Transcript ( A) and PVY-CP primers - KSU Faculty Member websites

‫صدق هللا العظيم‬
B.Sc. AGRIC. (PLANT PATHOLOGY ) FAC. OF AGRIC. , CAIRO UNIVERSITY, 1992
M.Sc. AGRIC. (PLANT PATHOLOGY ) FAC. OF AGRIC. AIN SHAMS UNIVERSITY , 1999
Prof. of Plant Pathology, Dep. of Plant Pathology, Fac.
of Agric ,.Ain Shams Universit.
Prof. of Plant Pathologv , ,Plant Pathology
Research Institute, Agricultural Research
Center
Prof. of Plant Pathology,
Dep.
of Pathologv
Plant Pathology,
Prof. of
Plant
, ,Plant Pathology Research
Fac . of Agric ,.AinInstitute,
Shams Universit
.
Agricultural
Research Center
■Is one of the most important and economic vegetable crop in Egypt. The
cultivated area in 2001 / 2002 reached about 200,000 feddan, which
yielded about 2000,000 tons with an average of 10 tons / feddan.
■ Potato usually subjected to numerous diseases caused by viruses, fungi,
bacteria, phytoplasma, and viroid. Virus diseases are mainly responsible
for the majority of the losses caused in potato production.
■ More than 25 different plant viruses are pathogenic to potato. i.e.
(PVY), (PVX), and (PLRV). These viruses may result in the losses of
certification of seed potato and also they highly affecting in the quality
and yield of potato in commercial production. (Beemester & Rozandaal,
1972, and Salazar, 1996)
VIRUSES INFECTING POTATO THEIR ABBREVIATION
AND MEANS OF SPREAD
VIRUS NAME
ABBREVIATION
VECTOR/ SPREAD
Potato virus Y
PVY
Aphids
Potato virus V
PVV
Aphids
Potato virus A
PVA
Aphids
Potato virus X
PVX
Mechanical
Potato leafroll virus
PLRV
Aphids
Potato virus M
PVM
Aphids
Potato virus S
PVS
Aphids
Potato virus T
PVT
True seed
Potato moptop virus
PMTV
Fungi
Alfalfa mosaic virus
ALMV
Aphids
Andean potato latent virus
APLV
Beetles
Andean potato mottle virus
APMV
Beetles
Beet curly top virus
BCTV
Leafhopper
Cucumber mosaic virus
CMV
Aphids
Potato yellow dwarf virus
PYDV
Leafhopper
Potato yellow vein virus
PYVV
Whiteflies
Potato deforming mosaic virus
PDMV
Whitefly
Solanum leaf curling virus
SLCV
?
Tobacco mosaic virus
TMV
Mechanical
Tobacco necrosis virus
TNV
Nematodes
Tobacco rattle virus
TRV
Nematodes
Tobacco ringspot virus
TRSV
Nematodes
TSV
Pollen / Thrips
Tomato black ring virus
TBRV
Nematodes
Tomato spotted wilt virus
TSWV
Thirps
Potato spindle tuber viroid
Potato witches broom disease
Potato stolpor disease
PSTVd
PWBD
PSD
Mechanical/ True seed
Leafhopper
Leafhopper
Tobacco streak virus
PVY is the type member of the potyvirus
group (Holling & Brunt, 1981; Matthews,
1985; and Barnett, 1991 & 1992).
PVY is considered one of the most
damaging potato viruses causing significant
yield depression and because the infection
with PVY spread easily and the yield losses
reached to 80 %
(deBokx & Huttinga, 1981).
Several groups of PVY strains according to the symptoms
reaction on Nicotiana tabacum cvs. White Burley and Samsun ,
Physalis floridana, and Solanum tuberosum
PVYO (common strain): induced mainly severe systemic crinkle
symptoms, rugosity of leaf drop, streak on potato, systemic
necrosis on P. floridana, and systemic mottling on tobacco plants.
