Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність
Нещодавно нами очищено ендогенний дефензин із проростків сосни звичайної, а також клоновано кДНК, яка кодує дефензин 1 (PsDef1, Pinus sylvestris дефензин 1). Фрагмент кДНК дефензину 1, який кодує зрілу форму цього білка, клоновано у вектор pET42а(+) та індуковано його експресію у бактеріальній систе...
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Інститут молекулярної біології і генетики НАН України
2008
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| Цитувати: | Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність / В.А. Ковальова, Р.Т. Гут, І.Т. Гут // Біополімери і клітина. — 2008. — Т. 24, № 5. — С. 377-384. — Бібліогр.: 26 назв. — укр., англ. |
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nasplib_isofts_kiev_ua-123456789-1577402025-02-23T17:41:29Z Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність Получение рекомбинантного дефензина 1 сосны обыкновенной и его антифунгальная активность Production of Scots pine recombinant defensin 1 and its antifungal activity Ковальова, В.А. Гут, Р.Т. Gout, I.T. Структура та функції біополімерів Нещодавно нами очищено ендогенний дефензин із проростків сосни звичайної, а також клоновано кДНК, яка кодує дефензин 1 (PsDef1, Pinus sylvestris дефензин 1). Фрагмент кДНК дефензину 1, який кодує зрілу форму цього білка, клоновано у вектор pET42а(+) та індуковано його експресію у бактеріальній системі Escherichia coli. Оптимізовано умови продукування рекомбінантного білка PsDef1, злитого з глутатіон-S-трансферазою, у розчинній формі. Після афінного очищення цього білка на глутатіон-сефарозі та протеолізу фактором Ха отримано біологічно активний препарат рекомбінантного PsDef1, антифунгальна активність якого співмірна з такою ендогенного дефензину 1 сосни звичайної. Recently we have purified an endogenous defensin from Scots pine germs, and cloned cDNA encoding defensin 1 (PsDef1, Pinus sylvestris defensin 1). The cDNA region encoding a mature form of Scots pine defensin 1 was cloned into a vector pET 42a(+), and the expression of recombinant GST/PsDef1 in the Escherichia coli bacterial system was induced. The conditions of production of soluble GST-proteins were optimized. After purification of the recombinant protein by affinity chromatography on Glutathione-Sepharose column and proteolytic cleavage with Factor Xa, the functionally active preparation of recombinant PsDef1 was obtained. Its antifungal activity is similar to that of endogenous Scots pine defensin 1. Ранее нами очищен эндогенный дефензин из проростков сосны обыкновенной, а также клонирована кДНК, кодирующая дефензин 1 (PsDef1, Pinus sylvestris дефензин 1). Фрагмент кДНК дефензина 1, кодирующий зрелую форму этого белка, клонирован в вектор pET42а(+) и индуцирована его экспрессия в бактериальной системе Escherichia coli. Оптимизированы условия продукции рекомбинантного белка PsDef1, слитого с глутатион-S-трансферазой, в растворимой форме. После аффинной очистки этого белка на глутатион-сефарозе и протеолиза фактором Ха получен биологически активный препарат рекомбинантного PsDef1, антифунгальная активность которого соизмерима з таковой эндогенного дефензина 1 сосны обыкновенной. 2008 Article Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність / В.А. Ковальова, Р.Т. Гут, І.Т. Гут // Біополімери і клітина. — 2008. — Т. 24, № 5. — С. 377-384. — Бібліогр.: 26 назв. — укр., англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0007B5 https://nasplib.isofts.kiev.ua/handle/123456789/157740 577.112.083 uk Біополімери і клітина application/pdf application/pdf Інститут молекулярної біології і генетики НАН України |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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Ukrainian |
| topic |
Структура та функції біополімерів Структура та функції біополімерів |
| spellingShingle |
Структура та функції біополімерів Структура та функції біополімерів Ковальова, В.А. Гут, Р.Т. Gout, I.T. Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність Біополімери і клітина |
| description |
Нещодавно нами очищено ендогенний дефензин із проростків сосни звичайної, а також клоновано кДНК, яка кодує дефензин 1 (PsDef1, Pinus sylvestris дефензин 1). Фрагмент кДНК дефензину 1, який кодує зрілу форму цього білка, клоновано у вектор pET42а(+) та індуковано його експресію у бактеріальній системі Escherichia coli. Оптимізовано умови продукування рекомбінантного білка PsDef1, злитого з глутатіон-S-трансферазою, у розчинній формі. Після афінного очищення цього білка на глутатіон-сефарозі та протеолізу фактором Ха отримано біологічно активний препарат рекомбінантного PsDef1, антифунгальна активність якого співмірна з такою ендогенного дефензину 1 сосни звичайної. |
| format |
Article |
| author |
Ковальова, В.А. Гут, Р.Т. Gout, I.T. |
| author_facet |
Ковальова, В.А. Гут, Р.Т. Gout, I.T. |
| author_sort |
Ковальова, В.А. |
| title |
Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність |
| title_short |
Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність |
| title_full |
Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність |
| title_fullStr |
Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність |
| title_full_unstemmed |
Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність |
| title_sort |
отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2008 |
| topic_facet |
Структура та функції біополімерів |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/157740 |
| citation_txt |
Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність / В.А. Ковальова, Р.Т. Гут, І.Т. Гут // Біополімери і клітина. — 2008. — Т. 24, № 5. — С. 377-384. — Бібліогр.: 26 назв. — укр., англ. |
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Біополімери і клітина |
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Production of recombinant Scots pine defensin 1 and
characterization of its antifungal activity
V. A. Kovaleva, R. T. Gout, I. T. Gout1
National University of Forestry, Henerala Chuprynky 103, Lviv, 79057, Ukraine
1University College London, Gower str., London, WCIE 6 BT, Great Britain
We have recently reported affinity purification and molecular cloning of defensin (PsDef1, Pinus sylvestris
defensin 1) from Scots pine germinating seeds. In this study, we describe subcloning of PsDef1 mature form
into bacterial expression vector pET 42à(+) and the expression of recombinant GST/PsDef1 in Escherichia
coli. The conditions for the expression and affinity purification of soluble GST/PsDef1GST were defined.
