Kлонування та аналіз кДНК дефензину 1 сосни звичайної
Антифунгальний білок з молекулярною масою 5,6 кДа очищено з коренів семиденних проростків сосни звичайної. Мас-спектрометричним аналізом показано його приналежність до рослинних дефензинів. кДНК дефензину 1 сосни (PsDef1) довжиною 252 п. н. отримано методом ПЛР-ампліфікіції з кДНК бібліотеки Pinus s...
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| Cite this: | Kлонування та аналіз кДНК дефензину 1 сосни звичайної / В.А. Ковальова, І.Т. Гут, Р.Г. Кіямова, В.В. Філоненко, Р.Т. Гут // Біополімери і клітина. — 2007. — Т. 23, № 5. — С. 398-404. — Бібліогр.: 25 назв. — укр., англ. |
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Ковальова, В.А. Гут, І.Т. Кіямова, Р.Г. Філоненко, В.В. Гут, Р.Т. 2019-06-20T04:14:15Z 2019-06-20T04:14:15Z 2007 Kлонування та аналіз кДНК дефензину 1 сосни звичайної / В.А. Ковальова, І.Т. Гут, Р.Г. Кіямова, В.В. Філоненко, Р.Т. Гут // Біополімери і клітина. — 2007. — Т. 23, № 5. — С. 398-404. — Бібліогр.: 25 назв. — укр., англ. 0233-7657 http://dx.doi.org/10.7124/bc.000779 https://nasplib.isofts.kiev.ua/handle/123456789/157498 577.112. Антифунгальний білок з молекулярною масою 5,6 кДа очищено з коренів семиденних проростків сосни звичайної. Мас-спектрометричним аналізом показано його приналежність до рослинних дефензинів. кДНК дефензину 1 сосни (PsDef1) довжиною 252 п. н. отримано методом ПЛР-ампліфікіції з кДНК бібліотеки Pinus sylvestris L. та клоновано у вектор рЕТ 23d(+). PsDef1 кДНК кодує білок з 83 амінокислотних залишків (а. з.), який містить N-кінцевий сигнальний пептид з 33 а. з. Зріла форма білка вирізняється наявністю специфічних консервативних залишків, властивих усім рослинним дефензинам. Показано структурну та функціональну подібність між PsDef1 та дефензинами групи І. Antifungal protein with a molecular weight 5.6 kDa was purified from 7-days old Scots pine seedlings. LC-MS/MS analysis revealed this protein to belong to plant defensins. A pine defensin 1 cDNA (PsDef1), 252 b. p. long, was obtained by PCR amplification from Pinus sylvestris L. cDNA library and was cloned into vector pET 23d(+). PsDef1 cDNA encodes 83-amino acid protein with 33-amino acid N-terminal signal peptide. The mature protein is characterized by the presence of specific conserved residues common to all plant defensins. Structural and functional similarity between PsDef1 and defensins of group 1 has been shown. Антифунгальный белок с молекулярной массой 5,6 кДа очищен из корней семидневных проростков сосны обыкновенной. Масс-спектрометрическим анализом показана його принадлежность к растительным дефензинам. кДНК дефензина 1 сосны (PsDef1) длиной 252 п. н. получена методом ПЦР-амплификации из кДНК библиотеки Pinus sylvestris L. и клонирована в вектор рЕТ23d(+). PsDef1 кДНК кодирует белок из 83 аминокислотных остатков (а. о.) с N-концевым сигнальным пептидом из 33 а. о. Для зрелой формы характерно наличие специфических консервативных остатков, свойственных всем растительным дефензинам. Показано структурное и функциональное сходство между PsDef1 и дефензинами группы І. uk Інститут молекулярної біології і генетики НАН України Біополімери і клітина Структура та функції біополімерів Kлонування та аналіз кДНК дефензину 1 сосни звичайної Kлонирование и анализ кДНК дефензина 1 сосны обыкновенной Cloning and analysis of defensin 1 cDNA from Scots pine Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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| title |
Kлонування та аналіз кДНК дефензину 1 сосни звичайної |
| spellingShingle |
Kлонування та аналіз кДНК дефензину 1 сосни звичайної Ковальова, В.А. Гут, І.Т. Кіямова, Р.Г. Філоненко, В.В. Гут, Р.Т. Структура та функції біополімерів |
| title_short |
Kлонування та аналіз кДНК дефензину 1 сосни звичайної |
| title_full |
