Field of Invention
The present invention relates to a salicylic acid inducible promoter DNA fragment obtained from Figwort
mosaic virus sub-genomic transcript promoter.
The present invention also relates to a method of producing such promoter DNA fragment from Figwort
mosaic virus sub-genomic transcript promoter.
BACKGROUND OF THE INVENTION:
One of the challenges of plant biotechnology is how to express a transgene in plant to produce crop with
increased productivity. The regulatory part of a gene, the promoter, plays an important role in this
regards. Wide spectrum of different promoters from plant, bacterial or viral- origin are currently being
used extremely for expressing transgene in plants [Yoshida and Shinmyo 2000, Lessard 2002] but there
are insufficient numbers of promoter available to resolve this task efficiently although. To resolve the
'promoter crisis' attempts are being made to develop useful promoters including synthetic, recombinant,
hybrid and synthetic-hybrid adopting different approaches [de Boer et al. 1983, Kay et al. 1987, Comai et
al. 1990, Last et al. 1991, Guerineau et al. 1992, Ni et al. 1995, Sawant et al. 2001, Xie et al. 2001,
Rushton et al. 2002, Bhullar et al. 2003, Sammarco and Grabczyk 2005, Chaturvedi et al. 2006, Zhang et
al. 2008 , Ranjan et al. 2011]. In plant biotechnology, these promoters were being used successfully to
tailor the expression pattern and inducibility of a 'gene-expressing cassette'. Designing new promoter
architecture through 'Cis-motif rearrangement' also became very promising for targeted manipulation of
transcriptional activation in context of understanding different metabolic and signaling pathways.
The promoter (control) region of a gene is modular and comprises of small sequence motifs (cis-
elements). The promoter's strength and tissue inducibility usually differed depending upon the copy
numbers and spacing among small sequence motifs [Last et al. 1991, Sawant et al. 2001, Xie et al. 2001,
Rushton et al. 2002, Bhullar et al. 2003, Sammarco and Grabczyk 2005, Gurr and Rushton 2005,

Chaturvedi et al. 2006, Zhang et al. 2008]. Combinatorial interaction between specific small sequence
motif/s with particular trans-acting nuclear protein factor determines the ultimate fate of transcription.
The overall expression and tissue specificity of the transgene could be customized by fine tuning of such
interactions [Berg and Hippel 1987, Rushton et al. 2002, Venter 2007].
Varying the numbers of following cis-elements Wl, W2, GCC, JERE several valuable synthetic
promoters were developed and the strength of such promoters were studied in planta [Rushton et al.
2002]. The performances of these promoters during pathogen attack were also investigated and these
modified promoters were tested to study the signaling and transcriptional activation of important genes
during plant-pathogen interaction [Rushton et al. 2002]. Similarly, the influence of copy-number and
spacing of the ACGT and GT cis elements in promoter sequence were studied in plants [Mehrotra et al.
2005]. Different value-added salicylic and abscisic acid inducible promoters were developed through
genetic manipulation of ACGT motifs in their core sequences [Mehrotra and Mehrotra 2010].
The small sequence motif, identified for the elevated-level of expression of the Cauliflower mosaic virus
35S promoter (CaMV 35S) [Ode11 et al. 1985, Benfey et al. 1989, Fang et al. 1989, Benfey and Chua
1990, Benfey et al. 1990a], octopine synthase (ocs) promoter [Kononowicz et al. 1992] and nopaline
synthase gene (nos) promoter [Ebert et al. 1987] contains the core 'TGACG' motif. Several transacting
factors binding to this TGACG motif have been reported earlier [Fromm et al. 1989, Lam et al. 1989,
Lam et al. 1990, Singh et al. 1989, Singh et al. 1990, Tabata et al. 1989, Tabata et al. 1991, Hartings et al.
1989, Prat et al. 1989, Tokuhisa et al. 1990, Lohmer et al. 1991]. Majority of these transcription factors
were found belonging to the leucine zipper (bZIP) class [Yamazaki et al. 1990, Katagirl et al. 1990,
McKnight 1991, Krawczyk et al. 2002]. The bZIP class of tobacco nuclear protein factors like TGA2.2,
TGA2.1 and ASF-1 (activation sequence factor-1) binds to the as-1 (-65 to -85) region of CaMV 35S
promoter. The as-1 contains two TGACG motifs [Lam et al. 1989] as imperfect palindromes with
palindromic center stretched by 12 nt. The spacing between the two palindromes of 'as-1' element also
influences the binding of respective TGA factors [Krawczyk et al. 2002]. It was reported that a single
copy of as-1 element was sufficient to confer root-expression when placed in up-stream of minimal

