Group Title: BMC Plant Biology
Title: Developmental changes in abundance of the VSPß protein following nuclear transformation of maize with the Soybean vspß cDNA
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Title: Developmental changes in abundance of the VSPß protein following nuclear transformation of maize with the Soybean vspß cDNA
Physical Description: Book
Language: English
Creator: Grando, Magali
Smith, Rex
Moreira, Cristina
Scully, Brian
Shatters, Robert
Publisher: BMC Plant Biology
Publication Date: 2005
Abstract: BACKGROUND:Developing monocots that accumulate more vegetative tissue protein is one strategy for improving nitrogen-sequestration and nutritive value of forage and silage crops. In soybeans (a dicotyledonous legume), the vspA and B genes encode subunits of a dimeric vegetative storage protein that plays an important role in nitrogen storage in vegetative tissues. Similar genes are found in monocots; however, they do not accumulate in leaves as storage proteins, and the ability of monocot leaves to support accumulation of an ectopically expressed soybean VSP is in question. To test this, transgenic maize (Zea Mays L. Hi-II hybrid) lines were created expressing soybean vspB from a maize ubiquitin Ubi-1 promoter.RESULTS:From 81 bombardments, 101 plants were regenerated, and plants from five independent lines produced vspB transcripts and VSPß polypeptides. In leaves from seven-week-old plants (prior to flowering), VSPß accumulated to 0.5% of the soluble leaf protein in primary transgenic plants (R0), but to only 0.03% in R1 plants. During seed-filling (silage-stage) in R1 plants, the VSPß protein was no longer detected in leaves and stems despite continued presence of the vspB RNA. The RNA transcripts for this peptide either became less efficiently translated, or the VSPß protein became unstable during seed-fill.CONCLUSION:Developmental differences in the accumulation of soybean VSPß when transgenically expressed in maize show that despite no changes in the vspB transcript level, VSPß protein that is readily detected in leaves of preflowering plants, becomes undetectable as seeds begin to develop.
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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BMC Plant Biology ioMed

Research article

Developmental changes in abundance of the VSPp protein following
nuclear transformation of maize with the Soybean vspfl cDNA
Magali F Grandol, Rex L Smith2, Cristina Moreira3, Brian T Scully4 and
Robert G Shatters Jr*3

Address: 'Instituto de Ciencias Biol6gicas/Faculdade de Agronomia e Medicina Veterinaria, Universidade de Passo Fundo, Passo Fundo RS,
99001-970, Brazil, 2Agronomy Department, Institute of Food and Agricultural Science, University of Florida, Gainesville FL, USA, 3United States
Department of Agriculture, Agricultural Research Service, United States Horticultural Research Laboratory, 2001 South Rock Road, Fort Pierce, FL,
USA and 4Indian River Research and Education Center, Institute of Food and Agricultural Science, University of Florida, Fort Pierce, FL, USA
Email: Magali F Grando; Rex L Smith; Cristina Moreira;
Brian T Scully; Robert G Shatters*
* Corresponding author