Affinity purification of the recombinant GST/PsDef1 on glutathione-sepharose column and proteolytic
removal of GST moiety with Factor Xa allowed us to generate functionally active preparations of
recombinant PsDef1. Moreover, the antimicrobial activity of recombinant PsDef1 was found to be
comparable to that of endogenous Scots pine defensin.
Keywords: recombinant defensin PsDef1, expression, affinity purification, antifungal activity.
In tro duc tion. Defensins are evo lu tion al ly con ser va -
tive mol e cules of in nate im mu nity of plants, mol lusks,
in sects and an i mals which are char ac ter ized by small
size (< 10 kD), and amphipathic, b-lay ers-rich struc -
ture and sta bi lized disulfide bridges [1, 2]. These se cre -
tory pro teins (pep tides) are syn the sized as pre cur sors
with fur ther re moval of sig nal pep tides. The ma ture
form of defensins from dif fer ent spe cies has
antimicrobial prop er ties [3].
Defensins are widely spread in plant king dom and
could be pu ri fied from seeds, veg e ta tive and gen er a tive
or gans of many an gio sperm spe cies. The genes of these
de fen sive pro teins are ex pressed con sti tu tively or un -
der the in flu ence of bi oti cal fac tors in the pe riph eral
cell lay ers, form ing the first de fen sive line against
patho gens [4, 5]. Plant defensins pos sess broad range
of bi o log i cal ac tiv i ties, in clud ing antifungal [3, 5, 6],
an ti bac te rial [7], in hib it ing of pro tein ases and
a-amylases [8, 9] and block ing of Ñà2+- and
Na+-channels [10].
Elu ci da tion of bi o log i cal prop er ties of plant
defensins by in vi tro stud ies has been well com pli -
mented by stud ies with the use of trans gen ic plants. Re -
search from sev eral lab o ra to ries pro vided the ev i dence
that overexpression of defensins re sults in the in crease
of the re sis tance to patho genic in fec tions which re mains
ef fec tive over sev eral gen er a tions. For ex am ples, con sti -
tu tive ex pres sion of rad ish defensin RsAFP2 in to bacco
in creases plant’s re sis tance against fun gal in fec tion by
Alternaria longipes and A. solani, which af fects the
leaves. More over, the ex pres sion of Medicago sativa
defensin in the po ta toes pro vided a ro bust re sis tance
against patho genic fun gus of Verti cilli um dahliae [11].
It is nec es sary to note the most stud ies on the func -
tion of plant defensins have been per formed us ing the
mod els of Arabidopsis thaliana and crops [12]. The
mo lec u lar mech a nisms of antimicrobial re sis tance in
long-lived plants, in clud ing wood spe cies, have not
been elu ci dated clearly so far [13].
The re cent stud ies from our lab o ra tory have pro -
vided the ev i dence that defensin from the Scots pine
377
ISSN 0233-7657. Biopolymers and cell. 2008. vol. 24. N 5. Translated from Ukrainian
Ó V. A. KOVALEVA, R. T. GOUT, I. T. GOUT, 2008
seed lings has the po ten tial to bind ef fi ciently and spe cif -
i cally to phosphotyrosine ma trixes. The re sults of mass
spec trom e try anal y sis of pu ri fied defensin pro vided us
the data re quired for the mo lec u lar clon ing of Pinus
sylvestris defensin, which we termed PsDef1. A cDNA
clone, cor re spond ing to PsDef1 (Acc. No. EF455616),
was iso lated from a cDNA li brary of seven-days old
Scots pine seed lings, which was gen er ated in our lab o ra -
tory. The mo lec u lar stud ies al lowed us to de duct the
amino acid se quence of defensin 1 and to carry out a
com par a tive anal y sis of its pri mary struc ture and prop er -
ties with defensins from var i ous plants [16, 17].