Kлонування та аналіз кДНК дефензину 1 сосни звичайної |
| title_fullStr |
Kлонування та аналіз кДНК дефензину 1 сосни звичайної |
| title_full_unstemmed |
Kлонування та аналіз кДНК дефензину 1 сосни звичайної |
| title_sort |
kлонування та аналіз кднк дефензину 1 сосни звичайної |
| author |
Ковальова, В.А. Гут, І.Т. Кіямова, Р.Г. Філоненко, В.В. Гут, Р.Т. |
| author_facet |
Ковальова, В.А. Гут, І.Т. Кіямова, Р.Г. Філоненко, В.В. Гут, Р.Т. |
| topic |
Структура та функції біополімерів |
| topic_facet |
Структура та функції біополімерів |
| publishDate |
2007 |
| language |
Ukrainian |
| container_title |
Біополімери і клітина |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Kлонирование и анализ кДНК дефензина 1 сосны обыкновенной Cloning and analysis of defensin 1 cDNA from Scots pine |
| description |
Антифунгальний білок з молекулярною масою 5,6 кДа очищено з коренів семиденних проростків сосни звичайної. Мас-спектрометричним аналізом показано його приналежність до рослинних дефензинів. кДНК дефензину 1 сосни (PsDef1) довжиною 252 п. н. отримано методом ПЛР-ампліфікіції з кДНК бібліотеки Pinus sylvestris L. та клоновано у вектор рЕТ 23d(+). PsDef1 кДНК кодує білок з 83 амінокислотних залишків (а. з.), який містить N-кінцевий сигнальний пептид з 33 а. з. Зріла форма білка вирізняється наявністю специфічних консервативних залишків, властивих усім рослинним дефензинам. Показано структурну та функціональну подібність між PsDef1 та дефензинами групи І.
Antifungal protein with a molecular weight 5.6 kDa was purified from 7-days old Scots pine seedlings. LC-MS/MS analysis revealed this protein to belong to plant defensins. A pine defensin 1 cDNA (PsDef1), 252 b. p. long, was obtained by PCR amplification from Pinus sylvestris L. cDNA library and was cloned into vector pET 23d(+). PsDef1 cDNA encodes 83-amino acid protein with 33-amino acid N-terminal signal peptide. The mature protein is characterized by the presence of specific conserved residues common to all plant defensins. Structural and functional similarity between PsDef1 and defensins of group 1 has been shown.
Антифунгальный белок с молекулярной массой 5,6 кДа очищен из корней семидневных проростков сосны обыкновенной. Масс-спектрометрическим анализом показана його принадлежность к растительным дефензинам. кДНК дефензина 1 сосны (PsDef1) длиной 252 п. н. получена методом ПЦР-амплификации из кДНК библиотеки Pinus sylvestris L. и клонирована в вектор рЕТ23d(+). PsDef1 кДНК кодирует белок из 83 аминокислотных остатков (а. о.) с N-концевым сигнальным пептидом из 33 а. о. Для зрелой формы характерно наличие специфических консервативных остатков, свойственных всем растительным дефензинам. Показано структурное и функциональное сходство между PsDef1 и дефензинами группы І.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/157498 |
| citation_txt |
Kлонування та аналіз кДНК дефензину 1 сосни звичайної / В.А. Ковальова, І.Т. Гут, Р.Г. Кіямова, В.В. Філоненко, Р.Т. Гут // Біополімери і клітина. — 2007. — Т. 23, № 5. — С. 398-404. — Бібліогр.: 25 назв. — укр., англ. |
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| fulltext |
Mo lec u lar clon ing and characterization of defensin 1
from Scots pine
V. A. Kovalyova, I.T. Gout1, R. G. K³yamova2, V. V. Filonenko2, R. T. Gout
De part ment of For estry, Na tional For estry Uni ver sity of Ukraine, Chuprynka
St., 103, Lviv, 79057, Ukraine
1Uni ver sity Col lage Lon don, Gower Street, Lon don WC1E 6BT, United King dom
2In sti tute of mo lec u lar bi ol ogy and ge net ics NAS of Ukraine
Academicain Zabolotnog str., 150, Kyiv, 03680 Ukraine
vakovaleva@mail.ru
A pro tein with antifungal ac tiv ity and a mo lec u lar weight of 5.6 kDa was pu ri fied from 7-day-old Scots pine
seed lings. The LC-MS/MS anal y sis re vealed that pu ri fied pro tein is highly ho mol o gous to plant defensins.