PVYC (stipple streak strain): Susceptible potato varieties might show
systemic mosaic or stipple streak. Tobacco plants reacted with
vein banding symptoms while the symptoms on P. foridana were
similar to those obtained by PVYO strain.
PVYN (tobacco venial necrosis strain): induced severe systemic venial
necrosis on tobacco, systemic motteling on P. floridana.
PVY NTN ( Potato tuber necrotic ringspot disease) associated with a
potato disease mentioned for the first time in Hungary in the 1980`s
(Beczner et al., 1984), caused by a strain of potato virus Y that
belongs to the tobacco veinal necrosis strain group (PVY N)
–
I - Isolation and identification of PVY-CP gene
1234-
Isolation and identification of PVY-CP gene using RT-PCR with Specific primers.
Molecular cloning and transformation into protein expression (PinPoint Xa-1) plasmid
Detection of PVY-CP expressed protein in E.coli
Sequencing of PVY-CP gene and computer analysis.
II- Detection and confirmation of the present isolate of PVY as
PVYNTN using RT-PCR, Nested , Hemi-nested-RT-PCR, and RFLP
Techniques.
III - Introduction of gene constructs into Agrobacterium vector.
1- Introduction of gene constructs into Agrobacterium vector and analysis of
transconjugants using PCR technique Southern hybridization and minipre and
restriction enzymes analysis.
2-Introduction of pBI121 –PVY- CP gene into potato plants and Gene integration analysis
using isolation of plant DNA, electrophoresis and Southern blot hybridization , PCR,
RT-PCR and Western blotting assays.
The Previously characterized Potato virus Y isolate (Amer, 1999) was isolated
from Potato (Solanum tuberosum L. cv. Nicola) plants, maintained in tobacco
(Nicotiana tabacum L.cv. Samsun) plants by mechanical inoculation.
Solanum tuberosum cv. Nicola leaves (A),
tuber (B) and Nicotiana tabacum cv. Samsun
(C) plants showing a typical sever veinal
necrosis Symptoms due to PVY infection
Fig (B) from FAO/IPGRI Technical
Guidelines for the Safe Movement of
Germplasm No 19 Potato
PART (1)
2-PVY –RNA molecules were required to
the introduction of preliminary reverse
transcription before the PCR
amplification, this done using
AMV-RTase
DNA primer specific for coat protein gene was used for cDNA
synthesis and amplification using PCR assay ( Sudarsono et al., 1993)
The `3- end (downstream) primer complementary to the viral RNA
sequence was partially degenerate, used to prime the cDNA synthesis
reaction.
5`-gga tcc aca tgt t(c/g) act cca ag ( t/c) ag-`3
A unique BamHI site ( underline ) was introduced adjacent to the viral
sequence to facilitate cloning of the amplified cDNA into expression
vectors.
The 5`-end (upstream) primer homologous was also partially
degenerate. This primer was designed to incorporate a unique BamHI
site (underline) an inframe initiation codon ( bold) and a unique NcoI
site ( italics) . The NcoI and BamHI sites partially overlap.
5`-gga tcc atg g(gc)aaa tga cac aat (t/c) gat gca-`3
Isolation and identification of coat protein gene using RT-PCR
Agarose gel (1%) electrophoretic analysis of RT-PCR amplification of the total RNA obtained by
SV-Total RNA isolation system from infected and uninfected plant materials with the present
isolate of PVY using PVY CP-H and PVY CP-C specific degenerate primer for PVY-CP. Infected
Nicola potato leaves (lane 2) and tuber (lane 3); infected tobacco leaves (lane 4); and infected
potato leaves (lane 6) and tubers (lane7) cv. Spounta. No amplified fragments of cDNA were
obtained from uninfected potato leaves cv. Nicola (lane 1), tobacco (lane 5) and potato cv.