In ad di tion, we have de vel oped a new pro to col for
the pu ri fi ca tion of en dog e nous defensin from Scots pine
seed lings and dem on strated its antifungal prop er ties [14,
15]. To fur ther ad vance our knowl edge on bi o log i cal
prop er ties of defensin 1, we have fo cused out re search
on gen er at ing large quan ti ties of re com bi nant PsDef1
and pro duc ing spe cific polyclonal and monoclonal an ti -
bod ies. The high level of pro tein pro duc tion can be
achieved by em ploy ing prokaryotic ex pres sion sys tem,
us ing GST or His-tag fu sions and Esch e richia coli. Here,
we de scribe for the first time con struc tion of an ex press -
ing plasmid cod ing for GST/ PsDef1 fu sion pro tein, af -
fin ity pu ri fi ca tion of re com bi nant pro tein and the re -
moval of GST moi ety by proteolytic cleav age with Fac -
tor X. The re sult ing prep a ra tion of ma ture form of
PsDef1 was shown to pos sess an ti bac te rial prop er ties
which are com pa ra ble to those of en dog e nous defensin
pu ri fied from Scots pine seed lings.
Ma te ri als and meth ods. The ex pres sion vec tor
pET42a(+) (‘Novagen’, USA) and E. coli strains XL-1
Blue and BL21 (DE3) have been used for clon ing and
ex pres sion stud ies. The phytopathogenic fun gus cul -
tures of Fusarium oxysporum YKM F-52897, F. solani
YKM F-50639, Bo try tis cinerea YKM F-16753,
Altermaria al ter nate YKM F-16752 and
phytopathogenic bac te ria of Erwinia carotovora YKM
B-1075 have been kindly pro vided by D.K. Zabolotny
In sti tute of Mi cro bi ol ogy and Vi rol ogy, Na tional
Acad emy of Sci ences of Ukraine. The necrotrophic
fun gus cul ture of Heterobasidion annosum has been
kindly pro vided by Dr. V. Kramarec (Lviv Na tional
For estry Uni ver sity of Ukraine). The cul ture of
oomicete Pythium dimorphum has been ob tained from
the In sti tute of Forestry Research (IBL, Poland).
Clon ing of ma ture form of Scots pine defensin into
pET42a(+) vec tor. A frag ment of cDNA cor re spond -
ing to ma ture form (ba sic do main) of Scots pine
defensin 1 (mPsDef1) was am pli fied by poly mer ase
chain re ac tion (PCR) us ing a tem plate cDNA clone for
defensin 1 and two prim ers: di rect primer CR765
(5’-CCATTCCATGGGAATGTGCAAAACCCCCA
G-3’) which con tains nu cle o tide se quences cor re -
spond ing to re stric tion endonuclease NcoI, fol lowed
by ini ti at ing Met and Gly in stead of Arg at the N-ter mi -
nus of ma ture form of Scots pine defensin 1; re verse
primer CR764 (5’-CATGAGAATTCTCAAGGGCA
GGGTTTGTA-3’) in cludes nu cle o tide se quences for
re stric tion endonuclease EcoRI, stop-codon and 3’-
cod ing frag ment of defensin 1.
The con di tions for PCR am pli fi ca tion have been
de scribed pre vi ously [17]. PCR prod ucts were an a -
lyzed by 1.5% agarose gel elec tro pho re sis in Tris-bo -
rate buffer, pH 8.3 (50 mM Tris-H3BO3, 2 mM EDTA)
un der 20 V/sm2. Am pli fied prod ucts were eluated from
the gel with the use of a gel ex trac tion kit from Qiagen
(USA). Pu ri fied DNA and pET42a(+) vec tor have been
hy dro lyzed with restrictases NcoI/EcoRI and then pre -
cip i tated us ing a stan dard method [18]. The T4
DNA-ligase (‘Fermentas’, Lith u a nia) was used for
link ing am pli fied cDNA frag ment of defensin 1 into
pET42a(+) vec tor in frame with the N-ter mi nally lo -
cated GST. The prod ucts of li ga tion were trans formed
in to XL-1 Blue com pe tent cells. The plasmid pu ri fi ca -
tion kit was used to pu rify plasmid DNA from an ti bi -
otic re sis tant col o nies. The pres ence of PsDef1 cDNA
in sert in pu ri fied plasmids was de tected by re stric tion
anal y sis with endonucleases XhoI and EcoRI as well as
by PCR anal y sis with prim ers of C765 and CR764. The
se quence of am pli fied PsDef1 in three re sult ing
plasmids was verified by sequence analysis on
automatic DNA sequencer ABI 73 TM.
The ex pres sion of re com bi nant PsDef1 in bac te rial
sys tem E. coli. BL21(DE3) com pe tent cells were trans -
formed with pET42a/mPsDef1 con struct and ob tained
col o nies were grown at 37oC in LB-me dium con tain ing
50 mg /ml kanamycin. When the op ti cal den sity of bac -
te rial cul tures reached A600 = 0.6, 0.4 mM
isopropyl-thiogalactosid (IPTG) was added to in duce
the ex pres sion of re com bi nant pro tein. Af ter the in cu -
ba tion of in duced cul tures for 2 hours, bac te rial cells
378
KOVALEVA V. A., GOUT R. T., GOUT I. T.
were col lected by centrifugation at 4000 g for 20 min -
utes. The pel let of bac te ria was washed with ice-cold
PBS, frozen and preserved at -70 Ñ.