The ob tained se quences and da ta base searches al lowed us to de sign a set of prim ers for mo lec u lar clon ing
of a full length cod ing se quence, cor re spond ing to Scots pine defensin 1 (PsDef1). This was achieved by a
PCR ap proach, us ing Pinus sylvestris ñDNA li brary as a tem plate. The spe cific prod uct of PCR am pli fi ca -
tion was cloned into ðÅÒ 23d(+) vec tor and se quenced. Bioinformatic anal y sis of gen er ated se quences
showed that the cod ing se quence of PsDef1 cDNA has the po ten tial to en code a pro tein of 83 amino ac ids in
length. The first 33 amino ac ids cor re spond to the N-ter mi nal sig nal pep tide, which is re moved af ter pro -
cess ing. The ma ture pro tein pos sesses con served res i dues which are com mon to all plant defensins. Based
on antifungal prop er ties and se quence sim i lar i ties PsDef1 was as signed to group 1 of plant defensins.
Key words: Scots pine, defensin PsDef1, mo lec u lar clon ing
In tro duc tion. In the last de cade, sig nif i cant prog -
ress has been made in un der stand ing the mech a nisms
which co or di nate the re sponse of plants to en vi ron men -
tal changes and patho genic or gan isms, in par tic u lar.
The ac ti va tion of tran scrip tion of antimicrobial pro -
teins (AMP), which in crease the re sis tance of plants to
patho genic or gan isms or in hibit the growth of patho -
gens, is con sid ered to be cru cial in driv ing the im mune
re sponse to patho genic fac tors [1]. The fam ily of plant
AMPs in cludes thionins, lipid-trans port pro teins,
heveins, snakins, defensins etc. AMPs are small (2–9
kDa) se cre tory pro teins, whose com pact struc ture is
sta bi lised by disulphide bridges. The num ber and lo -
cal isa tion of -Cys-Cys- pairs is a spe cific fea ture of
each AMPs group. Antimicrobial pro teins ex hibit high
ac tiv ity against many patho genic bac te ria and fungi
both in vi tro and in trans gen ic plants [2, 3].
Among AMPs, plant defensins pos sess a very broad
spec trum of bi o log i cal ac tiv ity. Their mo lec u lar weight
is 4–6 kDa, a ter tiary struc ture is formed by an a-he lix,
3 anti-par al lel b-sheets and 4 intramolecular disulphide
bridges [2, 4]. The pro tec tive func tion of plant
defensins is de fined by their antifungal [5, 6], an ti bac te -
rial [7], and in sec ti cidal [8] ac tiv i ties. It is im por tant to
note that some of these pro teins are known to act as pro -
398
ISSN 0233-7657. Biopolymers and cell. 2007. vol. 23. N 5. Translated from Ukrainian.
Ó V. A. KOVALYOVA, R. G. K²YAMOVA, I.T. GOUT, V. V. FILONENKO,
R. T. GOUT, 2007
te ase in hib i tors [7, 9]. The het er o ge ne ity of prop er ties
and struc ture/func tion de ter mi nants cre ated the ba sis
for the clas si fi ca tion of defensins into four groups [6].
The rep re sen ta tives of group I (morphogenic group) are
known to in hibit the growth of a wide range of
phytopathogenic fungi and in duce mor pho log i cal
changes in my ce lium. The antifungal ac tiv ity of group
II defensins is not as so ci ated with mor pho log i cal
changes in fungi. The an ti bac te rial fea tures of group I
and II defensins are ei ther not sig nif i cant or ab sent at
all. Plant defensins of group III are ac tive against bac -
te ria, but lack any fungistatic prop er ties. They were
found to in hibit a-amylases and pro teas es in vi tro as
well as pro tein syn the sis in a cell-free sys tem.
Defensins be long ing to group IV pos sess both
antifungal ac tiv ity with out any morphogenic ef fect and
high an ti bac te rial ac tiv ity against gram-pos i tive and
gram-neg a tive patho gens [10].