Published: 02 March 2005
BMC Plant Biology 2005, 5:3 doi: 10.1 186/1471-2229-5-3

Received: 21 May 2004
Accepted: 02 March 2005

This article is available from:
2005 Grando et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Developing monocots that accumulate more vegetative tissue protein is one
strategy for improving nitrogen-sequestration and nutritive value of forage and silage crops. In
soybeans (a dicotyledonous legume), the vspA and B genes encode subunits of a dimeric vegetative
storage protein that plays an important role in nitrogen storage in vegetative tissues. Similar genes
are found in monocots; however, they do not accumulate in leaves as storage proteins, and the
ability of monocot leaves to support accumulation of an ectopically expressed soybean VSP is in
question. To test this, transgenic maize (Zea Mays L. Hi-Il hybrid) lines were created expressing
soybean vspB from a maize ubiquitin Ubi-I promoter.
Results: From 81 bombardments, 101 plants were regenerated, and plants from five independent
lines produced vspB transcripts and VSPP polypeptides. In leaves from seven-week-old plants (prior
to flowering), VSPP accumulated to 0.5% of the soluble leaf protein in primary transgenic plants
(Ro), but to only 0.03% in R, plants. During seed-filling (silage-stage) in R, plants, the VSPp protein
was no longer detected in leaves and stems despite continued presence of the vspB RNA. The RNA
transcripts for this peptide either became less efficiently translated, or the VSPp protein became
unstable during seed-fill.
Conclusion: Developmental differences in the accumulation of soybean VSPP when transgenically
expressed in maize show that despite no changes in the vspB transcript level, VSPp protein that is
readily detected in leaves of preflowering plants, becomes undetectable as seeds begin to develop.

Although genetic variation for protein content has been
found in forage plants, this variability is narrower than
that observed for other traits such as digestibility [ 1 ]. Since
the major protein components in monocot forage and

silage crops are involved in metabolic activity, and hence
are not "true" storage proteins, it has been argued that it is
not feasible to make major changes in protein quality or
protein composition by conventional breeding [1]. How-
ever, genetic engineering may allow improvement in

Page 1 of 9
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protein quality and content through expression of a stor-
age protein not found in grass vegetative tissue.

Genes encoding seed storage proteins of various plant spe-
cies have been transgenically expressed to test for
improvement of nutritional quality. Most experiments
were conducted with tobacco and legume species includ-
ing alfalfa, soybean, canola, clover and lupins. For
nuclear-targeted genes, accumulation of these seed storage
proteins in vegetative tissue of transgenic plants was either
undetectable or very low. These included pea vicilin [2,3],
soybean conglycinin [4], sunflower seed agglutinin [5,6],
and phaseolin [7]. The instability of seed proteins in non-
seed tissues of transgenic plants was frequently attributed
to protein targeting to protease-rich vacuoles in the vege-
tative cells, and subsequent degradation [5,7,8].

The greatest accumulation of a seed storage protein from
a nuclear-targeted gene was achieved using zeins, a maize
seed storage protein that is targeted to "protein bodies"
directly from the endoplasmic reticulum (ER), thus avoid-
ing the secretary route to the cellular vacuole. Transgenic
expression from the CaMV 35S promoter in tobacco
resulted in the formation of these protein bodies contain-
ing the zein within vegetative tissues [8,9]. Alternatively,
"short-circuiting" the protein-targeting route by addition
of an ER retention signal to the storage-protein coding
region also increased protein accumulation up to 100x

In many legumes, accumulation of specific vegetative stor-
age proteins (VSPs) in leaves and stems is the main source
of increased nitrogen content [12-14]. Use of VSPs instead
of seed storage proteins to increase vegetative protein con-
tent in monocots may provide an advantage since they
have evolved to function in vegetative cell types. Legume
vegetative cells that accumulate VSP proteins contain mul-
tiple vacuole types and storage proteins are targeted to
specific vacuoles where they are not rapidly degraded
[15], and do not interfere with cellular metabolic proc-
esses. It is uncertain if monocots can produce similar vac-
uoles or successfully target a VSP to them. The most
studied VSPs are the soybean VSP(x and VSPP proteins,
which are lysine-rich glycosylated vacuolar proteins that
accumulate abundantly in leaves, stems and pods, but not
in seeds [12-14]. Recently, soybean vspA and/or vspB genes
fused to the 35S promoter were expressed in transgenic
tobacco to study their accumulation in a heterologous
dicot plant. Nuclear targeted genes produced VSP ranging
between 2 and 6% of the soluble protein in leaves of the
transgenic plants [16]; whereas, targeting to both the chlo-
roplast and the vacuole within the same plant resulted in
VSP comprising greater than 10% soluble protein [ 17,18].
Soybean VSP is therefore an excellent candidate for use in
transgenic improvement of plant protein status, particu-

larly grasses that contain limited levels of lysine [16].
However, it remains to be determined if VSPs can be
expressed and accumulated in monocot plants since stor-
age protein stability is dependent on post-translational
events that may differ between monocots and eudicots.
This manuscript presents the transgenic expression of soy-
bean VSPP in the leaves of transgenic maize and discusses
this expression in relation to the developmental stage of
the plant.

