Full text of DOI:10.1007/BF03183552

R
showed better growth condition in low-nitrogen medium
than the control.
Neurospora NADP-glutamate
dehydrogenases and its ex-
pression in E. coli and trans-
genic plants
1
Materials and methods
(ν) Neurospora sp. N. intermedia (Ni), N. crassa
(Nc) and N. sitophila (Ns) were purchased from China
Committee for Culture Collection of Microorganisms.
(ξ) GDH induction and total RNA preparation.
Conidia of Neurospora was grown at 30ć for 48 h,
in Vogel’s minimal medium. Mycelium was then trans-
ferred into inducible medium (containing 4% glucose,
0.02 mol/L NH4AC, 0.04 mol/L KNO3). After 3-h induc-
tion, mycelium was collected by centrifugation and was
ground to powder in liquid nitrogen. The total RNA was
extracted with guanidine isothiocyanate.
WANG Fang & TIAN Bo
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080,
China
Correspondence should be addressed to Tian Bo
Abstract
Genes of NADP-glutamate dehydrogenase
(NADP-GDH) were cloned from Neurospora intermedia (Ni),
N. crassa (Nc), and N. sitophila (Ns). The sequences showed a
high degree of homology at the cDNA and protein level. The
three GDH genes were cloned into pET30a and expressed in
E. coli. The activity assay of purified GDH showed that the
Ni-GDH had a higher activity and affinity to ammonia than
Ns-GDH, and Nc-GDH. The Km value of Ni-GDH ranges
from 0.3 to 0.45 mmol/L. Ni-gdh gene was transformed to
Nicotiana bethamiana plants. The transformed plants grew
much better in low nitrogen media than the only ROKII
vector transformed control.
(ο) RT-PCR and sequencing of GDH gene. The
primers were designed according to Nc-GDH gene[5].
Primer 1: 5Ągct cta gaa tgt cta acc ttc cct ctg ag3Ą
Primer 2: 5Ągcg agc tct tag ttc ttg gac cac cag tca
cc3Ą
PCR condition is at 94ć for 1 min, at 55ć for 1
min, 72ć 2 min, 25 cycles. The amplifications were in-
serted in pGEM-T easy vector and sequenced.
(π) Subcloning of GDH gene. GDH were sub-
cloned in pTE30a vector by appropriate restriction en-
donucleases. The positive clones were identified using
direct clone PCR and restriction endonuclease analyse.
Keywords: Neurospora, GDH, expression, transgenic tobacco.
GDH is a ubiquitous enzyme, present in the three
kingdoms of the eukaryotes, eubacteria and archaebacteria.
GDH catalyzes the reversible reductive amination of
2-oxoglutarate to form glutamate:
(ρ) GDH expression and purification. E. coli
BL21 (DE3) cells were transformed with pET30-gdh. The
cells were induced with 1 mmol/L IPTG for 3 h. GDH
was expressed as inclusion body. After sonification, pro-
tein was extracted from cell lysate and washed with
washing buffer (2 mol/L NaCl, 0.5% Triton X-100). The
inclusion body was denatured in 8 mol/L urea-MCAC-0
and loaded on Ni-NTA column. The eluted buffer was
MCAC-40, MCAC-60, MCAC-80, MCAC-100, MCAC-
200 and MCAC-500, each containing 8 mol/L urea. Col-
lect the eluted sample, and detect the purity by silver
staining on SDS-PAGE. The purified denatured GDH was
then dialyzed against two buffers, 0.1 mol/L Tris-HCl, 1
mmol/L EDTA, pH 7.4, and 0.1 mol/L Tris-HCl, 1
mmol/L EDTA, pH 8.5 respectively. Dialyze overnight,
and then collect the supernatant by centrifugation.
2-oxoglutarate+NADPH+
NH4
L-glutamate+NADP++H2O
ꢀ +H+
GDH was considered as a key route of ammonia as-
similation, but since 1974, the major route of ammonia
assimilation has generally been accepted to be the GS-
GOGAT cycle in high plants, because GS has a higher
affinity for ammonia than GDH[1]. So, the study of GDH
had lagged behind that of GS, and its function has not
been very clear. Recent research has found that GDH can
also contribute to ammonia assimilation and metabolism[2].