CaMV 35S promoter while a tetramer of as-1 element enhances the transcription in both leaf and root
[Lam et al. 1989]. In higher plants, salicylic acid (SA) induce'as-1' element (of CaMV 35S) mediated
enhanced transcriptional activation in plant tissues [Qin et al. 1994, Jupin et al. 1996].
OBJECTS OF THE INVENTION
An object of this invention is to propose a salicylic acid inducible engineered Figwort mosaic virus sub-
genomic transcript (FS) promoter DNA fragment.
Another object of this invention is to propose a method for obtaining an engineered promoter DNA
fragment.
Still another object of this invention is to propose a promoter DNA fragment inducible in response to
stress, particularly salicylic acid.
Further, object of this invention is to also propose expression of chimeric genes using the engineered
promoter DNA fragment in planta.
BRIEF DISCRIPTION OF THE INVENTION
According to this invention there is provided a salicylic acid inducible Figwort mosaic virus minimal
promoter [FS-(TGACG)2] having an engineered 131 base pair ( -100 to + 31) showing enhanced root-
activity was developed by inserting mutations at positions -84 (A→T) and -83(A→G) of the promoter.
In accordance with this invention there is provided A method for producing a promoter DNA
fragment comprising: Cloning of the 131 bp long FS-promoter sequence containing one TGACG
[FS-TGACG] motif from Figwort mosaic virus subgenomic transcript (FMV-sgt) promoter,
Constructing engineered promoter, FS(TGACG)2 by site directed mutagenesis.

The present invention provides an engineered salicylic acid inducible promoter DNA fragment [FS-
(TGACG)2] constructed by inserting an extra TGACG motif by site directed mutagenesis in the Figwort
mosaic virus sub-genomic transcript [Bhattacharyya et al. 2002] promoter DNA fragment (-100 to +31)
sequence by converting A at position -84 to T and A at position -83 to G.
The present invention also provides a method to induce a target gene which is based on the salicylic acid
inducible promoter DNA fragment.
♦ This engineered DNA clone was submitted, to Microbial Type Culture Collection and Gene
Bank (MTCC). Chandigarh. India under Budapest treaty.
MTCC Clone depository number; MTCC 5667.
BRIEF DESCRIPTIONS OF THE ACCOMPANYING DRAWINGS
Figure 1: The DNA sequence of the 131 base pair long (-100 to +31) engineered Figwort mosaic virus
sub-genomic transcript promoter [FS-(TGACG)2] DNA fragment.
Figure 2: Schematic maps of the expression cassette of the plant transformation vectors: pKYLX [only
vector], pKYLXGUS [harboring CaMV35S promoter], pKFS-(TGACG)GUS [harboring FS-(TGACG)]
and pKFS-(TGACG)2GUS [harboring the FS-(TGACG)2 promoter]. LB and RB refers to the left and right
T-DNA border. The arrows indicate the direction of transcription.
Figure 3: Shows typical example of GUS expression analysis in transgenic tobacco plants harboring
pKYLX, pKYLXGUS, pKFS-(TGACG)GUS and pKFS-(TGACG)2GUS. (A) shows GUS expression
levels estimated biochemically according to standard protocol (Jefferson et al. 1987, Bradford 1976). (B)
Shows histochemical staining of whole transgenic tobacco seedling with X-gluc. (C) 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 4: Shows typical example of spatial GUS expression analysis of the promoters pKYLX,
pKYLXGUS, PKFS-(TGACG)GUS and pKFS-(TGACG)2GUS in transgenic tobacco plants. (A) shows