Spounta leaves (lane 8). The arrow indicated an 801 bp for the RT-PCR products, lane M, PCR
marker
1- Amplification c DNA was ligated into TA Pin Point expression vector using
T4 DNA Ligase after Tag DNA polymerase was used for amplification whereas
this enzyme had a non- template dependent activity , which add a single
deoxadenosine (A) to the 3`- end of PCR products.
2- The linearized vector had a single 3`- deoxthyamidine (T) residues. This
allowed the PCR inserts to ligate efficiently with the vector
3- The ligation products were transformed into E. coli comptent cells ( JM109 strain)
PinPoint Xa-1 vector
White ampcilline resistance colonies were selected for screening
performed by minipreparation recombinant colonies which had
correct insert ( 801 bp) was validated by PCR and Restriction
analysis using BamHI
PCR
Restriction analysis using BamHI
Electrophoresis was performed on1 % agarose gel staining with ethedium bromide. Lane M,
PCR marker 50,150,300,500,750 and 1000 bp (Promega Co.).
Screening of of plasmid (Pin Point Xa-1 protein expression vector) transformed in E.coli. colonies by
plasmid minipreparation using restriction digestion enzymes by NocI and EcoRI. Electrophoresis wa
performed on agarose gel (1 %) staining with ethedium bromide to determine the orientation fragme
The left arrow indicated a 3.683 Kb; the other right bottom arrow indicated a 554 bp [ (colonies that
containing the right and orientation fragments), ( lanes (1, 2, 3 and 4)] , the other right high arrow ind
a 1.258 bp[ (colonies that containing the right size but not in orientation) (lane 5,6)] Lane M, PCR m
50,150,300,500,750 and 1000 bp ( Promega Co.)
A
B
(A) SDS PAGE (12 %) analysis showing rapid screening of small expression culture and
induced by adding 100 mM IPTG. silver nitrate stained SDS-PAGE the total cell proteins with a
major ~45 KD polypeptide bands (lanes :1, 2, 3, 4, 5, 6, 7 and 8 ) . B) Western Blot analysis
showing a prominent bands with an estimated molecular mass of approximately ~32 KD
representing expression coat protein for PVY which specifically reacted with goat anti rabbit
IgG . (lane 1, 2, 3, 4, 5, 6, 7 and 8). No PVY-CP expression was observed from uninduced
cultures (lane 9). Lane M: Protein Marker .
NCM- ELISA of representing expression coat protein for PVY that specifically reacted
with goat anti rabbit IgG. Lane A : 1,,3,4,5,6 and Lane B : 2,3,4, ), total cell protein
showing no PVY –CP expression ( lane A : 1 ) and Lane B : 1,5,6)
NUCLEOTIDE SEQUENCE AND DERIVED AMINO ACID SEQUENCES
OF THE PVY–CP EGYPTIAN ISOLATE
The CP sequence of PVY
isolate under study was
predicted to be
801 nucleotides in length,
encoding a protein of
267 amino acids
with a calculated Mr 29980
Dalton.
The nucleotide composition of
the cDNA CP sequence
revealed the highest contents
for (A) 35 %, followed by (G)
23 %, (T) 23 % and (C) 19
%
Amino acids
*Code
Name
The amino acid composition of the present isolate of PVY –
CP as determined by DNA Star Laser gene Software.