Results and Discussion
Development of primary (Ro) transgenic maize expressing
Out of 101 plants, twenty, belonging to five independent
lines (71-1, 45-1, 45-3, 44-1, and 4-1) were shown by
Southern blot analysis to contain a 1.5 kb hybridizing
band corresponding to the intact bar gene (Fig. 1). All 20
plants also contained the expected 1.9 kb band that
hybridized to the vspB gene. The same probe detected two
bands in EcoRI restricted Soybean genomic DNA, ~5.7 and
8.6 Kb, corresponding to the highly homologous genes
vspA and vspB [13].

Western blot analysis was used to detect the VSPp
polypeptide in leaf extracts from fourteen primary (R0)
transgenic maize plants at vegetative stage (7 weeks old).
A distinct VSPp band was not visible in silver-stained SDS-
PAGE separated maize extract proteins due to complexity
of the total protein pattern and the relatively low level of
VSPp expression (Fig. 2a). However, some of these plants
expressed VSPp at a level high enough to be detected by
Western blot analysis (Fig. 2b). Two plants (71-1-53 and
45-3-1) had the highest level of VSPP, while four plants
(71-1-23, 71-1-20, 45-1-4, and 45-1-7) accumulated a
lower level of VSPp. Although a faint lower MW peptide
band was visible in negative control maize extracts, VSP
was clearly only present in transgenic lines. Faint detec-
tion of bands in untransformed maize (none of identical
size to soybean VSPp) is consistent with previous reports
of cross-hybridizing of soybean VSP antibodies with pro-
teins from monocots [14]. In soybean extracts used as a
positive control, the antibody for VSPp recognized both
VSP( and VSPp polypeptides (Fig. 2b,c)

Computer analysis of digital images of the Western blots
was used to detect differences in relative band intensity of
the immunologically detected VSPp peptide. Because the
native soybean VSPs (VSP( plus VSPp) were easily visible
on total protein stained gels (Fig. 2a), relative quantifica-
tion of total stained proteins in the soybean samples indi-
cated that the VSPs represented about 10% of the total
soluble protein in young soybean leaves. This is close to
the previously reported value of 15 % [12]. The intensity
of the transgenic maize 45-3-1 VSP3 band on Western
blots was 44% of the soybean VSP's band (digital image

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BMC Plant Biology 2005, 5:3

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,- ^- ^- ^- lIn I- '-t In1 In1
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-' ci

o C

o 0

-ci c-i a
I O T In In
C. N- 03 0 '-t '-t

-t 0> 0
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bar 1.5 kb
:Gohmm onem 4l

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vspB 1.9 kb


^Fn ^
In 1 c1 C>' QC c
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Figure I
Southern blot analysis of primary (Ro) maize transformed with pAHC25 and pRSVP- I1. Twenty tgs of EcoRI restricted genomics
DNA were electrophoresed through 0.8% agarose and blotted to Hybond membrane. The membranes were probed with
either digoxigenin labeled bar (a) or vspB (b). Numbers and lines on sides of blots indicate location of molecular size markers
(the number represents size in kilobases). The "a" and "b" sections are aligned so that the same genomic DNA samples are ver-
tically aligned and represented by the same lane label. Plasmid lane is the plasmid containing the either the bar or the vsp/3
clones used as a positive control. Untransformed controls are lanes containing genomic DNA from untransformed Hi-11 maize.
Soybean indicates lanes containing restricted soybean genomic DNA. The two panels within each section represent separate
blots hybridized with the same probe.

