Title: Vascular tissue specific and stress inducible hybrid promoter ideal for plant derived
pharmaceutical production.
Field of Invention
This invention relates to a vascular tissue specific and stress inducible hybrid promoter DNA fragment
obtained from the Figwort mosaic virus full length and sub-genomic tianscript promoters.
This invention is also related to a method of obtaining such promoter DNA fragment from Figwort
mosaic virus full length and sub-genomic transcript promoters.
BACKGROUND OF THE INVENTION:
Transgene expression in plant is controlled by a number of factors. Among these, promoter, the primary
regulatory element, plays a major role in tuning the event of gene expression and function. The promoter
region of a gene expression cassette is modular comprising of several small sequence motifs (cis
elements) and combinatorial interactions among these cis-elements with the nuclear protein factors (trans-
factors) ultimately score the promoter strength and its tissue specificity(Atchison 1988, Dynan 1989).
Manipulating the architecture of the promoter through 'cis re-arrangement', the strength and tissue
specificity of a promoter can be optimized in desired manner. Using an ideal/ efficient promoter adjusted
to the plant's background (genotypic) and to the type of the transgene would always be a better choice
(over conventional promoter) for ectopic gene expression in plants.
Viruses under genus Caulimovirus and Badnavirus (Medberry et aL 1992; Bhattacharyya-Pakrashi et al.
1993) are belonging to the Caulimoviridae family - a group of plant double stranded DNA virus (of
approximately 8 Kb in length). In general there are two major typos of transcriptional promoters are
present in Caulimovirus genome, a full-length transcript promoter (FLt- promoter, equivalent to 35S) that
transcribes the whole viral genome and other is a sub-genomic transcript promoter (Sgt-promoter;
equivalent to 19S), it transcribes the viral ORF VI. Several Caulimoviriadae genomes have been fully
sequenced and their promoters have been characterized; these includes Cauliflower mosaic virus (CaMV

35S; Odell et al. 1985, CaMV 19S; Lawton et al. 1987), Figwori mosaic virus (FMV-FIt, Maiti et al.
1997, FMV-Sgt; Bhattacharyya et al. 2002), Peanut chlorotic streak virus (PCISV, Maiti and Shepherd.
1998),
Strawberry vein banding vims (SVBV, Petrzik et al. 1998; Pattanaik et al. 2004), Petunia vein banding
virus (PVCV, Richert-Poggeler and Shepherd 1997) and Mrabilis mosaic virus (MMV-Flt, Dey and
Maiti 1999a, 1999b, MMV-Sgt, Dey and Maiti 2003) etc. The CaMV 25S promoter is also equally
functional in both prokaryotic (Assaad and Signer, 1990) and eukaryotic plant and animal cells (Pobjecky
et al. 1990; Zahm et al. 1989 and Ballas et al. 1989).
OBJECTS OF THE INVENTION
An object of this invention is to propose a vascular tissue specific hybrid promoter DNA fragment
obtained from Figwort mosaic virus full length and sub-genomic transcript promoters;
Another object of this invention is to propose a stress inducible hybrid promoter DNA fragment obtained
from Figwort mosaic virus full length and sub-genomic transcript promoters;
Still another object of this invention is to propose a method for obtaining such a promoter DNA fragment;
Further, object of this invention is to propose a promoter DNA fragment inducible in response to stress;
Still Further object of this invention is to also propose expression of chimeric genes using the hybrid
promoter DNA fragment in planta.

BRIEF DESCRIPTION OF THE INVENTION
According to this invention there is provided a vascular tissue specific and stress inducible hybrid
promoter DNA fragment developed from the ligation of a upstream domain containing the activating
sequence of Figwort mosaic virus full length transcript promoter to another domain containing the
TATA box site of Figwort mosaic virus sub-genomic transcript (FMV-Sgt) promoter.
In accordance with this invention there is also provided a method of producing hybrid promoter DNA
fragment from Figwort mosaic virus comprising: isolating hybrid promoter (FUASFSCP) from Figwort
mosaic virus; subjecting the isolated FUASFSCP hybrid promoter to the step of molecular cloning and
determining the activities of the said hybrid promoter both in transient and transgenic assay.
** This promoter DNA clone FUASFSCP is submitted to Microbial Type Culture Collection and
Gene Bank (MTCC). Chandigarh. India under Budapest treaty.
MTCC clone depositary number: MTCC 5669
BRIEF DESCRIPTIONS OF THE ACCOMPANYINGS
Figure 1: The nucleotide sequence of the 381 base pair long hybrid Figwort mosaic virus promoter
(FUASFSCP) DNA fragment.
Figure 2: Schematic maps of the expression cassette of the plant transformation vectors: pKYLX (only
vector), pKYLXGUS (harboring CaMV35S promoter), pKFUAS, pKFSCP, and pKFUASFSCP. LB and
RB refers to the left and right T-DNA border. The arrows indicate the direction of transcription. E, H, Sm,
X and S represent EcolU, HiruttH, Smal, Xho\ and Sstl endonucleases sites respectively.
Figure 3: Transient expression analysis of the promoters CaMV35S, FUAS, FSCP, FUASFSCP coupled
to GUS reporter gene in tobacco protoplast system.