Abbr
No.count
% by weight
% by Frequency
**Order
A
Alanine
Ala
19
4.51
7.12
3
C
Cysteine
Cys
1
0.34
0.37
20
D
Aspartic acid
Asp
13
5.00
4.87
14
E
Glutamic acid
Glu
* 21
9.06
7.87
1
F
Phenylalanine
Phe
5
2.46
1.87
18
G
Glycine
Gly
18
3.43
6.74
5
H
Histidine
His
6
2.75
2.25
17
I
Isoleucine
Iie
14
5.29
5.24
11
K
Lysine
Lys
17
7.28
6.37
6
L
Leucine
Leu
18
6.80
6.74
4
M
Methionen
Met
13
5.70
4.87
12
N
Asparagine
Asn
16
6.10
5.99
8
P
Proline
Pro
13
4.22
4.87
15
Q
Glutamine
Gln
13
5.56
4.87
13
R
Arginine
Arg
14
7.30
5.24
10
S
Serine
Ser
16
4.65
5.99
9
T
Threonine
Thr
*21
7.09
7.87
2
V
Valine
Val
17
5.63
6.37
7
W
Trptophane
Trp
3
1.87
1.12
19
Y
Tyrosine
Tyr
9
4.91
3.37
16
Percent similarities of coat protein between the present isolate
and other PVY isolates and potyviruses a valuable from GenBank
Percent similarity ( %)
PVY isolates and potyviruse
No of nt of CP
Isolate used
Accession No.
88.8
801
PVY Chilean
X68221
88.8
801
PVY US
X68222
96.1
801
PVY Europe H
X68223
89.3
801
PVYO
X68226
98.4
798
PVY Sws N isolate
X97895
96.1
799
PVY NTN strain
AF321554
84.6
800
PVYO strain
AF345650
88.8
801
PVYN Egypt N strain
AF522296
88.6
801
PVY French N strain
D00441
99.5
828
PVY –T N strain
D12570
96.5
806
PVY Hungarian isolate
M95491
63.8
798
PeMV
NC_001517
59.7
799
ZYMV
L31350
60.0
779
TEV
M11458
63.0
807
PsbMV
NC_001671
Percentage sequence similarity between the PVY isolate under study and
different PVY isolates and potyviruses
EGYPT-Isolate
100 %
X97895 (SWS)
98.4 % 100 %
AF321554 (NTN)
96.1% 97.0 %100 %
AF345650 (O)
84.6 % 85.0% 86.2% 100 %
AF522296 (N E)
88.8% 93.0% 90.0% 93.0% 100 %
AJ253119 (PVV)
63.3% 63.0% 62.8% 61.2% 62.7% 100 %
D00441 (Fr)
88.6% 98.4% 90.7% 92.9% 99.9% 62.5% 100%
D12570 (T)
99.5% 98.6% 96.4% 86.4% 89.0% 62.3% 88.9% 100%
L31350 (ZYMV)
59.7% 60.2% 59.6% 57.0% 59.9% 62.6% 59.9% 57.2% 100%
M11458 (TEV)
60.0% 59.9% 60.1% 59.2% 60.7% 57.1% 60.6% 59.8% 61.3% 100%
M9549 (Hungaria)
96.5% 97.4% 98.9% 86.0% 90.8% 62.8% 90.6% 96.7% 58.9% 60.7% 100%
NC_001517(PeMv)
63.8% 63.5% 63.5% 60.7% 63.2% 64.7% 63.0% 62.5% 62.4% 58.3% 62.5% 100%
NC_001671( Psb)
63.0% 58.4% 57.6% 55.4% 62.4% 57.2% 62.5% 57.5% 52.1% 58.4% 57.4% 56.7% 100%
X68221 ( CH)
8.8% 89.7% 90.6% 86.1% 91.8% 61.7% 91.6% 89.1% 59.0% 60.3% 88.8% 90.5% 61.8% 56.7% 100%
X68222 (US)
88.3% 88.5% 90.4% 92.4% 97.4% 63.3% 97.3% 86.6% 63.6% 60.3% 90.3% 63.6% 62.2% 90.5% 100%
X68223(Europe)
96.1% 97.0% 98.7% 85.9% 90.6% 62.8% 90.5% 96.4% 59.1% 60.2% 98.6% 62.9% 57.6% 90.4% 90.2% 100%
X68226 (O)
89.3% 89.3% 90.4% 97.6% 62.8% 97.5% 89.3% 59.2% 60.3% 90.8% 69.8% 69.3% 60.3% 91.2% 96.9% 90.9% 100%
PHYLOGENETIC TREE
PVY –T
PVY NTN EG
PVY SWS
PVY Hungaria
PVY NTN
PVY Europe
PVY N Egypt
PVY Fr
PVY US
PVY O
PVY O
PVY Chilean
PVV
PeMV
ZYMV
.TEV
PsbMV
Phylogenetic tree between PV isolate under study and different PVY isolates
and potyviruses.