Defensins have been iden ti fied in var i ous tax o -
nomic groups of plants. The ex pres sion of four
defensin-like pro teins has been dem on strated in gym -
no sperm plants, in clud ing defensin pre cur sor (GbD)
Ginkgo biloba (Ginkgoaceae fam ily) [11], defensin
(PgD1) Picea glauca [12], pu ta tive plant defensin SPI1
and SPI1B Picea abies (Pinaceae fam ily) [13]. To date,
plant defensins were iso lated from seeds, veg e ta tive
and gen er a tive or gans of gym no sperm. Re cently, we
have pu ri fied defensin-like pro tein from gym no sperm
which was sim i lar to AMP in its antifungal prop er ties
[14]. Mo lec u lar clon ing and com par a tive anal y sis of
pri mary se quences of Scots pine defensin 1 (PsDef1)
with other plant defensins are pre sented in this study..
Ma te ri als and Meth ods. In this study we used se -
lected vi a ble seeds of Pinus sylvestris L. ob tained from
Busk State For estry, L’viv re gion, Ukraine. The cDNA
se quence of Pinus sylvestris Defensin 1 was cloned into
bac te rial ex pres sion vec tor pET 23d (+) (Novagen,
USA).
The pu ri fi ca tion of Defensin from Pinus sylvestris
seed lings was car ried out as pre vi ously de scribed
[14–16]. The pu rity of prep a ra tions was ana lysed by
gra di ent (5–22%) gel elec tro pho re sis at denaturating
con di tions in Laemmli sys tem [17] or in tris-tricine
buffer sys tem [18]. Sep a rated pro teins were visu al ised
by sil ver stain ing. The iden ti fi ca tion of pu ri fied pro -
teins was per formed by mass-spec trom e try at Uni ver -
sity Col lege Lon don. The bands of in ter est were cut out
from the gel, re duced with 10 mM dithiothreitol and
alkalined with 100 mM of io dine acetamide. The sam -
ples were then hy dro lysed with trypsin. The mix ture of
gen er ated proteolytic pep tides was frac tion ated on
PepMap C18 col umn (LC Packings, the Neth er lands)
for 30 min in den sity gra di ent (5–40%) of acetonitrile
and 0.1% for mic acid. Mass-spec trom e try anal y sis was
per formed us ing Q-TOF I (Micromass, UK). Pep tide
se quences gen er ated by Mas cot (Matrixscience, UK)
were used to search Swissprot and EST GenBank da ta -
bases.
The set of prim ers used for the am pli fi ca tion of
PsDef1 cDNA was de signed us ing the re sults of
mass-spec trom e try anal y sis and the align ment of nu cle -
o tide se quences cor re spond ing to Defensins from Pinus
pinaster (mar i time pine) and P. taeda (loblolly). The
am pli fi ca tion of PsDef1 cDNA was car ried out in two
sep a rate PCR re ac tions us ing as tem plates 1 ml of pri -
mary (1.2·106 pfu/ml), or am pli fied (2·109 pfu/ml) P.
sylvestris cDNA ex pres sion li brary. The li brary was
pro duced in our lab o ra tory from mRNA of Pinus
sylvestris root seed lings as pre vi ously de scribed [19]
For PCR am pli fi ca tion, we used oligonucleotides
CR763 (5'-CCATTCCATGGCGGGCAAGGGAGT-3')
and CR764 (5'-CATGAGAATTCTCAAGGGCAG-
GGTTTGTA-3'), which con tained NcoI and EcoRI
clon ing sites. PCR re ac tions were car ried out us ing
Pro teus am pli fier (Hel ena Bio Sci ences, UK), and Tag
poly mer ase from Fermentas (Lith u a nia). The fol low -
ing con di tions were used for am pli fi ca tion: 94°C for 3
min and then 30 cy cles (94°C, 1 min; 60°C, 1 min;
72°C, 1 min) and fi nally 5 min at 72°C.
PCR prod ucts were ana lysed in 1.5% agarose gel in
Tris-bo rate buffer, pH 8.3 (50 mM tris-H3BO3, 2 mM
EDTA) at 20 V/cm2. Am pli fied DNA of the ex pected
size was eluted from the gel us ing DNA ex trac tion kit
(Qiagen, USA).