pixel quantification of Fig. 2b). Accounting for the differ-
ences in total protein applied to the gel (less soybean total
protein was loaded), the VSPP protein was estimated to
have accumulated to 0.5 % of the total soluble protein.
This is similar to the highest level of seed storage protein
accumulation observed with the ectopic expression of
zein [8,9], but remains less than the 1% minimal expres-
sion level predicted by Wandelt et al. [ 11] to be needed to
directly alter the nutritional quality of the leaves.
Although the 0.5% of total soluble protein was too low to
alter nutritional value, detection of VSPp in 45-3-1
allowed monitoring ofVSPP level in leaves and stems dur-
ing plant development.

Presence of vspB in R, plants
R1 plants were produced by back-crossing the R0 plants
with Hi II control non-transformed pollen. Back-crossing
was performed because R0 plants directly regenerated

from tissue culture did not have synchronized production
of pollen and receptive female flowers. The R1 families
segregating for bar expression were analyzed for the pres-
ence of the vspB gene by Southern blot analysis. From 57
R1 plants analyzed, Southern blot analysis showed that 35
(61%) contained the vspB gene integrated into the
genome. Figure 3 shows examples for several R1 from five
R0 parental lines. The ~1.9 kb vspB band can be seen in
nine of the 16 R1 lines. There are also fainter bands one
slightly larger than the 1.9 kb band and one or two migrat-
ing between 4 and 5 kb. These are often observed as
incomplete restriction of all Eco RI sites internal to plas-
mid DNA that is integrated into the maize genome. Simi-
lar bands are observed even with the plasmid control.

Total RNA was isolated from leaves of young R1 plants at
the vegetative stage (7 weeks after planting). Eighteen
transgenic R1 plants, including the ones originating from

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BMC Plant Biology 2005, 5:3

- 1- on '-I-
F D C -

tt-~ ~c ~c -
I I I I -
--- on -
I I I I -

." by

:JWlw 4-

Figure 2
Western blot detection of VSPP in primary (Ro) transgenic maize. Thirty micrograms of protein from each sample were sepa-
rated by SDS-PAGE. (a) Silver-stained 12% SDS-PAGE polypeptide profile for six of the 14 analyzed Ro plants. (b) Immunode-
tection of VSP protein in SDS-PAGE separated transgenic Ro maize extracts transferred to Hybond-P membranes and
immunodetected using VSPp antiserum and the Reinascence kit chemiluminescent detection method (NEN Life Sciences Prod-
ucts, Inc). (c) Underexposed western blot showing two distinct bands corresponding to VSP( and VSPP polypeptides in soy-
bean leaves.

R0 plants representing different levels of VSP3 accumula-
tion (high VSPp accumulators: 45-3-1, 71-1-53; mid-level
VSPp accumulators: and 71-1-23, 71-1-20, 45-1-4, 45-1-7
and low VSPp accumulators: 44-1-1- 4-1-2), were ana-
lyzed for vspB transcripts. Because of high sequence simi-
larity (85%) between vspA and vspB cDNAs, the same
probe hybridized in soybean with both mRNAs as dem-
onstrated by Staswick [13]. The transgenic plants 71-1-
53A, 71-1-20A, 71-1-20E, and 71-1-201 produced a
hybridizing band of approximately the expected size of
1.1 kb indicating transgene expression (Fig. 4a); however,
most did not. Use of the Ubi-1 promoter for both the vspB
and bar gene probably led to a high number of transgenics
with the vspB silenced, and a higher level ofVSPP accumu-
lation will likely result in future work using a combina-
tion of different promoters.