PART (2)
PRIMERS USED
PRIMER
SEQUENCE 5`-3
Position
A
aac act cac aaa agc tttc a
43-62
B
t ( ct ) a(ct) aaa c(ag) ct ( ct ) att (ct) tcac
48-70
C
aat taa aac aac tca ata ca
1-21
D
tg( ct ) ga ( cta) cca cgc act atg aa
955-974
Primer D+ A =931 bp ( PVY N isolates )
Primer D+ C = 974 bp ( all PVY isolates )
Primer D+ B = 926 bp ( PVY O isolates)
Glias et al. (1996)
E
H -5` - tca tca aac aaa ctc ttt c -3
103
C- 5` - ttc caa agt gtc ctt tga g – 3`
919
Primer E specific for detection of PVY NTN ( 853 bp)
Weidemann et al. (1995)
Numbered in PVY NTN ( Thole et al. (1993)
RT-PCR
Isolate No 1 ( lane 11)
Isolate No 2 (lane 7)
Isolate No 3( lane 3)
Hemi-nested-PCR
Isolate No 1( lanes 9 &10 )
Isolate No 2 (lanes 5 & 6 )
Isolate No 3( lanes 1 & 2)
Agarose gel electrophoresis analysis (1 %) of RT-PCR amplified cDNA using
primers C and D (lanes 3, 7 and 11), and hemi-nested-RT-PCR using primer A
and D lanes ( 1 ,5 & 9) and primers B and D ( lanes ( 2, 6 &10)
for PVY isolates No.1 ( lanes 9,10 and 11) , No.2 ( Lanes 5,6 and 7) and No.3
( lanes 1,2 and 3). from tobacco plants infected with PVY. No fragment from
uninfected plant tissue (lane 4, 8 and 12) with primers A, B and C. The arrow
indicated a (974, 931 and 926 bp). Lane M : 50 bp step leader DNA marker.
The amplified fragment of PVY isolates using different
primers with RT-PCR and Hemi-nested- RT-PCR
Primers
PVY isolates
No.1 (NTN)
No. 2 (N)
No. 3 (O)
C+D
+
+
+
A+D
+
+
‫ــــ‬
B+D
‫ــــ‬
+
+
E
+
‫ــــ‬
‫ــــ‬
Ava II
B
Hinc II
A
Agarose gel 1 % electrophoresis analysis of restriction endonuclease digestion
of RT-PCR products amplified with primers C and D. Lane M : 50 bp Step leader
DNA marker ,lanes 1- 3 RT-PCR amplified from the present isolate of PVY (NO.1)
and restricted with Hinc II ( A ) and Ava II (B) . The arrow indicated a 660 and
808 bp.
PART (3)
pBI121 PLANT EXPRESSION VECTOR
Restriction map of the pBI121 plasmid containing NPT II gene with
Nos promoter and terminator and GUS gene with Cauliflower
mosaic virus 35 S promoter and terminator
1-The cloned virus coat protein gene ( Pin Point Xa-1 vector) was digested
with BamHI to release the CP gene fragment from the clone vector.
2- The CP gene fragment was separated from the vector by agarose gel
electrophoresis . Gene clean purification system (Promega ).was used to
purify DNA fragment from the agarose gel electrophorasis.
3-The pBI121 plant expression vector was digested with the same restriction
enzyme (Bam HI) , and the linearzed vector was purified from an agarose
gel using the Gene clean purification system.
4-The pBI121 vector and CP gene fragment were ligated using T4 DNA ligase.
