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(Last
modification: 03. May 2010)
Polyketide Synthase in Sorgoleone Biosynthesis (Sorghum bicolor)
RNAi Mediated
Repression of ARS1 and ARS2 in Sorghum bicolor
Functional identification with
recombinant enzymes is nice, but in vitro data in many cases cannot prove the
functions in the plants. These RNAi experiments were carried out to test this
rigorously. Constructs were established both for ARS1 and ARS2, and both were
used to obtain stable Sorghum transformants.
The results are shown below.
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Left: (A)
Relative ARS1 and ARS2 endogenous transcript levels in 10-d-old S.
bicolor hpRNA “+”
and hpRNA “-”
seedlings (representing six independent transformant events) were
determined by quantitative real-time RTPCR using gene-specific primers.
Data were normalized to an internal control (18S rRNA), and the
ΔΔCT method was used to obtain the relative
expression levels for each sequence, expressed as mean +/- SD from
assays performed in triplicate. Events numbered 1, 3, and 4 were
generated using the vector pARS1-RNAi, and events numbered 2, 5, and 6
were generated using pARS2-RNAi. |
Right: (C) Sorgoleone levels were
determined by GC-MS analysis of root exudates prepared from 10-d-old
hpRNA “+”
and hpRNA “-”
seedlings representing the six RNAi transformant events. Data are
expressed as mean +/- SD from four measurements. The limit of
quantitation (LOQ), determined to be ca. 0.003 µg/mg fresh weight, is
also indicated by a dashed line.
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Evaluation of S. bicolor
RNAi Transformant Events.
+ = positive hpRNA expression (i.e. one would expect
suppression of ARS transcripts),
- = no hpRNA
expression (i.e. one would expect no suppression of ARS transcripts),
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The expression levels for
endogenous ARS1 and ARS2 transcripts in “+”
and “-” individuals were independently
assayed for each event by quantitative real-time RT-PCR using ARS1- and
ARS2-specific primers showed: In all events, ARS1 and ARS2 expression levels
were substantially reduced in hpRNA “+”
individuals relative to hpRNA “-”,
reflecting the successful downregulation of ARS1 and ARS2 in hpRNA expressing
individuals. ARS1 silencing appeared somewhat Mehr effective overall than ARS2;
however, sufficient sequence identity existed between the 3'-coding regions of
the two genes to trigger silencing of both, as this was observed irrespective of
the vector used. Reduction of ARS1 transcript accumulation in hpRNA “+”
individuals ranged from about 66 to 96% relative to corresponding hpRNA “-”
individuals for each event, and ARS2 transcript levels were reduced from about
55 to 86%. Complete loss of ARS1/2 expression was not observed for any event.
However, relatively few studies have employed real-time RT-PCR to quantify
target inhibition in plant RNAi studies; thus, it is somewhat difficult to draw
direct comparisons at present. Additionally, DNA gel blot analyses, performed to
estimate the number of T-DNA loci in transformants, indicated approximately one
to two T-DNAs per event, with three of the six events (events 3, 4, and 6)
harboring a single T-DNA locus (not shown here).
Sorgoleone levels were also determined for hpRNA “+”
and “-” individuals by GC-MS from all
events, see above (right). Overall, a complete correlation was observed between
hpRNA expression and a dramatic reduction in sorgoleone accumulation. In all
cases where hpRNA expression was detected (“+”
samples), sorgoleone levels were reduced to amounts below the limit of
quantitation of the GC-MS analysis employed (about 0.003 µg/mg fresh weight).
This was observed in six independently transformed events, thus establishing
that the observed reduction in sorgoleone accumulation was dependent on the
expression of the hpRNA-generating transgene and was not transformation
event-specific.
Taken together, the results obtained
from ARS1/2-targeting RNAi experiments (this page), enzymatic assays using
recombinant ARS1 and ARS2 (Mehr...),
and the tissue-specific expression pattern determined for ARS1 and ARS2 (Mehr...)
strongly suggest that ARS1 and ARS2 represent the ARS enzymes proposed for the
biosynthesis of sorgoleone.
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Organisation der
Seiten über die Typ III PKS in Sorghum bicolor
-
Alkylresorcinol-Synthasen (ARS) in Sorgoleone Biosynthesis:
Mehr...
-
Typ III PKS
Transkripte
mit
präferentieller Expressin in Wurzelhaaren:
Mehr...
-
Aktivitäten und
Substratpräferenzens von ARS aus Sorghum bicolor:
Mehr...
-
Modellierung von Sorghum ARS:
Mehr...
-
ARS aus Reis (Oryza
sativa): Vergleich mit Sorghum ARS:
Mehr...
- Update July 2010: ARS or ARAS ?
Mehr...
-
ARS aus Sorghum und Reis (Oryza sativa),
Identifizierung von funktionell wichtigen Aminosäuren:
Mehr...
-
Diese Seite:
RNAi-vermittelte Repression
von ARS1 und ARS2 in Sorghum bicolor:
Mehr...
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