The prod uct of am pli fi ca tion and pET23d(+)
plasmid were di gested with NcoI and EcoRI, sep a rated
by gel elec tro pho re sis and pu ri fied as de scribed above.
Li ga tion re ac tion was per formed for 2 hours at room
tem per a ture us ing T4 phage DNA ligase (Fermentas) ac -
cord ing to the man u fac turer’s pro to col. XL-1 Blue com -
pe tent cells were trans formed with the li ga tion mix ture
by a stan dard method [20]. The pres ence of the DNA in -
MO LEC U LAR CLON ING AND CHARACTERIZATION OF DEFENSIN 1
399
sert in pET23d(+) plasmid was de tected by re stric tion
anal y sis with NcoI and EcoRI, and by PCR with prim ers
CR 763 and CR 764. Nu cle o tide se quence of the cloned
DNA frag ment was de ter mined by au to matic DNA se -
quenc ing, ABI 73TM (Ap plied Biosystems, UK).
Bioinformatic anal y sis and se quence align ments of
the nu cle o tide and amino acid se quences of Pinus
sylvestris defensin 1 were per formed us ing elec tronic
search ser vice BLAST 2.0, Na tional Cen tre of Bio tech -
no log i cal In for ma tion (NCBI), USA.
Re sults and Dis cus sion. Re cently we have pu ri fied
low mo lec u lar weight pro tein, spe cific for high in hib i tory
ac tiv ity to wards some phytopathogenic fungi, from
7-day-old root seed lings of Pinus sylvestris. Bio chem i cal
prop er ties and strong antifungal ac tiv ity clearly in di cated
that pu ri fied pro tein be longs to the group of plant
defensins [14]. The mo lec u lar weight of defensin-like
pro tein from Pinus sylvestris was es ti mated to be ap prox i -
mately 10 kDa when ana lysed by 5-22% SDS-PAAG
anal y sis (Fig.1, a). To de ter mine the size of pu ri fied pro -
tein more pre cisely, we em ployed tris-tricine buffer sys -
tem, which is op ti mal for the sep a ra tion of low-mo lec u lar
pro teins and pep tides. The sep a ra tion at this con di tions
re vealed that the molecular weight of pu ri fied pro tein is
ap prox i mately 5–6 kDa (Fig.1, b), which is sim i lar to
plant defensins.
To de ter mine the iden tity of pu ri fied pro tein with
strong antifungal ac tiv ity, the mass-spec trom e try anal -
y sis was car ried out. The pro tein of in ter est was treated
with trypsin and the amounts of gen er ated pep tides ana -
lysed by Q-TOF I. In this anal y sis, we found one pep -
tide of 19 amino ac ids in length,
TEGFPTGSCDFHVAGR (Fig.2, a). The search for
var i ous da ta bases in GenBank, us ing BLASTP
electronic sys tem
(www.ncbi.nlm.nih.gov/blast/BLAST.cgi) re vealed
cDNA clones with high level of homology (Fig.2, b).
The high est level of homology was ob served with gym -
no sperm defensins: a cor re spond ing pep tide from GbD
of G. biloba has 94% homology, while SPI1 of P. abies
and PgD1 of P. glauca ex hibit 87% homology. These
re sults clearly in di cate that a pro tein which was pu ri fied
from Pinus sylvestris seed lings and later shown to pos -
sess antifungal ac tiv ity be longs to the fam ily of plant
defensins. There fore, we named it Pinus sylvestris
Defensin 1 (PsDef 1).
Since nu cle o tide se quences of defensins from P.
abies and P. glauca are only 252 bp long, it could be
pos si ble to iden tify EST clones from Pinus sylvestris
cDNA li brar ies, which might en code the full length
cod ing se quence of PsDef 1, us ing the data of the
mass-spec trom e try anal y sis. Hav ing em ployed
TBLASTN soft ware to search var i ous da ta bases, we
iden ti fied 10 clones in cDNA li brar ies from P. pinaster
and 23 clones from P. taeda which pos sessed pro tein
se quences with very high level of homology to the pep -
tide iden ti fied by mass-spec trom e try. It is note wor thy
that the level of homology be tween their cod ing re gions
was 95%, while the N- and C-ter mi nal frag ments were
com pletely iden ti cal. These fea tures were taken into ac -
count when we de signed the prim ers for mo lec u lar
clon ing of a cDNA clone for PsDef 1.