GUS expression levels in root, leaf and stem of transgenic tobacco plants harboring above mentioned
promoter constructs estimated biochemically according to standard protocol (Jefferson et al. 1987,
Bradford 1976). (B) shows GUS transcript levels in the root, leaf and stem of transgenic tobacco plants
harboring 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 GUS expression analysis in transgenic Arabidopsis thaliana plants
harboring the promoter constructs pKYLX, pKYLXGUS, pKFS-(TGACG)GUS and pKFS-
(TGACG)2GUS. (A) shows GUS expression levels measured biochemically according to standard
protocol (Jefferson et al. 1987, Bradford 1976). (B) shows GUS staining of whole seedlings (21 days old)
with X-gluc.
Figure 6: Shows typical example for histochemical staining of roots of transgenic tobacco plants
harboring pKYLX, pKYLXGUS, pKFS(TGACG)GUS and pKFS-(TGACG)2GUS promoter constructs
with the fluorogenic substrate ImagenGreen (ImaGene Green™ GUS Gene Expression Kit; Invitrogen,
Oregon, USA). (A) shows superimposed images of transmitted and green fluorescent of ImageneGreen
treated roots captured with the help of confocal laser scanning microscopy. (B) shows bar diagram
denoting the fluorescent intensities (in Gray scale unit) of the ImageneGreen treated roots of the
transgenic plants harboring the above promoter constructs.
Figure 7: Shows typical example of GUS expression analysis in leaves of transgenic tobacco seedlings
(21 days old) harboring the promoter constructs pKYLX, pKYLXGUS, pKFS(TGACG)GUS and pKFS-
(TGACG)2GUS after induction with salicylic acid (24hours) (A) shows the GUS expression levels
measured biochemically according to standard protocol (Jefferson et al. 1987, Bradford 1976). (B) shows
the GUS transcript levels quantified by real-time PCR considering accumulation of GUS transcript driven
by CaMV35S promoter as 1.0.

DETAILED. DESCRIPTION OF THE INVENTION
The present invention provides an engineered salicylic acid inducible promoter DNA fragment developed
from Figwort mosaic virus sub-genomic transcript promoter. This 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 inducibility of the promoter DNA
fragment. The salient features of this invention are:
Molecular cloning of the 131 bp long FS-promoter sequence containing one TGACG motif [FS-
(TGACG)] (FMV coordinates 5233 to 5363, FSgt coordinates -100 to +31) from Figwort mosaic virus
subgenomic transcript (FMV-sgt) promoter.
Construction of an engineered promoter, FS-(TGACG)2 by site directed mutagenesis, to insert an extra
copy of TGACG motif into the promoter sequence. Site directed mutagenesis was carried out converting
A to T (at position -84) and A to G (at position -83).
The present invention also provides a method to express any gene in plants operatively linked to the
promoter.
The GUS expression directed by the FS-(TGACG)2 in the whole transgenic tobacco seedlings was found
to be 1.25 and 1.98 times higher compared to native [FS-(TGACG)] and CaMV 35S promoters
respectively (FIG. 3A). The GUS-transcript level in the whole tobacco transgenic seedling expressing
FS-(TGACG)2 construct was found to be 1.45 and 1.86 folds higher than that obtained from FS-(TGACG)
and CaMV35S promoters respectively (FIG. 3C).
The GUS expression directed by FS-(TGACG)2 was found to be 2.35 and 4.2 times stronger than FS-
(TGACG) and CaMV35S promoters respectively in roots and 1.22 and 1.6 times stronger expression than
FS-(TGACG) and CaMV35S promoters respectively, in leaves of transgenic tobacco plants (FIG. 4A).
The GUS transcript level directed by FS-(TGACG)2 was found to be 2.46 and 6.13 folds stronger than
the FS-(TGACG) and CaMV35S promoters respectively, in roots and 1.66 and 2.45 folds stronger than
the FS-(TGACG) and CaMV35S promoters respectively, in leaf of transgenic plants (FIG. 4B).
The GUS expression levels directed by FS-(TGACG)2 promoter in transgenic Arabidopsis thaliana plants
were 1.58 and 2.31 times stronger compared to that obtained from FS-(TGACG) and CaMV35S
promoters respectively (FIG. 5A).