Immunodetection of VSP3 protein in transgenic young
plant leaves showed variation in accumulation in compar-
isons between the parental (R0) and their R1 progeny, with
the greatest variation observed with the highest VSPp
expressing R0 plants (Fig 4b). The parental line 71-1-53
had a relatively high level of expression of VSPP, but the
only transgenic offspring from this line, 71-1-53A, had
almost undetectable VSPp; in contrast, a low level accu-
mulator, 71-1-20, produced R1 lines expressing different

levels of VSPP, although none of them expressed at higher
levels than the parent. Quantification of the relative level
of VSPp expressed in the R1 leaves showed that the highest
level measured was only 0.03% of total soluble protein.

Despite the overall low level of VSPp expression, for the
purposes of this work, VSPp accumulation in several of
the plants was high enough to study the relationship of
plant developmental stage and vspB/VSPP accumulation.
Both the transcript abundance and VSPp protein accumu-
lation were determined in the RI lines using real-time
quantitative RT-PCR and Western blot immunodetection,
respectively. Real-time RT-PCR is more sensitive that
Northern blot analysis and was able to detect transcripts
that were not seen with standard total RNA blotting meth-
ods. The vspB transcript was quantified in leaves from
immature plants (prior to tassel formation) and silage
stage plants (plants with developing seeds at the 18 DAP-
days after pollination stage), as well as, stems from the
silage stage plants, (Figure 5). The vspB transcript was
detected in all five transgenics (four of which had RNA not
detectable using standard Northern blot methods). The
relative level of RNA among the different plant samples
was not consistent across the different lines with some
having more transcripts in the young leaves while others
had more in the older leaves and stems.

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BMC Plant Biology 2005, 5:3

I 71-1-20









21.23 4 _
5.15 .15 "
4.97 S..-...
203r eej -i

1.90W m vspB
1. 58
1.38 -- k" .

Figure 3
Southern blot detection of vspB in R, transgenic maize. Twenty micrograms of EcoRI restricted genomics DNA was electro-
phoresed through 0.8% agarose and blotted to Hybond membrane. The membrane was probed with digoxigenin labeled vspB.
The alphabetical labels of each R, family represent individual R, plants. Control sample represent genomic DNA from untrans-
formed maize Hi-11. Two positive control samples are also included: plasmid, pRSVP- I restriction digested to liberate the vspB
cDNA clone (only partial digestion occurred as evidenced by the two bands at higher molecular weight than the vspB frag-
ment), and genomic DNA from the 45-1-2 Ro plant, previously shown to contain the vspB sequence. The Eco RI restricted soy-
bean genomic DNA is also included as a positive control for hybridization of the soybean vspB probe.

When vspB RNA abundance was compared to VSPP pro-
tein accumulation there was no correlation between the
two (Fig. 5a, b, c). Antiserum to VSPp detected VSPp only
in leaves from young maize plants that had not yet flow-
ered. No VSPp was detected in leaves and stems of silage
stage corn that had developing seed. The soybean VSPp3
peptide was the primary band reacting with the antiVSPf3
antiserum in young leaves of transgenic maize, however,
in silage stage stems and to a lesser extent the silage stage
leaves, there were multiple bands detected at a different
size than the VSPp. These were also detected in the non-
transgenic control plant samples and therefore, were not
due to different modifications of the soybean VSPp3.
Although the VSPp protein dropped below detectable lev-
els in the silage stage 71-1-20A, 45-3-1F and 45-3-1-G
plants, the vspfl transcript was still detectable. In fact, in
the 71-1-20A plants the transcript level was highest in the
silage stage leaves that had no detectable VSPp. Therefore,
post-transcriptional events (i.e., changes in either the
translational efficiency of the vspB transcript or the protein
stability of VSPp) were altered in the silage stage leaves
and stems as compared to the leaves of immature plants.