The pu ri fi ca tion of re com bi nant PsDef1 con ju -
gated with glutathion-S-transferase by af fin ity chro -
ma tog ra phy. All pu ri fi ca tion steps were per formed at 4
Ñ. Cell pel let ( 2.5 g) was re sus pend and lysed in 15 ml
buffer A, con sist ing of: 10 mM Tris-HCl, (pH 7.5), 150
mM NaCl, 50 mM NaF, 5 mM EDTA, 1% Tri ton
X-100, 1mM PMSF with proteinase cock tail of in hib i -
tors (‘Roche’, France). Cells were de stroyed by me -
chan i cal grind ing in a ho mog e nizer. The lysate of cells
was cen tri fuged at 17000 g for 20 min. The supernatant
was in cu bated on the wheel for 2 hours with a 50% sus -
pen sion of Glutathione-sepharose (‘Amersham’, Great
Brit ain), pre-washed in buffer A. The beads were
washed on the wheel with 50mM Tris-HCl, (pH 7.5),
150 mM NaCl with 0.05% of Twin-20 three times for 5
min utes and twice with 50 mM Tris-HCl, (pH 8.0).
Bound pro teins were eluated from
Glutathione-sepharose with 50 mM re duced
glutathione in 50 mM Tris-HCl, (pH 8.0), 150 mM
NaCl. The eluted frac tions were ana lysed by Brad ford
as say to mea sure pro tein con cen tra tions and by
SDS-PAGE for ex am in ing the qual ity of pu ri fied
GST-PsDef1. The frac tions, which con tained
GST-PsDef1 were com bined and di a lyzed against
buffer B, con tain ing 20 mM Tris-HCl (pH 7.5), 150
mM NaCl, 1 mM DTT, in or der to re move glutathione.
The con cen tra tion of gen er ated prep a ra tions of
GST-PsDef1 was mea sured by Brad ford as say. Pro -
duced GST-PsDef1 was stored at -20 Ñ in presence of
50% glycerol.
Pro duc tion of ma ture form of PsDef1. The Fac tor
Xa (‘Sigma’, USA) was used to cleave re com bi nant
Scots pine defensin (PsDef1) from the fu sion
polypeptide GST-PsDef1. In brief, GST-PsDef1 was
sorbed on the glutathione-sepharose and equil i brated
with buffer con tain ing 50 mM Tris-HCl (pH 8.0), 100
mM NaCl, 2mM CaCl2. Then, Fac tor Xa was added at
the con cen tra tion of 2 mg per 100 mi cro grams of
GST-PsDef1. The re ac tion was per formed un der room
tem per a ture for 5 hours on the wheel. Beads were then
col lected by centrifugation at 3000 g for 1 min ute. The
supernatant liq uid was col lected and loaded onto
Centricon YM 30 (‘Millipore’, USA) for con cen tra tion
at 10000 g. The fil trate was col lected and pro tein con -
cen tra tion mea sured by Brad ford as say and an a lyzed
by SDS-elec tro pho re sis in 15% PAAG in a Tris-tricine
buffer system [19].
Antimicrobial ac tiv ity of Scots pine re com bi nant
defensin 1 as say. To study the ac tiv ity of gen er ate prep a -
ra tions of re com bi nant defensin 1 the pieces of fun gus
my ce lium were placed into the cen ter of Petri dishes
(90x15 mm) with 1.8% po tato-dex trose agar. When the
di am e ter of fun gus col ony had reached 3 cm the ster ile
discs of fil ter pa per were placed onto the agar 0.5 cm
away from the col ony edge. The aliquots (100 ml) of dif -
fer ent pro tein con cen tra tions of PsDef1 were ap plied on
the pa per discs. Dis tilled wa ter has been used as a neg a -
tive con trol and dif fer ent con cen tra tions of en dog e nous
defensin pu ri fied from Scots pine seed lings were also
tested. The plates were then in cu bated at 23 °Ñ un til my -
ce lium en vel oped the disks with con trol so lu tion, while
growth-in hib it ing zones were formed around the discs
con tain ing recombinant defensin 1.
A quan ti ta tive anal y sis of the antifungal ac tiv ity of
Scots pine defensin was per formed as pre vi ously de -
scribed [20]. The spores were ex tracted from
sporulating fun gus cul tures, which have been grown on
the 1.8%-po tato-dextose agar. The spore sus pen sion
has been fil tered with dou ble layer of ster ile gauze. 80
microlitres of spore sus pen sion (2x104 spores/ml) in
po tato-dex trose broth and 20 microlitres of ster ile
defensin so lu tion (with con cen tra tions 0.2, 0.5, 1, 5,10
mg/ml) were added into the wells of 96-well microtiter
plate. The plates were in cu bated in dark at 23 Ñ. The
test sam ple con sisted of 20 milli litres of ster ile
distillated wa ter. Af ter 48 hours of in cu ba tion, the op ti -
cal den sity of fun gal sus pen sion was measured in each
well under 595 nm.
Re sults and dis cus sion. The pres ence of an
endoplasmic re tic u lum sig nal pep tide, which is re -
moved dur ing the pro tein pro cess ing, is a fea ture of all
plant defensins [3]. The se quence anal y sis of PsDef1
cDNA clones in di cated an open read ing frame of 83
amino acid res i dues. The first 33aa cor re spond to the
N-ter mi nal sig nal pep tide and re main ing 50aa form a
ba sic do main of ma ture defensin. To pro duce large
quan ti ties of re com bi nant ma ture form of defensin 1
we de cided to em ploy bac te rial ex pres sion sys tem of E.
coli, as it al lows ef fi cient, fast and rel a tively in ex pen -
379
PRODUCTION OF RECOMBINANT SCOTS PINE DEFENSIN 1
sive way of gen er at ing re com bi nant pro tein. Since the
eukaryotic sig nal se quence is not rec og nized in the
prokaryotic sys tem, we have cloned a DNA frag ment,
coding the mature form of PsDef1 into bacterial
expression vector.