Figure 4: Shows typical example of GUS expression analysis in transgenic tobacco plants harboring
pKYLX, pKYLXGUS, pKFUAS, pKFSCP, and pKFUASFSCP (A) shows GUS expression levels
estimated biochemically according to standard protocol (Jefferson et al. 1987, Bradford 1976). (6) Shows
relative GUS transcript accumulation levels of the above mentioned promoter constructs quantified by
real-time PCR considering accumulation of GUS transcript driven by CaMV35S promoter as 1.0.
Figure 5: Shows typical example of histochemical staining of different parts of plant expressing the
promoter constructs pKYLX, pKYLXGUS and pKFUASFSCP with X-gluc. (A) whole seedling (B)
transverse section of stem (C) transverse section of leaf petiole.
Figure 6: Shows typical example of histochemical staining of transgenic Arabidopsis thaliana plants
harboring the promoter constructs pKYLX, pKYLXGUS, pKFUASFSCP with X-gluc
Figure 7: Shows typical example for histochemical staining of stem cross sections and roots of transgenic
tobacco plants harboring pKYLX, pKYLXGUS, and pKFUASFSCPGUS promoter constructs with the
fluorogenic substrate ImagenGreen.
(A) shows bright field images of ImageneGreen treated stem cross sections captured with the help of
CLSM.
(B) shows fluorescent images of ImageneGreen treated stem cross sections captured with the help of
CLSM
(C) shows superimposed images of bright field and fluorescent images of ImageneGreen treated stem
cross sections captured with the help of CLSM
(D) shows bright field images of ImageneGreen treated roots captured With the help of CLSM.
(E) shows fluorescent images of ImageneGreen treated roots captured with the help of CLSM
(F) shows superimposed images of bright field and fluorescent images of ImageneGreen treated roots
captured with the help of CLSM

Figure 8: Shows typical example of GUS expression analysis in roots and leaves of transgenic tobacco
seedlings (21 days old) harboring the promoter constructs pKYLX, pKYLXGUS, pKFUASFSCPGUS
after induction with salicylic acid (24hours) measured biochemically according to standard protocol
(Jefferson et al. 1987, Bradford 1976). (A) Roots (B) Leaves.
Figure 9: Shows typical example of GUS expression analysis in roots and leaves of transgenic tobacco
seedlings (21 days old) harboring the promoter constructs pKYLX, pKYLXGUS, pKFUASFSCPGUS
after induction with jasmonic acid (24hours) measured biochemically according to standard protocol
(Jefferson et al. 1987, Bradford 1976). (A) Roots (B) Leaves.
Figure 10: Shows antibacterial activity of protoplast-derived human alpha defensin-1 (HNP-1) expressed
under control of CaMV35S and FUASFSCP using Escherichia coli cell (A) and Staphylococcus aureus
(B).
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides vascular tissue specific and stress inducible promoter DNA fragment
developed from Figwort mosaic virus full length transcript and sub-genomic transcript promoters. The
present invention describes the method of using the said promoter in plant system for expression of
transgene/s. The present invention also provides a method to induce a target gene based on the salicylic
acid and jasmonic acid inducibility of the promoter DNA fragment. The salient features of this invention
are:
1. Molecular cloning of upstream activation sequence (FMV genomic coordinates 6691 to 6885, Fit
promoter coordinates -259 to -54 from TSS) from Figwort mosaic virus full-length transcript
(FMV-Flt) promoter.
2. Molecular cloning of FSCP (FMV genomic coordinates 5184 to 5363, FSgt promoter coordinates
-151 to +31) from Figwort mosaic virus sub-genomic transcript (FMV-Sgt) promoter.
3. Construction of hybrid promoter FUASFSCP by intermolecular hybridization of UAS sequence
from Figwort mosaic virus full-length transcript promoter with core promoter sequence of
Figwort mosaic virus sub-genomic transcript promoter containing TATA element