The histochemical staining of the transgenic seedlings, Tobacco (FIG. 3B) and Arabidopsis (FIG. 5B)
expressing the above promoter constructs clearly demonstrates that the FS-(TGACG)2 promoter activity
was much stronger compared to FS-(TGACG) and CaMV35S promoters.
The observation from the fluorescent images obtained from transgenic roots treated with ImageneGreen
also confirmed above results.
After 24 hours of induction by salicylic acid (SA) the inducible promoter, FS-(TGACG)2 showed 3.41 and
4.1 folds higher GUS activity compared FS-(TGACG) and CaMV35S promoters respectively.
EXAMPLE;
Example 1 Preparation of transformation vectors.
Construction ofFS-(TGACG)i promoter fragment.
The 131 bp (-100 to +31) long Figwort mosaic virus sub-genomic transcript promoter fragment was PCR
amplified using a plasmid clone of Figwort mosaic virus sub-genomic transcript promoter (pFS3GUS,
Bhattacharyya et al., 2002) as template and specific synthetic forward primers; FP- FS-(TGACG) [for
FS-(TGACG)] and FP-FS-(TGACG)2 [for FS-(TGACG)2]. The FP-FS-(TGACG)2 primer contains
respective mutation (Table 1), so as to insert an extra TGACG motif in the promoter sequence. The PCR
amplifications were carried out under the following standard conditions: 33 cycles of denaturation (94° C
for 3 min), annealing (57° C for 45 sec), and extension (72° C for 45sec). The PCR products was gel
purified, restriction digested with EcoRI and HindIII, and cloned into the corresponding sites of pUC119
to generate pUFS-(TGACG) and pUFS-(TGACG)2.
Construction of plant expression vector for assaying promoter actvitiv.
The promoter fragments FS-(TGACG) and FS-(TGACG)2 were isolated as EcoRI and HindIII fragment
from above described pUC119 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 pKFS-(TGACG)GUS and pKFS-(TGACG)2GUS respectively.

Example 2 Transgenic plant development
Tobacco plant transformation and evaluation of transgenic plants
The plant expression vectors pKFS-(TGACG)GUS, pKFSKTGACG)2GUS and pKYLXGUS were
introduced into Agrobacterium tumifaciens strain C587Cl:pGV3850 by freeze thaw method (Hofgen and
Willmitzer 1988). Transgenic Tobacco plants (Nicotiana tabacum cv samsun NN) were raised following
protocol as described earlier (Marti et al. 1993). Transgenic plants were characterized following standard
protocol.
Arabidopsis plant transformation and evaluation of transgenic plants
Transgenic Arabidopsis plants were generated using the engineered agrobacteria transformed with pKFS-
(TGACG) GUS, pKFS-(TGACG)2GUS and pKYLXGUS individually according to Zhang et al. 2006.
Transgenic plants were characterized following standard protocol.
Example 3
Biochemical GUS assay
GUS enzyme activity of whole seedling, root, leaf and stem of transgenic tobacco plants (21 days old)
and whole seedling of Arabidopsis plants (21 days old) expressing FS-(TGACG), FS-(TGACG)2 and
CaMV35S were measured according to the standard protocols (Jefferson et al., 1987, Bradford MM 1976)
Histochemical GUS assay
Transgenic seedlings (21 days old) of tobacco and Arabidopsis harboring the FS-(TGACG), FS-
(TGACG)2 and CaMV35S promoter constructs were subjected to histochemical GUS staining using 1%
X-gluc solution.
Example 4
Confocal laser scanning microscopy study
Roots of the transgenic tobacco plants harboring the above mentioned promoter constructs were incubated
for 2 hrs with 55µM ImaGene Green C12FDGIcU substrate (ImaGene Green™ GUS Gene Expression
Kit; Invitrogen, Oregon, USA.) which produces a fluorescent product 5-dodecanoylaminofluorcscein