A DNA frag ment, cor re spond ing to ma ture form of
mPsDef1, was am pli fied by PCR us ing PsDef1 cDNA
as a tem plate and two spe cific prim ers. Agarose gel
elec tro pho retic anal y sis of PCR prod ucts re vealed a
ma jor band of ap prox i mately 200bp which closely cor -
re lates to ex pected size of 174 bp. (Fig ure 1, a). The
prod uct of am pli fi ca tion was di gested with NcoI and
XhoI and li gated into linearized pET42a(+) vec tor. The
XL-1 Blue com pe tent cells were trans formed with the
li ga tion mix and plated for an ti bi otic se lec tion. Plasmid
DNA was pu ri fied from col o nies grown on agar plates
con tain ing kanamycin. The pres ence of PsDef1 in sert
in pu ri fied plasmid DNAs was ana lysed by PCR am pli -
fi ca tion (Fig ure 1b) and re stric tion anal y sis with NcoI
and XhoI endonuclease (Fig ure 1c). The ob tained re -
sults clearly in di cate that PsDef1 cDNA is present in 4
out of 6 plasmid preparations.
To study ex pres sion the BL21 (DE3) com pe tent
cells were trans formed by the gen er ated
pET42a/mPsDef1 plasmid . In par al lel, the vec tor
(pET42a alone) was also in tro duced into the BL21
(DE3) com pe tent cells. The ex pres sion of re com bi nant
GST-PsDef1 was in duced in BL21 (DE3) by the ad di -
tion of 1mM IPTG. The SDS-PAGE anal y sis of to tal
lysates from in ducted cells re vealed the pres ence of re -
com bi nant pro tein of an ex pected mo lec u lar weight,
35.5 kD (Fig ure 2). This anal y sis also showed that
GST-PsDef1 fu sion pro tein is sol u ble in buffer con -
tain ing 1%Tri ton X-100. We also found that to tal cell
lysate con tained ap prox i mately 15% of GST-PsDef1
fu sion pro teins. No ta bly, some amounts of
GST-PsDef1 were also observed among
detergent-insoluble proteins.
Tak ing into ac count that re com bi nant GST-PsDef1
was found in both Tri ton sol u ble and in sol u ble frac -
tions, in the form of in clu sion bod ies, we fo cused our
ef forts on op ti miz ing the con di tions for gen er at ing cul -
tur ing max i mum level of tri ton-sol u ble GST-PsDef1.
By test ing var i ous tem per a tures for the ex pres sion of
GST-PsDef1 we found that the op ti mal tem per a ture for
380
KOVALEVA V. A., GOUT R. T., GOUT I. T.
Fig.1. Cloning of a cDNA fragment encoding
mature form of the Scots pine defensin 1: (a) 1 –
the products of PCR amplification of cDNA
PsDef1; Ì – 1 kb Plus DNA Ladder GibñoBRL;
(b) the analysis of pET42a/mPsDef1 plasmid by
PCR (lane 1); Ì – 1 kb DNA Ladder Fermentas;
(c) restriction analysis of pET42a/mPsDef1
plasmid using endonucleases Xho 1 and Nco 1
(lane 1); Ì – 1 kb DNA Ladder Fermentas.
Fig.2. SDS-PAAG results of GST-PsDef1 exspession in E. coli
BL21(DE3) strain: 1, 2, 3, 4 – fractions of the total cell lysates; 5, 6,
7, 8 – triton-soluble protein fractions; Ì – broad-range protein
standards BioRad. Lanes 1, 2, 5, 6 –uninduced bacterial cultures; 3,
4, 7, 8 – proteins from the cells induced by the addition of IPTG; 1,
3, 5, 7 – the cells transformed by plasmid without insert; 2, 4, 6, 8
–the cells, transformed by recombinant vector. Amount of proteins
on each of the lines is equivalent to the 100 µl cell suspension.
gen er at ing the high est level of re com bi nant
GST-PsDef1 is 37 °C, while low er ing the in duc tion
tem per a ture to 28-30°C(of ten used in or der to fold
polypeptide chain of re com bi nant prod uct cor rectly)
re sulted in no tice able re duc tion of tri ton-sol u ble re -
com bi nant pro tein. The same ef fect was no ticed with
the in crease of the in duc tion time. In sum mary, the op -
ti mal ex pres sion of sol u ble GST-PsDef1 was achieved
by in cu bat ing trans formed cells with 0.4mM IPTG for
2 hours at 37°Ñ.