5- The ligation mixture was transformed into Agrobacterium cells
A- PCR TECHNIQUES AND SOUTHERN BLOT HYBRIDIZATION FOR ANLYSIS OF
TRANSCONJUGATES USING PVY-CP PRIMER
A
B
A: PCR product profiles of the A. tumefacience strain LBA 4404 transformed by pBI121 plasmi
containing PVY CP gene. PCR products were fractionated on 1 % agarose gel and stained with
ethedium bromide .B) Souther blotting hybridization with DIG labeled PVY CP probe. Lane M,
PCR marker. Lane1,2,3,4,6,7,8,9,10,11,12and13), PCR from pBI121 plasmid after minipreparati
producing PVY-CP gene fragment (801bp), No fragment from plasmid pBI 121 without PVY -CP
gene. (Lane 5). The arrow indicated a 801 bp (PVY- CP),
B- The transformed colonies were screened using the PCR strategy
using specific primer for GUS gene (Worall, 1998) .
5`- agc atc tct tca gcg taa gg –`3
(Forward primer)
5`- tga aca acg aac tga act gg –`3 (Reverse primer
PCRproduct profiles of the A. tumefacience strain LBA 4404 transformed by
pBI121 plasmid containing GUS gene. PCR products were fractionated on 1 %
agarose gel and stained with ethedium bromide .Lane M : 50 bp step leader DNA
marker .Lane (1, 2 , 3 ,4 , 5, 6 ,7 ,8 , 10 , 11 , 12 and13 ) PCR from pBI121 plasmid
after minipreparation producing GUS gene fragment ( 610bp) , No fragment from
plasmid pBI121 ( lane 9). The arrow indicated a 610 bp (PVY CP).
C- Validation of cloning of PVY-CP gene into Agrobacterium cells using
restriction analysis
Electrophoresis separation of the digested products of plasmid pBI121 with BamHI
restriction enzyme. Separation was carried out on 1 % agarose gel and stained with
ethedium bromide. Lane M, PCR marker. Lane ( 1, 2 ,3 ,4, 5 , 7 , 9 and10): digested
plasmid pBI121 with BamHI after minipreparation producing PVY CP gene fragment
(801bp) and vector fragment (13 kp) , no PVY –CP gene fragment from plasmid pBI121
without PVY CP gene. Lane (6 and 8). Lane (11): control pBI121 plasmid uncut. The
bottom arrow indicated a 801 bp (PVY CP) whereas the high arrow indicated to 13 KB
(pBI121 vector).
The use of primers for PCR resulted in
amplification of 801 bp fragment of the CP of PVY
Southern blot hybridization and restriction analysis
using BamHI are confirming the presence of PVY
in pBI121 plasmid
The use of primers for PCR resulted in
amplification of 610 bp fragment of the GUS GENE
is confirming the presence of GUS gene in pBI121
plasmid
Agrobacterium mediated transformation has been
employed successfully with a number of potato
plants using leaf disks as explant tissue
Using
A- Specific primer for GUS gene
B- Specific primer for PVY-CP
PCR products of selected transformed potato plantlets. Total genomic DNA was extracted from
12 transformed potato plants and used as a template for PCR using GUS primers (GUS 1 and
GUS 2) ( A) and PVY-CP primers ( B) . PCR products were fractionated on 1 % agarose gel
containing ethedium bromide. Lane (1and 6): PCR products of different putative transformed
plantlets , lane M : PCR marker, no PCR amplified from non successfully transformed potato
plantlets ( Lanes 2,3,4,5,7,8,9,10,11 and 12).
GUS probe
Using
PVY probe
Southern blot hybridization analysis of genomic DNA extracted from 12 transformed potato
plantlets. The total genomic DNA were electrophoresis on 1 % agarose gel, and transferred to
a nitrocellulose membranethen hybridized with GUS gene (A) and PVY-CP (B) probes . Lane
(1and 6): DNA extraction from different transformed plants that had GUS and PVY –CP genes
integraded into potato plantlets, whereas No hybridization reaction between the genomic DNA
from non successfully transformed potato plantlets and GUS and PVY-CP probe (Lanes
2,3,4,5,7,8,9,10,11 and 12).