For mo lec u lar clon ing of P. sylvestris defensin 1,
we used a cDNA li brary cre ated in our lab o ra tory from
the 7-day-old root seed lings [19]. Pri mary and am pli -
fied li brar ies were used as tem plates dur ing PCR am pli -
fi ca tion with a set of prim ers, spe cific to P. sylvestris
defensin 1. Elec tro pho retic anal y sis of PCR prod ucts
re vealed the pres ence of a ma jor band with an ex pected
size of 270 bp (Fig.3, a). The prod uct of am pli fi ca tion
KOVALYOVA V. A. ET AL.
400
Fig.1 SDS-PAGE anal y sis of antifungal pro tein from Pinus
sylvestris: sep a ra tion of pro teins in 5–22% gra di ent gel in
Laemmle’s sys tem (a); sep a ra tion of pro teins in 15% gel with
tris-tricine buffer (b). Sep a rated pro teins were vi su al ized by silver
staining.
was cloned into pET 23d(+) vec tor which was then
trans formed into XL-1 Blue com pe tent cells. The pres -
ence of the cDNA in sert in the re sult ing plasmid was
con firmed by both PCR (Fig.3, b) and re stric tion anal y -
sis (Fig.3, c). DNA se quenc ing re vealed the in sert of
252 bp in length (Fig.4, a), which we de pos ited in
GenBank, No.EF455616. Bioinformatic anal y sis
showed that de ducted amino acid se quence con tains a
pep tide which is iden ti cal to that iden ti fied by
mass-spec trom e try. No ta bly, cDNA PsDef1 shows
MO LEC U LAR CLON ING AND CHARACTERIZATION OF DEFENSIN 1
401
Fig.2 The iden ti fi ca tion of antifungal pro tein from Pinus sylvestris by mass-spec trom e try: (a) the se quence of gen er ated tryptic pep tide –
TEGFPTGSCDFHVAGR; (b) and se quence align ment of the tryptic pep tide with cor re spond ing pep tides from gym no sperm defensins.
Fig.3 Clon ing of Pinus sylvestris defensin 1 cDNA: (a) the
prod ucts of PCR am pli fi ca tion from pri mary (1) and am pli fied
cDNA li brary (2); M – 1 kb Plus DNA Lad der GibcoBRL; b –
the anal y sis of pET23d-PsDef plasmid by PCR (1); M – 1 kb
DNA Lad der Fermentas; c – re stric tion anal y sis of
pET23d-PsDef1 plasmid us ing endonucleases EcoRI and NcoI
(1); M – 1 kb DNA Lad der Fermentas.
84% iden tity with nu cle o tide se quences of defensins
from Pinacea fam ily and 77% iden tity with G. biloba
from Ginkgoaceae fam ily.
Anal y sis of de ducted amino acid se quence of Pinus
sylvestris defensin 1 (83aa) us ing SignalP soft ware
(www.cbs.dtu.dk/ser vices/SignalP) re vealed the pres -
ence of the N-ter mi nal sig nal pep tide of 33aa, which is
a spe cific fea ture of plant defensins. In ad di tion, all se -
cre tory pro teins are known to pos sess the sig nal pep -
tide. The ma ture form of PsDef1 is 50aa in length,
which is the same for other gym no sperm defensins
(Fig.4, b). Cal cu lated mo lec u lar weight of PsDef1 is
5601.6 Da, which is in good cor re la tion with the re sults
of SDS-PAGE anal y sis (Fig.1, b). The value of
isoelectric point of 8.9 was ob tained on the ba sis of
amino acid se quence, us ing pI/Mw soft ware [21].
The search in amino acid se quence of PsDef1 for
known do mains us ing Mo tif Scan soft ware
(scansite.mit.edu/motifscan) re vealed that se quences be -
tween amino acid res i dues 35–85 form the struc ture,
known as gamma-thionin, which pro vides the pri mary
name of plant defensins – g-thionins. This do main con -
sists of an a-he lix and three anti-par al lel b-sheets, con -
nected by four disulphide bridges [2]. The spe cific fea -
KOVALYOVA V. A. ET AL.