4. The GUS expression directed by the FUASFSCP in tobacco protoplast is 4.48 times higher
compared to CaMV35S promoter (FIG. 3)
5. The GUS expression directed by the FUASFSCP in the whole transgenic tobacco seedlings is
4.17 times higher compared to CaMV 35S promoter (FIG. 4A). The GIS-transcript level in the
whole tobacco transgenic seedling expressing FUASFSCP construct was 6.14 folds higher than
that obtained from CaMV35S promoter (FIG. 4B).
6. The histochemical staining of the transgenic tobacco seedlings (FIG. 5 A) expressing the above
promoter constructs clearly demonstrates that the FUASFSCP promoter activity was much
stronger compared to the CaMV 3SS promoter.
7. The histochemical staining of the transgenic Arabidopsis seedlings (FIG. 6) expressing the above
promoter constructs clearly demonstrates that the FUASFSCP promoter activity was much
stronger compared to the CaMV 35 S promoter.
8. The histochemical staining of transverse sections of stem and leaf petiole of transgenic plants
expressing the above promoter constructs demonstrates the FUASFSCP promoter shows
maximum expression in vascular tissue as compared to other promoters.
9. The observation from the fluorescent images obtained from transgenic stem sections and roots
treated with ImageneGreen also confirms the above results.
10. After 24hours of induction in presence of salicylic acid the promoter FUASAFSCP showed 3.1
and 4.5 times stronger activity than CaMV35S promoter in leaf and root respectively.
11. After 24hours of induction in presence of jasmonic acid the promoter FUASAFSCP showed 3.2
and 3.5 times stronger activity than CaMV35S promoter in leaf and root respectively.

EXAMPLE;
Example-1: Preparation of transformation vectors.
Construction ofFUASFSCP promoter fragment.
The 195 bp long FUAS (-249 to -54, upstream activation sequence of F promoter) and 182 bp long FSCP
(-151 to +31, TATA box containing core-promoter sequence of FS promoter) were PCR amplified using
specific primer pairs (Table 1) having appropriate sequence to generate EcoRI and HincU sites at the 5'
end and Sma\ and Hindis, sites at the 3' end as per protocol described earlier (Dey and Maiti 1999). PCR-
amplified fragments were digested with EcoBl and HindBl, gel-purified and cloned into the
corresponding sites of the sequencing vector pUC119. The resulting plasmids were designated as
pUFUAS and pUFSCP respectively. The sequences of these clones were verified.
The FSCP promoter fragment was isolated from pUFSCP clone as HincH-HindUl fragment; and inserted
into the Smal and HmdDl sites of pUFUAS to generate pUFUASFSCP clone.
Construction of transient expression vector for assaying the promoter wi tobacco protoplast system.
The promoter fragments FUAS, FSCP and FUASFSCP were isolated from the respective
pUC119 based clones as Ecoffl and HmdHl fragments and subcloned into, the corresponding sites of
protoplast expression vector pUCPMAGUS (Dey and Maiti, 1999) replacing the CaMV35S promoter.
The resulting plasmids were designated as pUPFUASGUS, pUPFSCPGUS and pUPFUASFSCPGUS
respectively.
Construction of plant expression vectors for assaying the promoter activity in plants.
The promoter fragments FUAS, FSCP and FUASFSCP were isolated as EcoM and Hindttl fragment
from the respective pUCl 19 based clones and sub-cloned into the corresponding sites of the plant
expression vector pKYLXGUS (Dey and Maiti 1999a) replacing the CaMV35S promoter. The resulting
plasmids were designated as pKFUASGUS, pKFSCPGUS and pKFUASFSCPGUS respectively.

Example-2:
Transient assay of the promoters in tobacco protoplast system.
Protoplasts from tobacco cell suspension cultures (Xanthi 'Brad') were purified on 20% sucrose gradient
after being digested by cellulase (Sigma Cat # C0615-1 G) and pectinase (Sigma Cat # P4300-5 KU).
Ten ug aliquot of each of the pUPFUASGUS, pUPFSCPGUS and pUPFUASFSCPGUS promoter
constructs were electroporated into 2 X 106 protoplasts at 200V, 965 uF capacitance for 40-50 ms using
the GenePulser II Apparatus (Bio-Rad) with Capacitance Extender II (model 165-2107). Electroporated
protoplasts were incubated at 28°C dark for 20 hours. For GUS expression analysis the protoplasts were
harvested and biochemical GUS assay was performed according to Jefferson et al. 1987.
Example 3:
Transgenic plant development
Tobacco plant transformation and evaluation of transgenic plants
The plant expression vectors pKYLX71, pKYLXGUS, pKFUASGUS, pKFSCPGUS and
pKFUASFSCPGUS were introduced into Agrabacterium tumifaciens strain C587Cl:pGV3850 by freeze
thaw method (Hofgen and Willmitzer 1988). Transgenic Tobacco plants (Nicotiana tabacum cv samsun
NN) were raised using the engineered agrobacteria and kanamycin resistance as selective marker.
Transgenic plants were characterized following standard protocol.
Arabidopsis plant transformation and evaluation of transgenic plants
The engineered Agrobacteria containing the plant expression vectors pKYLX71, pKYLXGUS,
pKFUASGUS, pKFSCPGUS and pKFUASFSCPGUS were used for raising transgenic Arabidopsis
plants according to Zhang et al. 2006. Transgenic plants were characterized following standard protocol.