Total cellular leaf RNA from transformed plants was used in the RT-PCR
experiments using the same primers used for amplification of PVY-CP gene
transcription analysis of PVY- CP expressed in transformed plants. RT-PCR was
done from total RNA of transformed plants using specific primers for PVY- CP
gene.
Western immunoblot analysis for protein expressed in leaves of positively RNA
transcripts tested of potato plants transformed with PVY-CP gene. Protein
extracted from leaves (200 mg) was separated on 12 % SDS- Polyacrylamid gel
electrophoresis and electroblotted on to a nitrocellulose membrane.CP was
identified by immunoblotting. .Lane (1and6) represents the transformed potato
plants
● RT-PCR assay was developed for the detection, isolation and
identification of PVY-CP gene from nucleic acid extracts of infected
potato and tobacco plants using DNA primers specific for PVY –CP
gene . The size of the major RT-PCR product from PVY-CP infected
plants was 801bp.
● The PVY- CP gene was ligated into the protein expression vector
(Pin Point Xa-1), and transformed into E.coli strain JM 109 and
expressed in E.coli using IPTG fusion protein. SDS- PAGE assays
was used as a rapid screening of small expression culture and
revealed the presence of a prominent band with estimated molecular
mass approximately 45 KD (including the 13 KD from plasmid
expression as a tac for fusion constructions blus ~32 KDa from protein
expressed PVY CP gene) in the total proteins isolated from induced
culture.
● Western blotting and Dot-ELISA assays were used for detecting
and confirming representing the expression of PVY-CP in E.coli which
specifically reacted with antibodies of PVY.
● Complete nucleotide sequence of PVY-CP gene was
determined from Egyptian isolate of PVY NTN. The size of
the coat protein gene 801 nucleotides in length, encoding a
deduced protein of 267 amino acids with calculated
molecular weight about 29.98 KD.
● Nucleic acid sequence analysis revealed a range of
84.6 – 99.5 % sequence identity among the PVY isolate
under study and other 11 PVY isolates, and 59.7 –64 %
sequence identity among 5 different potyviruses a
valuable from Gene Bank. The highest sequence
similarity was found between the present isolate and
PVY –T isolate (99.5%) , while the lowest was found
between the present isolate and PVY O isolate ( 84.6 %).
● Detection and confirmation of the present isolate (NO.1) of PVY
as PVYNTN using RT- PCR,Nested , Hemi nested RT-PCR, and
Restriction fragment length polymorphism (RFLP) techniques
.● The PVY-CP gene was cloned into plant expression vector
( pBI121 ) and transformed into Agrobacterium strain LBA4404
by direct transformation.
Recombinant Agrobacterium (Transcongugate) was confirmed
and screened by PCR using specific primers for PVY-CP and
GUS genes and restriction analysis using BamHI. The obtained
results indicating and confirming the presence the PVY-CP
gene in pBI121 plasmid.
● Transformation of leaf explants of potato plant cv. Nicola
was carried out by co-cultivation with the recombinant
Agrobacterium cells. Regenerated shoots and roots were
produced on MS medium supplemented with 100 mg/l
kanamycin.
The regenerated transformed potato plants were tested for the
presence of the PVY-CP gene though different molecular
analysis techniques including PCR, Nucleic acid hybridization,
RT-PCR and Western blot assays. These results confirmed that
the PVY-CP gene was integrated and expressed into the
genome of potato plants.
Prof. Dr. M. El-Hammady
Prof. Dr. M. E. Khalil
Prof. Dr. H.M.Mazyad
Prof. Dr .A. S. Gamal El-Din
Prof. Dr. F.Abo-El-Abbas
Dr. Steen Lykkke Nielsen
Prof. Dr. A.A. Shalaby