The use of pET42a ex pres sion plasmid im plied the
pro duc tion of re com bi nant PsDef1, con ju gated with
GST. The ex pres sion of PsDef1 fused to GST al lowed
us to em ploy af fin ity chro ma tog ra phy on
glutathione-sepharose. As shown in Fig ure 3a, af fin ity
pu ri fi ca tion from cells ex press ing plasmid alone or
pET42a/mPsDef1 re sults in highly en riched prep a ra -
tions of GST and GST-PsDef1 re spec tively. Both prep -
a ra tions con tain some mi nor bands which ex hibit sim i -
lar pat tern in both sam ples. In or der to elim i nate the
pos si bil ity of non-spe cific pro tein sorbtion onto
glutathione-sepharose, we in cu bated af fin ity beads
with Tri ton-sol u ble pro teins of uninduced cells. The re -
sults pre sented in Fig ure 3b (lane 3), show very lit tle of
non-spe cific bind ing to Glutatione sepharose from
lysates of uninduced cells. We have also elim i nated the
pos si bil ity that ob served mi nor bands are bind ing part -
ners of re com bi nant GST and GST-PsDef1, as they
were not eluted from the af fin ity ma trixes in the pres -
ence of 0.5 M NaCl, but were readily eluated with 50
mM of re duced glutathione. As seen in the spec trum of
tri ton-sol u ble pro teins (Fig ure 3b, lanes 1 and 2) mi nor
bands ap pear only af ter the in duc tion of re com bi nant
pro teins. There fore, the mi nor pro teins are the prod ucts
of ei ther deg ra da tion or in com plete trans la tion of
GST-PsDef1. The affinity purified preparations of
recombinant GST-PsDef1 contained approximately
75% the fusion protein.
The GST-PsDef1 fu sion pro tein has mo lec u lar
mass of ap prox i mately 35.5 kDa. The fu sion con sists of
220 amino ac ids of GST at the N-ter mi nus and 51aa
cor re spond ing to ma ture form of PsDef1. There is also
a 58aa in sert be tween GST and PsDef1, which con tains
Fac tor X re stric tion site and some se quences from
pET42a vec tor. Fac tor Xa is a serine proteinase, which
rec og nizes and hydrolyzes the se quences of Ile Glu Gly
Arg af ter cer tain amino acid se quences. The clon ing
strat egy of pET42a/PsDef1 ex pres sion vec tor al lows
proteolytic re moval of the ma ture form of PsDef1, con -
sist ing of 52 amino-ac ids, where the first two amino ac -
ids are the ini ti at ing methionine and then glycine
instead of arginine.
To re move GST moi ety from the
GST-PsDef1fusion pro tein, we tested two cleav age
pro to cols. Vari ant A: the cleav age of re com bi nant
GST-PsDef1with Fac tor Xa was car ried out in so lu tion.
381
PRODUCTION OF RECOMBINANT SCOTS PINE DEFENSIN 1
Fig.3. Isolation of the recombinant Scots pine defensin 1 from the E. coli BL21(DE3) cell lysates: (à) purification of recombinant
GST?PsDef1 onto affinity sorbent: 1, 2 – triton-soluble proteins from the cells transformed by pET-42à and pET-42à-ÐsDef1 plasmids,
respectively; 3 – GST preparation; 4 – GST-ÐsDef1 preparation; (b) 1, 2 – triton-soluble proteins from the uninduced and induced cells
transformed by pET-42à/mDef1 plasmid, respectively; 3, 4 – proteins that bound to glutathione-Sepharose from lysates of the uninduced and
IPTG-induced cells, respectively. Proteins were separated by 12% SDS-PAAG electrophoresis in Laemmle’s system; (c) ? fractions after the
proteolytic digestion of GST- ÐsDef1 with Õà factor: 1, 2 – immobilized proteins on glutathione-Sepharose before and after digestion,
respectively. 15% Tricine-SDS-PAAG was performed for separation of each fraction. Gels were stained by Coomassie G-250.
The re moval of cleaved GST was per formed on
Glutathione-sepharose. The sep a ra tion of Fac tor Xa
from cleaved PsDef1was car ried out by centrifugation
on Centricon YM 30 col umns (‘Millipore’).
Vari ant B: pro te ol y sis of the GST-PsDef1 fu sion
pro tein was per formed on Glutathione sepharose. Here,
Fac tor Xa was added to the Glutathione-sepharose sus -
pen sion with im mo bi lized re com bi nant GST-PsDef1.
Af ter the cleav age, PsDef1 was re leased into so lu tion,
which was then pro cessed for the re moval of Fac tor Xa
proteinase as de scribed above. Hav ing both pro to cols
com pared, we found that the cleav age on beads is
faster, more eco nom i cal and al lows greater output of
PsDef1 protein.
Fur ther stud ies in di cated that op ti mal cleav age of
GST-PsDef1 re quires 20mkg of Fac tor Xa per 1 mg of
fu sion pro tein. The time-course anal y sis of Fac tor Xa
cleav age showed that full cleav age of GST-PsDef1 re -
quires 5 hours at room tem per a ture (Fig ure 3, c). As
seen in lane 2, the band cor re spond ing to the
GST-PsDef1fusion dis ap pear af ter 5 hrs of di ges tion,
while the ma jor band of ap prox i mately 30kDa, which
cor re spond to GST alone, is clearly detected.
So, the pro to col de signed for Fac tor Xacleav age of
GST-PsDef1 al lows the pro duc tion of ho mo ge neous
prep a ra tions of re com bi nant PsDef1. The fi nal out put
of PsDef1was ap prox i mately 10% from to tal
GST/PsDef1 fu sion pro tein, im mo bi lized on
Glutathione-sepharose.