Lightfoot[3] found that plant nitrogen metabolism in corn
and tobacco was altered by transformation with a highly
active assimilatory GDH gene from E. coli. And plants
transformed with GDH of alga Chlorella sorokiniana
showed increased growth and improved stress tolerance[4].
They all applied for patents. Neurospora is a group of well
studied fungi. GDH plays a key role in their nitrogen as-
similation. We chose three species: Neurospora interme-
dia, Neurospora crassa and Neurospora sitophila, and
cloned their GDH gene, expressed in E. coli. After purifi-
cation and a series of enzyme assay, Ni-GDH showed
higher activity and affinity for ammonia, its Km value is
around 0.3ü0.45 mmol/L. Gene of Ni-GDH was trans-
formed into Nicotiana bethamiana and the transformants
(ς) GDH activity assay. Two assay systems were
used.
System A measured the rate of reductive amination
of 2-oxoglutarate: 2.55 mL of 0.1 mol/L Tris-HCl , pH 7.4
(containing 1 mmol/L EDTA), 0.1 mL of 1 mol/L NH4Cl,
0.15 mL of 2 mol/L 2-oxoglutaric acid (adjust to pH 7.4
with NaOH), 0.2 mL of 0.15% NADPH, 2 PLꢀpurified
GDH at 25ć.
System C measured the rate of oxidative deamina-
tion of glutamate: 2.8 mL of 0.16 mol/L glutamate, 0.1
Chinese Science Bulletin Vol. 46 No. 1 January 2001
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Fig. 1. The sequences of Ni-, Ns- and Nc-gdh genes.
mol/L Tris-HCl, pH 8.5 (containing 1 mmol/L EDTA), 0.2
mL of 0.2% NADP+, 2 PL GDH at 37ć.
After 10-min incubation, changes in absorbance at
340 nm were measured. Enzyme activity (initial rate) was
expressed in units of Pmol of NADPH oxidized per min
or Pmol of NADP+ reduced per min.
(σ) Affinity of NADP-GDH for ammonia. Km
values for three GDH were determined using dou-
ble-reciprocal (1/v against 1/[S]) plots according to
Lineweaver-Burk. Control assays showed no significant
background NADPH oxidation in the absence of enzyme.
The [NH4+] were 1, 1.25, 2 and 5 mmol/L respectively.
The reaction velocity was measured as Pmol of NADPH
oxidized per min.
2
Results
(ν) GDH sequence. Three gdh genes are all 1365
bp long and the open reading frame encoded 455 amino
acid residues. The deduced amino acid sequences of
Ni-GDH and Ns-GDH were similar to the Nc-GDH. The
homology reaches 98%. They are strongly selective use of
codons, that is pyrimidines preferred to purines and gua-
nine to adenine in the third positions. The sequences of
Ni-gdh and Ns-gdh were submitted to GenBank, Bankit
345811 and Bankit 345812 respectively (fig. 1).
(
ξ
) Expression in E. coli. BL21(DE3) was trans-
formed with pET30-gdh. After IPTG induction, the cell
lysate were loaded on SDS-PAGE (fig. 2). The molecular
weight is identical to the deduced MW 50 ku. After soni-
fication and SDS-PAGE, we found that the GDH existed
as the inclusion body.
(τ) Plant transformation. Ni-GDH gene was in-
serted into pROKĊ and transformed into Agrobacterium
tumifeciens LBA4404. N. bethamiana leaf discs from
seedlings were transformed with LBA4404 using standard
tobacco transformation procedures. Transformed shoots
were selected by Southern blotting. The positive trans-
formants and the pROK Ċ alone transformants then
transferred to low-nitrogen MS medium (containing only
5 mmol/L [NH4+]) to observe the growth of transformed
shoots with the control plants.
