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.

Isolation of a cationic peroxidase from cultured peanut cells

1980 ◽  
Vol 58 (21) ◽  
pp. 2280-2284 ◽  
Author(s):  
B. A. Maldonado ◽  
R. B. van Huystee
Keyword(s):  
Ion Exchange ◽  
Suspension Culture ◽  
Cell Suspension ◽  
Ammonium Sulfate ◽  
Cell Suspension Culture ◽  
Specific Activity ◽  
High Specific Activity ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Single Peptide

Medium that had supported the growth of a peanut cell suspension culture was found to be a rich source of peroxidase. Precipitation with acetone and ammonium sulfate, followed by a pH shift and ion exchange chromatography, led to the isolation of a single cationic peroxidase isozyme of high specific activity and high RZ of 2.91. The isolated peroxidase was shown to be a single peptide chain.

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.

scholarly journals Citrus Cell Suspension Culture Establishment, Maintenance, Efficient Transformation and Regeneration to Complete Transgenic Plant

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 664
Author(s):  
M. Moniruzzaman ◽  
Yun Zhong ◽  
Zhifeng Huang ◽  
Huaxue Yan ◽  
Lv Yuanda ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Suspension Culture ◽  
Cell Suspension ◽  
Cell Suspension Culture ◽  
Culture System ◽  
Transformation Rate ◽  
Suspension Cell Culture ◽  
Citrus Reticulata ◽  
Agrobacterium Mediated Transformation ◽  
Solid Media

Agrobacterium-mediated transformation of epicotyl segment has been used in Citrus transgenic studies. The approach suffers, however, from limitations such as occasionally seed unavailability, the low transformation efficiency of juvenile tissues and the high frequency of chimeric plants. Therefore, a suspension cell culture system was established and used to generate transgenic plants in this study to overcome the shortcomings. The embryonic calli were successfully developed from undeveloped ovules of the three cultivars used in this study, “Sweet orange”-Egyptian cultivar (Citrus sinensis), “Shatangju” (Citrus reticulata) and “W. Murcott” (Citrus reticulata), on three different solid media. Effects of media, genotypes and ages of ovules on the induction of embryonic calli were also investigated. The result showed that the ovules’ age interferes with the callus production more significantly than media and genotypes. The 8 to 10 week-old ovules were found to be the best materials. A cell suspension culture system was established in an H+H liquid medium. Transgenic plants were obtained from Agrobacterium-mediated transformation of cell suspension as long as eight weeks subculture intervals. A high transformation rate (~35%) was achieved by using our systems, confirming BASTA selection and later on by PCR confirmation. The results demonstrated that transformation of cell suspension should be more useful for the generation of non-chimeric transgenic Citrus plants. It was also shown that our cell suspension culture procedure was efficient in maintaining the vigor and regeneration potential of the cells.

scholarly journals Stimulatory effect of methyl jasmonate and squalestatin on phenolic metabolism through induction of LOX activity in cell suspension culture of yew

2014 ◽  
Vol 38 ◽  
pp. 76-82 ◽  
Author(s):  
Zohreh JALALPOUR ◽  
Leila SHABANI ◽  
Ladan AFGHANI ◽  
Majid SHARIFI-TEHRANI ◽  
Sayed-Asadollah AMINI
Keyword(s):  
Suspension Culture ◽  
Methyl Jasmonate ◽  
Cell Suspension ◽  
Cell Suspension Culture ◽  
Phenolic Metabolism

The reduction of starch accumulation in transgenic sugarcane cell suspension culture lines

2008 ◽  
Vol 3 (11) ◽  
pp. 1398-1406 ◽  
Author(s):  
Stephanus J. Ferreira ◽  
Jens Kossmann ◽  
James R. Lloyd ◽  
Jan-Hendrik Groenewald
Keyword(s):  
Suspension Culture ◽  
Cell Suspension ◽  
Cell Suspension Culture ◽  
Starch Accumulation ◽  
Transgenic Sugarcane
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