The ac tiv ity of re com bi nant pro teins de pends on
cor rect fold ing of their polypeptide chain. Ter tiary
struc ture of plant defensin con sists of three antiparallel
b-sheets and an a-he lix, sta bi lized with 4 disulfide
bridges. So, the ac tiv ity of re com bi nant defensin could
be lost from in ad e quate for ma tion of -Cys-Cys pairs.
The study of mech a nisms of ac tion of plant defensins,
per formed in the Braeckert group [23], re vealed sig nif -
i cant role of two re gions in Rs-AFP2 which me di ated
the in ter ac tion with re cep tors on the fun gus mem brane.
These re gions are formed by amino acid res i dues, lo -
cated in dif fer ent parts of the polypeptide chain. For ex -
am ple, one re gion in volves Thr10, Ser12, Leu28 and
Phe 49. The site-di rected sub sti tu tion of these amino
ac ids with amino res i dues which disturbe the 3D struc -
ture of defensin, leads to the loss of antifungal activity.
Next, we tested the antifungal ac tiv ity of re com bi -
nant GST-PsDef1 and cleaved PsDef1. In this study,
we used the fungi from the ge nus of Fusarium. No
antifungal ac tiv ity was ob served in the pres ence of re -
com bi nant GST/PsDef1 (even, when the con cen tra tion
382
KOVALEVA V. A., GOUT R. T., GOUT I. T.
Fig.4. Inhibitory effect of purified recombinant ÐsDef1 on the growth of Heterobasidion annosum (à) and Fusarium solani (b): K - the sterile
distilled water; 1 – 5 µg of preparation; 2 – 10 µg of preparation.
of GST/PsDef1 in the cul ture me dium was 50 mg/ml).
When, the ac tiv ity of PsDef1 was tested in the growth
in hib it ing as say, we reproducibly ob served strong
antifungal prop er ties of gen er ated prep a ra tions of
PsDef1. The antifungal ac tiv ity was mea sured by the
for ma tion of zones, where the growth of my ce lium of
H. annosum and F solani was inhibited (picture 4).
These find ings pro vided the vi sual ev i dence for the
in hi bi tion of fun gal growth by re com bi nant PsDef1.
The next task was to de velop the as say which would al -
low the quantitation of PsDef1 antifungal ac tiv ity. To
do so, we em ployed the microspectrophotometric
method in which the op ti cal den sity of fungi spore sus -
pen sion was mea sured af ter 48 hours of in cu ba tion in
po tato-dex trose broth, con tain ing var i ous con cen tra -
tions of re com bi nant defensin. When max i mum con -
cen tra tion (4 mi cro grams/mil li li ter) of PsDef1 was
used, the in hi bi tion of fun gal growth was: for F solani
by 81%, for F oxysporum by 50%, for H annosum by
71%, for Pythium dimorphum by 84%. These re sults
in di cate dif fer en tial ac tiv ity of re com bi nant PsDef1 to -
wards dif fer ent fungi. Dif fer en tial in hib i tory ef fect of
plant defensins to wards dif fer ent fungi spe cies has
been pre vi ously de scribed [24].
The ac tiv ity of antimicrobial prep a ra tions is mea -
sured by the value of IC50. We have es ti mated IC50 of re -
com bi nant PsDef1 for F solani, F oxysporum, B.
cinerea, and P. dimorphum. Their rates are 1.4, 4.0,
2.6, 1.2, 2.0 mi cro grams per mil li li ter re spec tively. The
defensin PgD1 from Picea glauca, which ex hib its 80%
iden tity to PsDef1, has been re cently ex pressed in bac -
te rial sys tem and its antimicrobial ac tiv ity has been
stud ied. The pu ri fied re com bi nant prep a ra tions of
PgD1 in hib ited the growth of F. oxysporum by 95.2%,
which is sim i lar to that of Scots pine defensin 1 [25].
In pre vi ous stud ies, we re searched the antifungal
ac tiv ity of en dog e nous defensin pu ri fied from Scots
pine seed lings [15, 26]. These re sults al lowed us to
com pare the val ues of IC50 for re com bi nant and en dog -
e nous defensin. The re sults of this com par a tive anal y -
sis are rep re sented in the di a gram of Fig ure 5. We
found that the ac tiv ity of re com bi nant PsDef1 is 1.5-2
times lower then that of en dog e nous defensin from
seed lings. This is prob a bly the re sult of par tial pro te ol -
y sis of PsDef1 at Gly-Arg44 site, which might be re -
cog nised by Fac tor Xa.
In sum mary, we de scribed for the first time the ex -
pres sion and af fin ity pu ri fi ca tion of re com bi nant
defensin 1 from Scots pine pos sess ing a strong
antifungal ac tiv ity. The avail abil ity of re com bi nant
PsDef1 pro vides us with an ex cel lent op por tu nity to
study bio chem i cal and func tional prop er ties of plant
defensins and to elu ci date mo lec u lar mech a nisms of
their ac tion. In ad di tion, re com bi nant prep a ra tion of
PsDef1 will be used for mak ing spe cific polyclonal and
monoclonal an ti bod ies and to study the ex pres sion pro -
file of defensin 1 in re sponse to var i ous
growth-regulatory agents and pathogenic organisms.
