Agriculture Reference
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“Celebrity” tomatoes. In particular, with the aim of minimizing possible effects of PLD alpha
gene disruption on plant growth and development, the fruit-specific
E
8 promoter was used
instead of the constitutive
CaMV35S
promoter. The
E
8 promoter is responsive to ethylene,
but high-level expression in tomato fruit appears to be regulated by an ethylene-independent,
ripening-related
cis
element (Deikman et al., 1998). Aside from fully developed fruit tissues,
E
8 is expressed mainly in anthers of mature, stage 4 flowers. An antisense construct of
LePLD
2
was cloned into the binary vector pBI121 (with 35S and GUS excised) for use in
Agrobacterium
-mediated transformation of tomato cotyledon explants. The entire construct
included a copy of the npt II (neomycin phosphotransferase) antibiotic-resistance gene under
control of the
NOS1
(nopaline synthase) promoter and terminator, and a 2.0-kb fragment of
the
LePLD
α
2
cDNA (nucleotides 880-2,846, constituting 70% of the coding region plus
the 3
-UTR) in antisense orientation regulated by the
E
8 promoter and
NOS1
terminator.
Following selection based on kanamycin resistance, 25 putative antisense
LePLD
α
2
transformants were identified by DNA gel blot (Southern) analysis using leaf tissue ge-
nomic DNA and probes for npt II and
E
8. Of these, plants representing 16 lines were raised
to maturity, and pericarp tissue from mature green fruit (40 days after pollination) was tested
for suppression of
LePLD
α
2
by semiquantitative RT-PCR using isolated total RNA. The
Ambion QuantumRNA
TM
quantitative RT-PCR kit was used to determine levels of
LePLD
α
α
2
cDNA relative to control 18S cDNA. The amplified
LePLD
2
and 18S cDNA fragments
produced by RT-PCR were separated on agarose gels and quantified by densitometry mea-
surements after staining with ethidium bromide. Among the sixteen lines tested, four showed
clear suppression of
LePLD
α
α
2
, whereas three surprisingly exhibited overexpression of the
α
gene. The four
LePLD
2
suppressed lines (designated 8-4-A, 10-3-B, 10-5-C, and 10-6-C),
plus line 8-2-E that showed the highest level of
LePLD
2
overexpression, were propagated
through two subsequent generations by self-fertilization for further analyses of PLD alpha
gene expression and activity, as well as fruit physiology and quality attributes.
α
9.7.6 Levels of
LePLD
α
transcripts in fruit of
LePLD
α
2
antisense lines
LePLD
2
transcript abundance in pericarp of wild type and antisense transgenic fruit har-
vested 40 days after pollination (DAP) was determined by both RNA gel blots (Northern
blots) and the semiquantitative RT-PCR 18S competimer system (Ambion QuantumRNA)
described earlier (Fig. 9.16). For the Northern blots, a 646-bp segment of the
LePLD
α
2
cDNA coding region (nucleotides 1,080-1,725) was used as the radiolabeled probe, whereas
in the RT-PCR experiments a 0.6-kb cDNA fragment of
LePLD
α
2
(coding region nu-
cleotides 735-1,323) was amplified. The two methods gave similar results, and averaging
the two values, transcript levels in the four suppressed lines ranged from about 20 to 45%
of that in wild-type controls, and transcript in line 8-2-E was roughly 75% higher than that
in wild type.
Concerning possible suppression or overexpression of the other two tomato PLD al-
pha isogenes, because of the high percentage of sequence identity between
LePLD
α
α
2
and
LePLD
α
3
(91% in the open reading frame), it seemed likely that
LePLD
α
2
antisense would
3
isogene. However, perhaps because the 3
-UTR was
also affect expression of the
LePLD
α
included in the 2-kb antisense
LePLD
2
fragment, this does not appear to be the case. Semi-
quantitative RT-PCR analysis indicated that in pericarp tissue of fruit harvested at 40 DAP
there was little if any reduction (or increase) in
LePLD
α
α
3
transcript in the five
LePLD
α
2
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