402
Fig.4 Nu cle o tide and de ducted amino acid se quences of PsDef1: (a) sig nal pep tide is un der lined; the po si tion of the tryptic peptided iden ti -
fied by mass-spec trom e try is in di cated by a dashed line; pro tein se quence align ment of PsFef1 with amino acid se quences of gym no sperm
defensins; (b) and the rep re sen ta tives of dif fer ent groups of plant defensins (c) the se quences of sig nal pep tides were elim i nated; the ver ti cal
line is made for max i mal align ment of se quences; stars in di cate iden ti cal amino ac ids; con ser va tive cysteins are boxed; join ing lines cor re -
spond to disulphide bridges. Ab bre vi a tions used: Psdef1 – defensin 1 Pinus sylvestris (ac ces sion no.EF455616); GbD – defensin pre cur sor
Ginkgo biloba (ac ces sion no.AY695796); SPI1 – pu ta tive gamma-thionin pro tein Picea abies (ac ces sion no.X91487); PgD1 – defensin
Picea glauca (ac ces sion no.AY494051); SPI1B – pu ta tive plant defensin Picea abies (ac ces sion no.AF548021); RsAFP2 – antifungal pro -
tein 2 Raphanus sativus (ac ces sion no.U18556); DmAMP1 – defensin Dahlia merckii (ac ces sion no.AAB34972); SIa2 – in hib i tor of in sect
a-amylases 2 Sor ghum bicolour (ac ces sion no.P21924); So-D2 – antimicrobial pep tide D2 Spinacia oleracea (ac ces sion no.P81571)
ture of this do main is the fact that only 23% of amino
acid res i dues are con ser va tive, in clud ing all cysteins (po -
si tions 4, 15, 21, 25, 36, 45, 47, and 51), two glycines (13
and 34), serine (8), an ar o matic res i due in po si tion 11
and glutamic acid in po si tion 29 (start ing point is
RsAFP2 – the most stud ied plant defensin) (Fig.4, c).
De tailed anal y sis of the pri mary struc ture of
defensin 1 and known Picea defensins al lowed us to re -
veal 81% iden tity be tween these pro teins. The high
level of homology points out pos si ble sim i lar ity in their
bi o log i cal ac tiv ity. The overexpression of SPI1 in em -
bry onic cells of P. abies L. is known to in crease the re -
sis tance of these cells to in fec tion by Heterobasidion
annosum [22]. Re cently, we have dem on strated high
antifungal ac tiv ity of en dog e nous PsDef1 to wards
phytopathogenic fungi Fusarium, Alternaria, and Bo -
try tis in vi tro [14, 23].
Com par a tive anal y sis of PsDef1 amino acid se -
quences with that of dif fer ent groups of defensins,
namely RsAFP2 (group I), DmAMP1 (II), SIa2 (III),
and So-D2 (IV) re vealed high level of homology with
RsAFP2 (58%) and So-D2 (59%). Lit er a ture data tes -
tify in fa vour of high antifungal ac tiv ity of these plant
defensins, the IC50 value (pro tein con cen tra tion, which
shows 50% in hi bi tion of fun gal growth) in re gards of
Fusarium fam ily is lower than 1 mM [7, 23]. The IC50
for en dog e nous PsDef1 in the same ex per i men tal set up
was found to be 0.7 mM [14, 24]. One spe cific fea ture
of these defensins is a high pos i tive charge at pH 7.0:
PsDef1 has the charge of +6.7 and sim i lar val ues are
found for RsAFP2 (group I) – +5.8 and So-D2 (IV) –
+7.6. It has been pre vi ously dem on strated that
antifungal ac tiv ity of AMP is de ter mined by the value
of pos i tive charge of mol e cule [24].