(ο) Purification and refolding. Under denaturing
condition in 8 mol/L urea, the inclusion body was loaded
on the Ni-NTA column. After imidazole gradient eluting,
we got the purified GDH (fig. 3). The GDH was collected
and dialyzed against two different pH buffer, 0.1 mol/L
Tris-HCl, 1 mmol/L EDTA, pH 7.4 and 8.5 respectively.
(π) Enzyme assay. In reductive amination system
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Chinese Science Bulletin Vol. 46 No. 1 January 2001

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A, the activities were Ni-GDH 109.92 U/mg, Nc-GDH
100.25 U/mg, and Ns-GDH 95.73 U/mg, while in the
oxidative deamination system, they are Ni-GDH 72.93 U/
mg, Nc-GDH 65.00 U/mg and Ns-GDH 63.13 U/mg. The
results showed that Ni-GDH activity expressed in E. coli
is a little higher than the other two (figs. 4 and 5).
Fig. 3. The purification of GDH using Ni-NTA affinity column (silver
Fig. 2. GDH expression in E. coil (10% SDS-PAGE). 1, Protein MW
marker; 2, BL21(DE3); 3, pET30a in BL21(DE3); 4ü6, Ni-GDH,
Nc-GDH and Ns-GDH expression in BL21(DE3).
stained). 1, The total protein; 2ü7, eluted samples using imidazole gra-
dient elution (MCAC-40, MCAC-60, MCAC-80, MCAC-100, MCAC-
200 and MCAC-500).
Fig. 4. Changes in absorbance at 340 nm in system A of Ni-GDH
Fig. 5. Changes in absorbance at 340 nm in system C of Ni-GDH
(
), Ns-GDH(
üƵü
) and Nc-GDH ( ).
üƷü
üƹü
(
), Ns-GDH (
üƵü
) and Nc-GDH ( ).
üƷü
üƹü
much better than the control.
(ρ) Affinity for ammonia. According to Linewea-
ver-Burk double-reciprocal protocol, we deduced that the
Km value of Ni-GDH is around 0.3ü0.45 mmol/L, and
Nc-GDH 0.4ü0.5 mmol/L, and Ns-GDH 0.46ü0.52
mmol/L. The affinity of Ni-GDH for ammonia is also
higher than Nc-GDH and Ns-GDH.
(ς) Ni-GDH can increase tobacco growth in low-
nitrogen medium. N. bethamiana containing Ni-GDH
gene can grow well in low-nitrogen medium (5 mmol/L
[NH4+]), while the control showed significant nitro-
gen-deficient symptom, leaf yellow, growth retarded, etc.
(fig. 6). Details will be reported in another paper.
3
Discussion
We cloned Ni-GDH and Ns-GDH for the first time. And
according to its higher enzyme activity and affinity for
ammonia character, Ni-GDH has been used to transform
tobacco N. bethamiana. The positive transformants grew
Fig. 6. Ni-GDH can increase tobacco growth in low-nitrogen medium
(5 mmol/L [NH4+]). Left: Transformed N. bethamiana expressing Ni-
GDH; right: pROKĊ alone transformed N. bethamiana.
Nitrogen is often the rate-limiting element in plant
Chinese Science Bulletin Vol. 46 No. 1 January 2001
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PAPER
growth and all field crops have a fundamental dependence
on inorganic nitrogenous fertilizer. Nitrogenous fertilizer,
which is usually supplied as ammonium nitrate or urea,
typically accounts for 40% of the total costs. On the other
hand, unassimilated nitrogen is converted to nitrate and is
leached into groundwater, and it is an enviromental con-
tamination. Increasing the use efficiency of nitrogen in
plants is the major field of agriculture biotechnology. At
these attempts, GDH can play a good role in increasing
nitrogen assimilation and decreasing pollution. And be-
cause GDH is a ubiquitous enzyme existing in almost all
the creatures, it can be applied to all the crops theoreti-
cally. Because it is a new biotechnology to increase nitro-
gen assimilation, until now, no publications are available.
Our research on GDH application will appear in another
paper.
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(Received December 1, 2000)
Acknowledgements Thanks are due to Prof. Qian Shijun of the Insti-
tute of Microbiology of the CAS for valuable suggestion.
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