rnai mediated curcin precursor gene silencing in jatropha (jatropha curcas l.)

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RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.) Vikas Yadav Patade Deepti Khatri Kamal Kumar Atul Grover Maya Kumari Sanjay Mohan Gupta Devender Kumar Mohammed Nasim Received: 25 June 2013 / Accepted: 14 February 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract Curcin, a type I ribosomal inhibiting protein- RIP, encoded by curcin precursor gene, is a phytotoxin present in Jatropha (Jatropha curcas L.). Here, we report designing of RNAi construct for the curcin precursor gene and further its genetic transformation of Jatropha to reduce its transcript expression. Curcin precursor gene was first cloned from Jatropha strain DARL-2 and part of the gene sequence was cloned in sense and antisense orientation separated by an intron sequence in plant expression binary vector pRI101 AN. The construction of the RNAi vector was confirmed by double digestion and nucleotide sequencing. The vector was then mobilized into Agrobac- terium tumefaciens strain GV 3101 and used for tissue culture independent in planta transformation protocol optimized for Jatropha. Germinating seeds were injured with a needle before infection with Agrobacterium and then transferred to sterilized sand medium. The seedlings were grown for 90 days and genomic DNA was isolated from leaves for transgenic confirmation based on real time PCR with NPT II specific dual labeled probe. Result of the transgenic confirmation analysis revealed presence of the gene silencing construct in ten out of 30 tested seedlings. Further, quantitative transcript expression analysis of the curcin precursor gene revealed reduction in the transcript abundance by more than 98 % to undetectable level. The transgenic plants are being grown in containment for further studies on reduction in curcin protein content in Jatropha seeds. Keywords Physic nut in planta Genetic transformation Curcin Introduction Jatropha (Jatropha curcas L.), also known as physic nut, is an important non-edible tropical and subtropical oilseed deciduous plant with great potential as a petro crop. The plant belongs to euphorbiaceae family and is indigenous to Mexico and Central America. Multiple including medicinal uses of the plant as a whole are reported [1]. The plant has attracted attention as a potential biofuel crop owing to its hardiness, easy propagation, drought endur- ance, high oil content, and suitability of seed oil for biofuel conversion [2, 3]. Better performance of Jatropha biodiesel compared with petro-diesel has been demonstrated [4]. Therefore, large-scale cultivation of Jatropha is promoted for biodiesel production. At present, the varieties being used to establish plantations outside Central American region are toxic preventing use of the Jatropha seed meals as animal feed or as a fertilizer. Besides, the extensive prolonged exposure to crushed seeds or oil increases the risk among the growers, processors and consumers [4]. The toxicity of the whole seed of Jatropha has been attributed to a protein component, designated as ‘‘curcin’’, a type I ribosome inhibiting protein-RIP [5]. Curcin is a phytotoxin found mainly in the seeds of Jatropha, however, its induced expression has been reported in leaves on exposure to biotic and abiotic stress factors [6, 7]. A range of methods like extraction with polar organic solvent and/or heat/NaHCO 3 treatment have been proposed Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3301-8) contains supplementary material, which is available to authorized users. V. Y. Patade (&) D. Khatri K. Kumar A. Grover M. Kumari S. M. Gupta D. Kumar M. Nasim Defence Institute of Bio-Energy Research, Haldwani-263 139, Nainital, Uttarakhand, India e-mail: [email protected] 123 Mol Biol Rep DOI 10.1007/s11033-014-3301-8

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Page 1: RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.)

RNAi Mediated curcin precursor gene silencing in Jatropha(Jatropha curcas L.)

Vikas Yadav Patade • Deepti Khatri • Kamal Kumar •

Atul Grover • Maya Kumari • Sanjay Mohan Gupta •

Devender Kumar • Mohammed Nasim

Received: 25 June 2013 / Accepted: 14 February 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Curcin, a type I ribosomal inhibiting protein-

RIP, encoded by curcin precursor gene, is a phytotoxin

present in Jatropha (Jatropha curcas L.). Here, we report

designing of RNAi construct for the curcin precursor gene

and further its genetic transformation of Jatropha to reduce

its transcript expression. Curcin precursor gene was first

cloned from Jatropha strain DARL-2 and part of the gene

sequence was cloned in sense and antisense orientation

separated by an intron sequence in plant expression binary

vector pRI101 AN. The construction of the RNAi vector

was confirmed by double digestion and nucleotide

sequencing. The vector was then mobilized into Agrobac-

terium tumefaciens strain GV 3101 and used for tissue

culture independent in planta transformation protocol

optimized for Jatropha. Germinating seeds were injured

with a needle before infection with Agrobacterium and

then transferred to sterilized sand medium. The seedlings

were grown for 90 days and genomic DNA was isolated

from leaves for transgenic confirmation based on real time

PCR with NPT II specific dual labeled probe. Result of the

transgenic confirmation analysis revealed presence of the

gene silencing construct in ten out of 30 tested seedlings.