(λex/λem:495/ 518) on enzymatic hydrolysis. The green fluorescent images were taken by exciting at 488
nm and emissions were collected between 501 and 536 nm with detector (PMT) gain set at 1150V using a
CLSM (TCS SP5; Leica, D-68165 Mannheim, Germany).
Example 5 GUS transcript assay for transgenic plants.
RNA isolation
Total RNA was isolated from the whole seedling, root, leaf and stem of transgenic seedlings expressing
pKYLX (vector), pKFS-(TGACG)GUS, pKFS-(TGACG)2GUS and pKYLXGUS using the RNeasy plant
mini Kit (Qiagen, Tokyo, Japan) according to manufacturer's protocol. Subsequently extracted RNA was
treated with DNase I at 37°C for 10 minutes and purified by phenol-chloroform extraction. The mRNA
fraction was purified from the total RNA pool using the PolyATract mRNA isolation system (Promega,
Madison, WI, USA).
Real-time PCR analysis
A 100ng of mRNA was used to synthesize the first strand cDNA using AMV reverse transcriptase and
oligo (dT) primers at 42°C for 1.5 hours. Real time PCR reactions were performed using SYBR Premix
Ex Taq™ II (Perfect Real Time, Takara Bio Inc.) according to manufacturer's instruction on an Opticon-2
qRT-PCR instrument (MJ Research, Bio-Rad; Model CFD-3220). GAPDH cDNA was used as an internal
control for normalization of GUS mRNA levels in the real time PCR. The PCR cycling program was
conducted as follows; 94°C for 30 sec followed by 40 cycles of 94"C for 2 sec, 57°C for 5 sec, 72°C for 30
sec. and sequences of GAPDH and the GUS specific primers were shown in Table 1. The GUS mRNA
levels in whole seedlings, roots, leaves and stems of transgenic plant expressing above promoter
constructs were calculated individually using the 2ΔΔCT method [Livak and Schmittgen 2001, Pfaffl
2001].

Example 6
Salicylic acid treatment
Transgenic tobacco seeds expressing pKYLX (vector), pKFS-(TGACG)GUS, pKFS-(TGACG)2GUS and
pKYLXGUS promoter constructs individually were germinated on half MS plate (containing 300 mg/liter
Kanamycin) and allowed to grow under tissue culture conditions as mentioned above. The leaves of
transgenic seedlings (21 days old) expressing above promoter constructs were collected individually and
treated in presence of 150 uM of sodium salicylate (pH 6.8) 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. Total RNA was isolated from same treated samples and real
time PCR was performed.
Table 1: List of oligonucleotide primers used for amplifying different promoter fragments and
genes

Information for sequence:
(i) Sequence characteristics: Genomic DNA
(ii) Molecule type: Engineered DNA
(iii) Original source: Figwort mosaic virus (FMV)

WE CLAIM:
1. A salicylic acid Figwort mosaic virus minimal promoter [FS-(TGACG)2] having an engineered
131 base pair ( -100 to + 31) showing enhanced root-activity was developed by inserting
mutations at positions -84 (A→T) and -83(A→G) of the promoter.
2. The promoter as claimed in claim 1, comprises of a TATA box sequence from Figwort mosaic
virus sub-genomic transcript promoter between co-ordinates -46 to -40.
3. The promoter as claimed in claim 1, wherein the said promoter is coupled to GUS reporter gene is
found on an average 1.25 and 1.98 times stronger in tobacco and 1.58 and 2.31 times stronger in
Arabidopsis as compared to FS-(TGACG) and CaMV35S promoters respectively.
4. The promoter as claimed in claim 1 wherein the said promoter is coupled to GUS reporter gene is
found to be 2.35 and 4.2 times stronger in root than FS-(TGACG) and CaMV35S promoter
respectively.
5. The promoter as claimed in claim 1 wherein the said promoter is coupled to GUS reporter gene is
found to be expressing 1.22 and 1.6 times higher level of GUS expression in leaf compared to FS-
(TGACG) and CaMV35S promoters respectively.
6. The promoter as claimed in claim 1 wherein the said promoter is coupled to GUS reporter gene
showed 3.41 and 4.1 times stronger in leaf than FS-(TGACG) and CaMV35S promoter
constructs respectively under SA stress (24 hr).
7. A method for producing a promoter DNA fragment comprising:
Cloning of the 131 bp long FS-promoter sequence containing one TGACG [FS-TGACG] motif
from Figwort mosaic virus subgenomic transcript (FMV-sgt) promoter,
Constructing engineered promoter, FS-(TGACG)2 by site directed mutagenesis.
8. The method as claimed in claim 1, wherein the said engineered promoter is prepared by inserting
an extra copy of TGACG motif into the promoter sequence site directed mutagenesis to convert A
to T (at position -84) and A to G (at position -83).

9. The engineered promoter as claimed in claim 1 controls the transcription and translate of a
chimeric gene in plant cells.
10. The engineered promoter as claimed in claim 1 A method is used for producing a heterologous
protein in plant cells.

A salicylic acid inducible Figwort mosaic virus minimal promoter [FS-(TGACG)2] having an engineered
131 base pair ( -100 to + 31) showing enhanced root-activity was developed by inserting mutations at
positions -84 (A→T) and -83(A→G) of the promoter.