We have pre vi ously shown that en dog e nous
PsDef1 causes mor pho log i cal changes in fungi my ce -
lium, which pro vided the ground for its as sign ment to
morphogenic group I of plant defensins [14]. Ex per i -
ments with site-di rected mu ta gen e sis re vealed the bi o -
logic ac tiv ity of each group of defensins to be de ter -
mined by short con ser va tive se quences, which are spe -
cific for each group only, or might be de fined by a
sin gle amino acid. Com par a tive anal y sis of pri mary
amino acid se quences be tween PsDef1 and RsAFP2 al -
lowed us to de tect a highly ho mol o gous re gion
(2–10aa), a hy dro pho bic re gion (38–41 a.r.), which are
im por tant de ter mi nants of their antifungal ac tiv i ties,
pos si bly func tion ing as bind ing sites for spe cific re cep -
tors on fun gal mem branes. The sub sti tu tion of Tyr38
for Gly in RsAFP2 re sulted in the change of pro tein
con for ma tion and the loss of its ac tiv ity. No ta bly, this
res i due is highly con served in plant defensins, which
be long to groups II and III. In PsDef1 this po si tion is
oc cu pied by ho mol o gous ar o matic amino acid
phenylalanine. An other con served amino acid in both
groups of defensins is Lys44 and its sub sti tu tion for a
neu tral res i due de creases antifungal ac tiv ity of RsAFP2
sig nif i cantly [25]. Tak ing into ac count that PsDef1 and
RsAFP-2 ex hibit sig nif i cant struc tural sim i lar i ties and
be long to the same fam ily of plant defensins, one might
ex pect the sim i lar ity of mo lec u lar mech a nisms co or di -
nat ing their antifungal ac tiv i ties.
There fore, in this study we pres ent mo lec u lar clon -
ing of Pinus sylvestris defensin 1 for the first time. The
find ings pre sented open for us the op por tu ni ties for the
fol low ing: i) pro duc ing re com bi nant PsDef1 and in ves -
ti gat ing its bio chem i cal, struc tural, and func tional
prop er ties; ii) gen er at ing spe cific an ti bod ies against
PsDef1, which would be par tic u larly use ful to study its
ex pres sion in Pinus sylvestris tis sues at var i ous con di -
tions; iii) elu ci dat ing antifungal action of plant
defensins.
Â. À. Êî âà ëå âà, È. Ò. Ãóò, Ð. Ã. Êèÿ ìî âà, Â. Â. Ôè ëî íåí êî,
Ð. Ò. Ãóò
Këî íè ðî âà íèå è àíà ëèç êÄÍÊ äå ôåí çè íà 1 ñî ñíû
îá ûê íî âåí íîé
Ðå çþ ìå
Àíòèôóíãàëüíûé áå ëîê ñ ìî ëå êó ëÿð íîé ìàñ ñîé 5,6 êÄà î÷è ùåí
èç êîð íåé ñå ìè äíåâ íûõ ïðî ðîñ òêîâ ñî ñíû îá ûê íî âåí íîé.
Ìàññ-ñïåê òðî ìåò ðè ÷åñ êèì àíà ëè çîì ïî êà çà íà éîãî ïðè íàä -
ëåæ íîñòü ê ðàñ òè òåëü íûì äå ôåí çè íàì. êÄÍÊ äå ôåí çè íà 1 ñî -
ñíû (PsDef1) äëè íîé 252 ï. í. ïî ëó ÷å íà ìåòîäîì
ÏÖÐ-àì ïëè ôè êà öèè èç êÄÍÊ áèá ëè î òå êè Pinus sylvestris L. è
êëî íè ðî âà íà â âåê òîð ðÅÒ23d(+). PsDef1 êÄÍÊ êî äè ðó åò áå ëîê
èç 83 àìè íî êèñ ëîò íûõ îñòàò êîâ (à. î.) ñ N-êîí öå âûì ñèã íàëü -
íûì ïåï òè äîì èç 33 à. î. Äëÿ çðå ëîé ôîð ìû õà ðàê òåð íî íà ëè -
÷èå ñïå öè ôè ÷åñ êèõ êîí ñåð âà òèâ íûõ îñòàò êîâ, ñâî éñòâåí íûõ
âñåì ðàñ òè òåëü íûì äå ôåí çè íàì. Ïî êà çà íî ñòðóê òóð íîå è
ôóíê öè î íàëü íîå ñõî äñòâî ìåæ äó PsDef1 è äå ôåí çè íà ìè ãðóï -
ïû ².
Êëþ ÷å âûå ñëî âà: ñî ñíà îá ûê íî âåí íàÿ, äå ôåí çèí PsDef1, ìî -
ëå êó ëÿð íîå êëî íè ðî âà íèå.
MO LEC U LAR CLON ING AND CHARACTERIZATION OF DEFENSIN 1
403
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Re ceived 02.07.07
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