulated 2-3-fold by fluoride salts and 1.4-fold by NaCl, whereas iodide salts caused substantial inhibition of enzyme activity. The effect of the various 1:1 electrolytes on enzyme activity appeared to be related more to their differential effects on solution structure rather than ionic strength. DGAT was solubilized from membranes of MD embryos and the cell suspension culture by about 80 and 50% respectively, using 2 M NaCl in 1% (w/v) octanoyl-N-methyl-glucamide (MEGA-8) (pH 8.0 buffer) at a detergent to protein ratio of 2:1. The specific activity of solubilized DGAT was about 2-fold greater than that of the particulate enzyme. The mechanism of solubilization appeared to be related to the lowering of the critical micellar concentration of MEGA-8 in the presence of NaCl. DGAT, solubilized from MD embryos, eluted with an M(r) of about 2 x 10(6) during gel-filtration chromatography on a Superose 6 column equilibrated in buffer containing 0.1% (w/v) MEGA-8. The solubilized enzyme exhibited optimal activity at pH 7. At concentrations above 2 microM acyl-CoA, the specificity of solubilized DGAT for oleoyl-CoA and palmitoyl-CoA was considerably greater than for stearoyl-CoA.

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