The vspB gene was successfully introduced into R0 regener-
ated maize and transferred to the R, progeny, of which
vspB transcript and VSPp protein were detected and stud-
ied. This is the first report on introduction and expression
of a legume vegetative storage protein in a monocot plant.
The inability to detect VSPp from the maize vegetative tis-
sue at the time of seed development, even when the vspB
transcript was still expressed, must have arisen from either
reduced translational efficiency of the vspB transcript or a
decrease in the stability of the VSPp protein. The reduction
of seed storage protein level in leaves of transgenic eud-
icots was also observed with expression of vicillin in
alfalfa [11] and tobacco [2]; however, it was not observed
with the expression ofVSP(o in tobacco [16] or ovalbumin
in alfalfa [30]. These data suggest that factors controlling
developmental change in vegetative tissue protein accu-
mulation are a combination of host plant traits and innate
characteristics of the ectopically expressed protein. It is
interesting to speculate that if, in maize (a monocot), soy-
bean VSPp was degraded in a manner that provided
amino acids that were translocated to the seed to support
seed development, then development of high level VSP
expressing monocots may be a way to improve nitrogen
content of the seed/grain produced by the plant.

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BMC Plant Biology 2005, 5:3








Soybean leaves

__j IC

S. .-. ,.
.' .i .

:f T
, ,. 4 ,-.
.'. i-^.

'- 'z, t"

Untransformed control
young soybean
old soybean
Untransformed control
71-1-20 A
71-1-20 E
71-1-20 G
71-1-20 I
71-1-23 C
71-1-23 F
71-1-23 B
45-1-2 B
45-1-2 C
45-1-3 E
45-1-3 A
45-1-3 B
45-1-3 D
young soybean
old soybean

Figure 4
(a) Northern blot detection of vspB transcripts in R, transgenic maize. Thirty micrograms of total RNA were separated on 1.2%
agarose formaldehyde gels and blotted to Hybond N+ membranes. The vspB transcripts were detected by hybridized with a dig-
oxigenin labeled vspB probe. (b) Western blot detection of VSP in Ro and their progeny (Ri) transgenic maize. Thirty micro-
grams of protein in extracts from leaves of 7 weeks old plants were separated on 12% SDS-PAGE, blotted onto Hybond-P
membrane, and VSP was immunodetected using VSPP antiserum and the Renascence kit chemiluminescent detection method
(NEN Life Sciences Products, Inc).

Plant material and tissue culture methods
Plants of hybrid "Hi-II" maize were established in a green-
house and immature tassels were used for embryogenic
type II callus production, as described by Armstrong [19].

Transgenic plant development
Microprojectile bombardment of callus was performed
using the procedure of Somers et al. [20]. Calli were

cobombarded with equal amounts of pRSVP-1 (Shatters
Jr., unpublished) and pAHC25 [21]. Plasmid pRSVP-1
was constructed by restricting the soybean vspB cDNA
clone (998 bp) from pKSH3 [22] with EcoRI, blunt ends
of this fragment were produced using S1 nuclease and the
fragment was cloned into similarly blunt ended BamHI
restricted pAHC17 [21]. As a result, the vspB coding region
was inserted downstream of the Ubi-1 promoter and a 5'
untranslated region (exon) and intron; and upstream of

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BMC Plant Biology 2005, 5:3

Leaves from preflowing plants
Leaves from silage stage plants
Stems from silage stage plants

0 40



- o

s 0

Control 45-3-1F 45-3-1G

R Cotrol 45-3-1F 45-3-G

b)L-.^MN __ ME_0_____________

Figure 5
a) Real-time RT-PCR quantification of vspB transcripts in R, transgenic maize tissue. Two hundred micrograms of total RNA
from the indicated tissue was used as the template source for real-time RT-PCR detection of vspB transcripts. Reactions were
performed in 15 uL volume using the Qiagen Quantitect SYBR Green kit. Expression values are calculated by normalizing all
threshold cycles (Ct) for vspB to the 18S rRNA Ct and converting this value to fold-increase over the value for the lowest
expressing tissue, *44- I I-stem, which was arbitrarily set at 1.0. b,c) Developmental changes in VSPP level in transgenic maize
vegetative tissues. Soybean VSPp was immunodetected in Western blots of 30 tgs of total protein from crude extracts sepa-
rated by 12% SDS-PAGE and blotted to Hybond-P membranes. Crude Extracts were prepared from: YL, leaves from preflow-
ering plants; SL, leaves from silage stage plants; and SS, stems from silage stage plants. (b) Coomassie blue stained SDS-PAGE
separated proteins from crude extracts; (c) VSP was immunodetected using VSPp antiserum and the Renascence kit chemilu-
minescent detection method (NEN Life Sciences Products, Inc). The arrows indicate the VSP-P protein band.