Further, quantitative transcript expression analysis of the

curcin precursor gene revealed reduction in the transcript

abundance by more than 98 % to undetectable level. The

transgenic plants are being grown in containment for

further studies on reduction in curcin protein content in

Jatropha seeds.

Keywords Physic nut � in planta � Genetic

transformation � Curcin

Introduction

Jatropha (Jatropha curcas L.), also known as physic nut, is

an important non-edible tropical and subtropical oilseed

deciduous plant with great potential as a petro crop. The

plant belongs to euphorbiaceae family and is indigenous

to Mexico and Central America. Multiple including

medicinal uses of the plant as a whole are reported [1]. The

plant has attracted attention as a potential biofuel crop

owing to its hardiness, easy propagation, drought endur-

ance, high oil content, and suitability of seed oil for biofuel

conversion [2, 3]. Better performance of Jatropha biodiesel

compared with petro-diesel has been demonstrated [4].

Therefore, large-scale cultivation of Jatropha is promoted

for biodiesel production. At present, the varieties being

used to establish plantations outside Central American

region are toxic preventing use of the Jatropha seed meals

as animal feed or as a fertilizer. Besides, the extensive

prolonged exposure to crushed seeds or oil increases the

risk among the growers, processors and consumers [4]. The

toxicity of the whole seed of Jatropha has been attributed to

a protein component, designated as ‘‘curcin’’, a type I

ribosome inhibiting protein-RIP [5]. Curcin is a phytotoxin

found mainly in the seeds of Jatropha, however, its induced

expression has been reported in leaves on exposure to

biotic and abiotic stress factors [6, 7].

A range of methods like extraction with polar organic

solvent and/or heat/NaHCO3 treatment have been proposed

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11033-014-3301-8) contains supplementarymaterial, which is available to authorized users.

V. Y. Patade (&) � D. Khatri � K. Kumar � A. Grover �M. Kumari � S. M. Gupta � D. Kumar � M. Nasim

Defence Institute of Bio-Energy Research, Haldwani-263 139,

Nainital, Uttarakhand, India

e-mail: [email protected]

123

Mol Biol Rep

DOI 10.1007/s11033-014-3301-8

Page 2: RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.)

to detoxify the defatted seed meal, however economically

feasible method for large-scale detoxification is yet to

develop [4, 8]. Thus, adoption of varieties lacking curcin

and other toxins will improve economic feasibility of

Jatropha cultivation as well as eliminate any potential risk

associated with prolonged exposure. However, the genetic

improvement through conventional breeding may be

hampered due to limited genetic diversity in Jatropha

germplasm. Non-toxic/edible Mexican Jatropha is known

to lack phorbol esters, but its curcin content is not lesser

than the other toxic genotypes [9]. Therefore, great scope

exists for genetic improvement of the Jatropha with

reduced curcin content through biotechnological interven-

tions. Genetic engineering approach of RNA mediated

gene silencing [10–12] may be employed for reducing the

toxic protein content in the seed meal. Agrobacterium

tumefaciens mediated genetic transformation of plants has

become a method of choice for transgenic development

owing to its advantages such as lower copy stable genomic

integration of transgene and fewer DNA rearrangements

leading to its stable inheritable expression as compared to

direct DNA delivery methods [13–15]. An efficient genetic

transformation is an important pre-requisite for transgenic

development. Though, in vitro regeneration from various

explants have been reported in Jatropha [16–19], applica-

tion of these regeneration systems in tissue culture

dependent genetic transformation have been restricted due

to lower transformation efficiency and several handling

steps. Further, more time is required for transgenic devel-

opment which is another serious concern in the perennial

plant. As against, tissue culture independent in planta

Agrobacterium mediated genetic transformation method is

easy to perform, fast and having higher transformation

efficiency [20]. Feasibility of in planta genetic transfor-

mation is reported in other crops [21], but such reports are

missing in Jatropha. We report herewith the designing of

RNAi construct for curcin precursor gene and optimization

of in planta Agrobacterium mediated genetic transforma-

tion to develop Jatropha with low curcin transcript

expression.

Materials and methods

Seed material

Seeds of Jatropha strain DARL-2 were cleaned by washing

with tap water for multiple times, and soaked overnight in a

solution (0.1 %, w/v) of carbendazim based broad spec-

trum systemic fungicide. Seeds were then allowed to dry in

shed for 24 h. The dried seeds were then germinated on

filter paper placed in glass plates for 4 days at room

temperature.

Cloning curcin precursor gene

Genomic DNA was isolated from young leaves using plant

genomic DNA isolation kit (BioServe-India, India). Prim-

ers were designed based on the sequence information [6]

available in NCBI GenBank (http://ncbi.nlm.nih.gov/gen

bank). The PCR master mix and thermal cycling were

optimized to amplify the curcin precusor gene. The puri-

fied PCR product was cloned in pGEMT cloning vector

and sequenced commercially (Eurofins, India). The

sequence was analyzed for homology using Blast program

[22]. The gene structure was predicted using FGENESH

2.6 tool (http://linux1.softberry.com/berry.phtml). The

Motif scanning of the predicted amino acid sequence was

performed using MyHits program [23].