Â. À. Êî âàëü î âà, Ð. Ò. Ãóò, ². Ò. Ãóò
Îòðè ìàí íÿ ðå êîìá³íà íòíî ãî äå ôåí çè íó 1 ñî ñíè çâè ÷àé íî¿ òà
éîãî àí òè ôóí ãàëü íà àê òèâí³ñòü
Ðå çþ ìå
Íå ùî äàâ íî íàìè î÷è ùå íî åí äî ãåí íèé äå ôåí çèí ³ç ïðî ðîñòê³â
ñî ñíè çâè ÷àé íî¿, à òà êîæ êëî íî âà íî êÄÍÊ, ÿêà êîäóº äå ôåí çèí
1 (PsDef1, Pinus sylvestris äå ôåí çèí 1). Ôðàã ìåíò êÄÍÊ äå ôåí -
çè íó 1, ÿêèé êîäóº çð³ëó ôîð ìó öüî ãî á³ëêà, êëî íî âà íî ó âåê òîð
pET42à(+) òà ³íäó êî âà íî éîãî åêñïðåñ³þ ó áàê òåð³àëüí³é ñèñ -
òåì³ Escherichia coli. Îïòèì³çî âà íî óìî âè ïðî äó êó âàí íÿ ðå -
êîìá³íà íòíî ãî á³ëêà PsDef1, çëè òî ãî ç
ãëó òàò³îí-S-òðàíñ ôå ðà çîþ, ó ðîç ÷èíí³é ôîðì³. ϳñëÿ àô³ííî -
ãî î÷è ùåí íÿ öüî ãî á³ëêà íà ãëó òàò³îí-ñå ôà ðîç³ òà ïðî òå îë³çó
ôàê òî ðîì Õà îò ðè ìà íî á³îëîã³÷íî àê òèâ íèé ïðå ïà ðàò ðå -
êîìá³íà íòíî ãî PsDef1, àí òè ôóí ãàëü íà àê òèâí³ñòü ÿêî ãî
ñï³âì³ðíà ç òà êîþ åí äî ãåí íî ãî äå ôåí çè íó 1 ñî ñíè çâè ÷àé íî¿.
Êëþ ÷îâ³ ñëî âà: ðå êîìá³íà íòíèé äå ôåí çèí PsDef1,
åêñïðåñ³ÿ, àô³ííå î÷è ùåí íÿ, àí òè ôóí ãàëü íà àê òèâí³ñòü.
383
PRODUCTION OF RECOMBINANT SCOTS PINE DEFENSIN 1
Fig.5. Comparative analysis of the antifungal activity of the
endogenous PsDef1(1) with that of its recombinant analog (2).
Â. À. Êî âà ëå âà, Ð. Ò. Ãóò, È. Ò. Ãóò
Ïî ëó ÷å íèå ðå êîì áè íàí òíî ãî äå ôåí çè íà 1 ñî ñíû îá ûê íî âåí íîé
è åãî àí òè ôóí ãàëü íàÿ àê òèâ íîñòü
Ðå çþ ìå
Ðà íåå íàìè î÷è ùåí ýí äî ãåí íûé äå ôåí çèí èç ïðî ðîñ òêîâ ñî ñíû
îá ûê íî âåí íîé, à òàê æå êëî íè ðî âà íà êÄÍÊ, êî äè ðó þ ùàÿ äå ôåí -
çèí 1 (PsDef1, Pinus sylvestris äå ôåí çèí 1). Ôðàã ìåíò êÄÍÊ äå -
ôåí çè íà 1, êî äè ðó þ ùèé çðå ëóþ ôîð ìó ýòî ãî áåë êà, êëî íè ðî âàí â
âåê òîð pET42à(+) è èí äó öè ðî âà íà åãî ýêñ ïðåñ ñèÿ â áàê òå ðè àëü -
íîé ñèñ òå ìå Escherichia coli. Îïòè ìè çè ðî âà íû óñëî âèÿ ïðî äóê -
öèè ðå êîì áè íàí òíî ãî áåë êà PsDef1, ñëè òî ãî ñ
ãëó òà òè îí-S-òðàíñ ôå ðà çîé, â ðàñ òâî ðè ìîé ôîð ìå. Ïîñ ëå àô -
ôèí íîé î÷èñ òêè ýòî ãî áåë êà íà ãëó òà òè îí-ñå ôà ðî çå è ïðî òå î -
ëè çà ôàê òî ðîì Õà ïî ëó ÷åí áè î ëî ãè ÷åñ êè àê òèâ íûé ïðå ïà ðàò
ðå êîì áè íàí òíî ãî PsDef1, àí òè ôóí ãàëü íàÿ àê òèâ íîñòü êî òî -
ðî ãî ñî èç ìå ðè ìà ç òà êî âîé ýí äî ãåí íî ãî äå ôåí çè íà 1 ñî ñíû îá -
ûê íî âåí íîé.
Êëþ ÷å âûå ñëî âà: ðå êîì áè íàí òíûé äå ôåí çèí PsDef1, ýêñ -
ïðåñ ñèÿ, àô ôèí íàÿ î÷èñ òêà, àí òè ôóí ãàëü íàÿ àê òèâ íîñòü.
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UDC 577.112.083
Received 16.05.08
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KOVALEVA V. A., GOUT R. T., GOUT I. T.
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