the nos terminator sequence. The plasmid pAHC25 carried
the bar gene and the uidA reporter gene, both under the
control of the Ubi-1 promoter. Bombardments were
performed with a Biolistic& PDS-1000/He Particle Deliv-
ery System (Bio-Rad Laboratories, Hercules, CA) and an

osmotic treatment was applied to reduce the cell damage
caused by the gene transfer method [231. Putative trans-
genic maize were regenerated from glufosinate resistant
callus as described by Armstrong [19 and grown in five-
gallon pots containing sterile sand and Metromix-350

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71-1-20A 45-3-1F 45-3-1G 44-1-1L 44-1-1G
Transgenic plant

BMC Plant Biology 2005, 5:3

(1:1). Plants were fertilized weekly with Peter's 20-20-20
with micronutrients (Division of United Industry Corp.,
St. Louis, MO).

Southern blot analysis
One gram of frozen young leaf tissue was ground in liquid
nitrogen and genomic DNA was extracted using the Del-
laporta procedure [24]. Twenty micrograms of genomic
DNA were digested with EcoRI, which released a 1.9 Kb
fragment containing the vspB gene, the nos terminator and
part of the Ubi-1 promoter. DNA was separated on a 0.8%
agarose gel, blotted onto Hybond N+ membrane (Amer-
sham Pharmacia Biotech, Inc. Piscataway, NJ) by capillary
blotting [25], and UV cross-linked. The non-radioactive
digoxigenin system (Roche Molecular Biochemicals, Indi-
anapolis, IN) was used for labeling and detection of the
transgene. Blotted DNA was probed with either a 611 or a
843 bp of vspB gene segment amplified from pRSVP-1 and
gel purified. The forward and reverse primers 5'-GTTCT-
were used, respectively, to amplify a 611 bp segment, and
the primer pair 5'-GCAGGCTACCAAAGGT-3' and 5'-
TAGGTGACITACCCACAT-3' was used to amplified the
product of 843 bp.

For identification of bar transgenic plants, the DNA was
digested with EcoRI, which released a fragment of ~ 1.5 Kb
that contained part of the Ubi-1 promoter, the bar gene,
and the nos terminator, and was identified with a 419 bp
digoxigenin labeled probe produced by PCR amplifica-
tion of pAHC25 using the forward and reverse primers: 5'-
CCGTGCTTGA-3', respectively.

Northern blot analysis
Total RNA was isolated from 2 g of tissue using acid gua-
nidine isothiocyanate-phenol-chlorophorm extraction
[261, resuspended in T10E1 and treated with 2 tl RNasin
(4U/[tl) RNAse inhibitor (Promega, Madison, WI) and
stored at -70 C until use. Thirty micrograms of total RNA
were separated on 1.2% agarose formaldehyde gels and
transferred to Hybond N+ membrane by capillary blotting
overnight or by pressure blotting for one hour using a
PosiBloft 30-30 pressure blotter (Stratagene, La Jolla, CA)
according to the manufacture's instructions. Membranes
were UV-crosslinked, and probed with the same probes as
described for Southern blot analysis.
Chemilluminescence was captured using a Kodak Digital
Science Image Station 440 CF (Eastman Kodak Company,
Rochester, NY).