Example 4:
Biochemical GUS assay
GUS enzyme activity of transgenic tobacco seedling (21 days old) expressing GUS under the
control of FUAS, FSCP, FUASFSCP and CaMV35S were measured according to the standard protocols
(Jefferson et al. 1987, Bradford MM 1976)
Histochemical GUS assay
Transgenic tobacco seedlings (21 days old), transverse sections of stem and leaf petiole harboring
die FUASFSCP and CaMV35S promoter constructs were subjected to histochemical GUS staining using
1% X-gluc solution.
Example 5:
Real-time PCR analysis
Total RNA was extracted from 21 days old transgenic seedlings expressing; pKYLXGUS, pKFUASGUS,
pKFSCPGUS and pKFUASFSCPGUS individually and treated with DNasel to remove the DNA
contamination. The first strand cDNA sysnthesis was carried out using cDNA synthesis Kit (Fermentas,
USA) and 7 ug of total RNA. Real-time PCR were performed using the corresponding cDNA templates
and SYBR Premix Ex Taq™ II (Perfect Real Time, Takara Bio Inc., Japan) employing Opticon-2 Real-
time PCR machine (MJ Research, Bio-Rad; Model; CFD-3220). Gene specific primers for GUS and
GADPH (Table 1) were used to carry out the Real-Time-PCR reactions as per the kit's instructions. Cycle
parameters were 94°C for 30 sec followed by 37 cycles of 94°C for 5 sec, 57°C for 30 sec, 72° C for 30
sec and finally 72°C for 5 minute. The absence of genomic DNA contamination was confirmed using
minus-reverse-transcriptase controls. Threshold cycle (CT) value for each reaction was obtained with the
help of the software attached with the machine and the difference in the transcript level (in fold) between
CaMV 35S and FUASFSCP promoters were calculated using 2**" method (Livak and Schmittgen 2001,
Pfaffl2001).

Example 6:
CLSMstudv
Stem cross sections and roots of the transgenic tobacco plants harboring the FUASFSCP and
CaMV35S promoter constructs were treated with 55uM ImaGene Green C12FDGlcU substrate (ImaGene
Green™ GUS Gene Expression Kit; Invitrogen, Oregon, USA,), kept under vacuum infiltration for 10
mins initially and then incubated at room temperature for 2-3 hours in the dark. The green fluorescent
images were taken by exciting with a 488nm diode laser and emissions collected between 501 and 536
tun with detector (PMT) gain set at 1150V using a CLSM (TCS SP5; Leica, D-68165 Mannheim,
Germany).
Example 7:
Salicylic acid and Jasmonic acid treatment
Transgenic tobacco seeds expressing pKYLX (vector), pKYLXGUS and pKFUASFSCPGUS promoter
constructs individually were germinated on half MS plate (containing 300 mg/liter Kanamycin). The
leaves and roots of the transgenic seedlings (21 days old) were collected individually and treated in
presence of 150 uM of sodium salicylate (pH 6.8) and methyl jasmonate separately for 0 hr and 24 hr
according to Jupin et al.1996. After treatments, GUS activities from leaves of above mentioned promoter
constructs were measured according to Jefferson et al. 1987.
Example S:
Antimicrobial activity of chimeric promoter driven protoplast-derived HNP-1
Human a-defensin-1 (HNP-1) was amplified using gene specific primer pair (Table 1) so as to
generate Xhol site at 5' end and Sacl at 3'end using HNP-1 cDNA clone as a template. PCR
amplifications were carried out under the following standard conditions; denaturation (94°C for 1 min),
annealing (57°C for 45sec) and extension (72°C for 30 sec) for 35 cycles. Amplified product was gel-
purified and digested with Xhol and Sacl, cloned into the corresponding sites of the sequencing vector
pBSK+ to form pBSHNP-1. The defensin gene (HNP-1) was isolated as Xhol and Sacl fragment and
cloned into corresponding sites of pUCPMAGUS and pUPFUASFSCPGUS by replacing the GUS gene.