Real-Time RT-PCR
Total RNA extractions for real-time RT-PCR were per-
formed using 500 mg of tissue ground to fine powder
using mortar and pestle in the presence of liquid nitrogen,

then processed with the RNeasy midiprep Kit (Qiagen,
Germany), following the manufacturer's protocol. Trace
DNA contamination was removed from total RNA by a
combination of acid phenol: chloroform 5:1 pH= 4.7
extraction and Dnase I treatment (Ambion, Texas). Real-
time RT-PCR was performed on a Rotor-Gene RG-3000
(Corbett Research, Australia) using the Quantitect SYBR
Green real-time RT-PCR kit (Qiagen, Germany), and the
manufacturers protocols with 300 ng of Dnase I treated
total RNA. Primers were designed to amplify a 108 bp
fragment of the soybean vspB using the following primers:
GAGCGTTC1TC-3'. Reverse transcription was performed
for 30 min at 50C followed by a 15 min denaturing at
950C, and 40 cycles of 40 s at 95 C, 40 s at 580C and 40
s at 720C. Quantification was based on relative abun-
dance to maize 18S RNA by amplifying a 174 bp fragment
with primers: 5'-CCTGCGGCTTAATTGACTC-3' and 5'-
GTTAGCAGGCTGAGGTCTCG-3', and using the compar-
ative quantification function of the Rotor-Gene RG-3000
software. All real-time RT-PCR experiments were con-
ducted in triplicate and on triplicate RNA preparations for
each sample. Melting curve analysis and agarose gel elec-
trophoresis were performed to verify single product

Western blot analysis
Protein was extracted from 100 mg of leaves and stems
with 0.5 ml of phosphate-buffered saline (137 mM NaC1,
2.7 mM KC1, 10 mM Na2HPO4, 2 mM KH2PO4) supple-
mented with 1 tablet/10 ml buffer of the Complete-Mini
protease inhibitor cocktail (Roche Molecular Biochemi-
cals, Indianapolis, IN) by homogenizing in the presence
of zirconia/silica 0.1 mm dia. beads. Centrifuged extract
supernatants were removed and used for protein concen-
tration determination [27]. Because of the low protein
yields in stem extracts, they were precipitated with 10%
trichloroacetic acid (TCA), washed with ice-cold acetone
and resuspended prior to SDS-PAGE analysis.

Thirty micrograms of protein were separated on 12% SDS-
polyacrylamide gels. Proteins in the gel were either
stained with silver nitrate [28] or transferred to Hybond-P
(PVDF) membranes (Amersham Pharmacia Biotech, Inc.
Piscataway, NJ) using Trans-Blot SD Semi-Dry Transfer
Cell blotter and recommended protocols (Bio-Rad Labo-
ratories). Soybean VSP was immunologically detected on
the PDF membranes with anti VSP[3 serum (provided by
P. Staswick, University of Nebraska, produced as previ-
ously described [29] used at a 1:5,000 dilution. Detection
was performed using a luminol substrate and the NEN
Life Science Products, Inc. (Boston, MA) Reinascence kit.
Chemiluminescent signal was captured by a Kodak Digital
Science Image Station 440 Cf, and analyzed with Kodak
ID Scientific Image Software. Quantification of band

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BMC Plant Biology 2005, 5:3

intensity was always compared relative to samples from
the same gel.

List of abbreviations
RT-PCR, reverse transcriptase-polymerase chain reaction;
VSP, vegetative storage protein

Authors' contributions
MFG participated in experimental design, carried out the
transgenic plant development, plant crosses, and molecu-
lar blotting/detection methods, and participated in man-
uscript draft preparation. RLS participated in
experimental design, and provided guidance and training
in development of transgenic maize. CM performed RT-
PCR experiments. BTS participated in experimental design
and provided expertise and training in plant crosses. RGS
conceived of the study, participated in experimental
design, coordinated the experimental plan, and wrote the
draft manuscript. All authors read and approved the final

This work was supported, in part, by grants from the Florida Dairy
Research Council.

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