The resulting constructs were designated as pUCPMAHNP-1 and pUPFUASFSCPHNP-1 respectively.
Tobacco protoplasts were electroporated with 10 ug of each of the pUCPMAHNP-1 and
pUPFUASFSCPHNP-1 individually according to protocol described earlier (Dey and MaM 1999a).
Untransformed protoplast was used as control. After 20 hours of incubation protein isolation was carried
out by homogenizing the protoplast in a buffer containing 50mM Tris-HCL 5mM EDTA and protease
inhibitor cocktail(Sigma, USA). The homogenate was centrifuged at 10,000 X g for 15 minutes.
Supernatant containing protein was collected in a fresh tube and protein concentration was quantified
according to Bradford 1976. A lOOul PBS containing 10 ug of protein extract from protoplasts
transformed with each of the above constructs were coated into a 96 well plate. The concentration of
protoplast derived HNP-lwas estimated following indirect ELISA protocol (Vazquez et al. 1996) using
anti-HNP-1 antibody (Santacruz, USA)
Antimicrobial assay of the recombinant peptide was performed according to Nitschke et al. 2002 with
slight modification using two bacterial culture namely E. colt (TBI, non-pathogenic) and Staphylococcus
aureus (pathogenic). In brief, an aliquot of 1.0 ml PBS containing approximately 107 CFU of bacteria]
cells of E. coli (TBI) and Staphylococcus aureus individually were centrifuged and resuspended in
Mueller-Hinton broth containing 100 ug of protein extract in a final volume of 1.0 ml. These were
incubated at 37° C for 2 hours. An aliquot of 100 ul from 105 dilutions was spread on LB Agar plate,
incubated overnight at 37° C and Colony Forming Unit (CFU) was counted.

WE CLAIM:
1. A vascular tissue specific and stress inducible hybrid promoter DNA fragment developed from the
ligation of the upstream activating sequence of Figwort mosaic virus full length transcript promoter to
another domain containing the TATA box site of Figwort mosaic virus sub-genomic transcript (FMV-
Sgt) promoter.
2. The vascular tissue specific and stress inducible hybrid promoter as claimed in claim 1, wherein said
domain containing the 195 bp fragment of UAS of Figwort mosaic virus full length transcript (FMV-FIt)
promoter, coordinates -249 to -54 from transcription start site (TSS).
3. The hybrid promoter as claimed in claim 1, wherein said domain containing the TATA box site of
Figwort mosaic virus sub-genomic transcript (FMV-Sgt) promoter is about 182 bp, coordinates -151 to
+31 from TSS.
4. The hybrid promoter (FUASFSCP) as claimed in claim 1, wherein said promoter is coupled to GUS
showed 4.48 and 4.17 times stronger activity than that of CaMV35S promoter in transient protoplast
system and transgenic tobacco plants respectively.
5. The hybrid promoter as claimed in claim 1 showed 3.1 and 4.5 times stronger activity than CaMV35S
promoter after treatment with elicitor salicylic acid for 24hrs in leaf and root respectively.
6. The hybrid promoter as claimed in claim 1 showed 3.2 and 3.5 times stronger activity than CaMV35S
promoter after treatment with elicitor jasmonic acid in leaf and root respectively.
7. A method of producing hybrid promoter DNA fragment from Figwort mosaic virus comprising:
isolating hybrid promoter (FUASFSCP) from Figwort mosaic virus; subjecting the isolated FUASFSCP
hybrid promoter to the step of molecular cloning and determining the activities of the said hybrid
promoter both in transient and transgenic assay.

8. The method as claimed in claim 7, wherein for the step of molecular cloning, the upstream activation
sequence (-249 to -54 from TSS) from Figwort mosaic virus mil length transcript (FMV-FIt) promoter is
used and downstream sequence containing TATA element (-151 to +31 from TSS) of Figwort mosaic
virus sub-genomic transcript (FMV-Sgt) promoter is used.
9. A chimeric gene which is transcribed and translated in plant cells under the control of the said hybrid
promoter as claimed in claim 1.
10. A method of producing a heterologous protein using the hybrid promoter as claimed in claim 1 in
plant cells.

Vascular tissue specific and stress inducible hybrid promoter ideal for plant derived
pharmaceutical production.
A vascular tissue specific and stress inducible hybrid promoter DNA fragment developed from the
ligation of a upstream activation sequence of Figwort mosaic virus full length transcript promoter (FMV-Flt)
to another domain containing the TATA box site of Figwort mosaic virus sub-genomic transcript
(FMV-Sgf) promoter.