Design of RNAi construct

Part of the curcin precursor gene sequence (334 bp at

position 601–934) was cloned in sense and antisense ori-

entation separated by 192 bp intronic sequence (X04753)

under the regulation of constitutive promoter CaMV 35S in

plant expression binary vector pRI101 AN (Takara Bio,

Inc.). The silencing construct (pRI101 AN–CurSil-1)

preparation was confirmed by double digestion with

restriction enzymes (Eco RI/Sac I) as well as by

sequencing.

Establishment of in planta genetic transformation

The germinating seeds (5 days after sowing) were used for

in planta genetic transformation studies. Seed coats were

removed and the seeds were washed with tap water. Further

steps were carried out in laminar air flow. The seeds were

first washed with sterilized distilled water and blot dried on

autoclave sterilized tissue paper. The embryonic axes of

the germinating seeds were exposed with a sterilized blade

for infection.

Agrobacterium tumefaciens strain GV 3101 harboring

plant expression vector pRI101 AN carrying selectable

marker gene NPT II was used for standardization of the

genetic transformation experiment. The single isolated

colony was inoculated in yeast extract mannitol-YEM

(MgSO4�7H2O, 0.2 g/L; yeast extract, 1.0 g/L; mannitol

10.0 g/L; NaCl 0.1 g/L; K2HPO4, 0.5 g/L; pH adjusted to

7.0) broth medium with appropriate Kanamycin (50 mg/L)

and Rifampicin (25 mg/L) selection. The cultures were

incubated at 28 �C at 200 rpm for 24 h. The growth of the

culture was estimated based on spectrophotometric

absorption at 600 nm. The cells were precipitated by cen-

trifugation at 22 �C for 3 min at 8,0009g. The cells were

washed with Acetosyringone solution (100 lM) by cen-

trifugation at 8,0009g at 22 �C for 3 min to remove traces

Mol Biol Rep

123

Page 3: RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.)

of the antibiotics. The washed cells were re-suspended in

the Acetosyringone solution to adjust final optical density

of 1.0–2.0.

The prepared seeds were then injured and infected with

needle (21 G X 1.5) dipped in the Agrobacterium suspen-

sion. The infected seeds were incubated with the Agro-

bacterium suspension in flask at 28 �C for up to 60 min in

an incubator shaker at 100 rpm (Table 1). Another set of

the seeds after infection was subjected to vacuum

(80 K Pa) treatment. The seeds were placed in an ultrafil-

tration assembly with the Agrobacterium suspension and

vacuum was applied for up to 20 min. The seeds were then

blot dried and transferred to sterilized sand medium satu-

rated with water in pots. The grown seedlings were given

Hoagland medium and grown in a contained laboratory for

90 days. Transformation experiment was repeated to con-

firm the reproducibility of the results. Upon standardiza-

tion, the same set of protocol and bacterial strain was used

for genetic transformation with silencing vector.

Transgenic validation

Genomic DNA was isolated from the young leaves using

plant genomic DNA isolation kit (BioServe–India, India).

The genomic DNA was quantified based on spectropho-

tometric absorbance at 260/280 nm. The isolated genomic

DNA (50 ng/reaction) was used in transgenic confirmation

using an indirect assay of real-time detection of NPT II

gene. In brief, real time PCR reaction (50 lL) was set up

with the master mix components including molecular

biology grade water, 109 PCR buffer, dNTP mix

(10 mM), primers (10 lM) and dual labeled probe (3 lM)

specific to NPT II gene sequence, and Taq DNA poly-

merase (0.2 U). At the end, genomic DNA was added to

the master mix. Two step PCR reaction was performed in

real time PCR (Mx3005P; Stratagene, Germany) with ini-

tial denaturation for 5 min, followed by 28 cycles of

denaturation at 95 �C for 30 s and primer annealing/

extension at 60 �C for 30 s. Fluorescence specific to the

reporter dye was collected at the end of annealing/exten-

sion step of each cycle. The threshold fluorescence was set

by the instrument and the obtained Ct values were recor-

ded. Further, to avoid false negative results in the putative

transgenic samples, the isolated samples were amplified

with primers specific to internal control gene, b Actin. The

transgenic confirmation reactions were performed in trip-

licate and the experiment was repeated twice to check the

reproducibility of the results.

Validation of gene silencing by qRT PCR analysis

All the glassware and plastic ware used in RNA isolation

were made RNase free by treating with 0.1 % (v/v) diethyl

pyrocarbonate (DEPC; SIGMA Chemicals, USA) for 16 h

followed by autoclaving for 1 h and oven drying at 80 �C

for 48 h. The leaves were mechanically wounded before

the RNA isolation from five independent confirmed (as

above) transformed and wild type seedlings using TRIzol

reagent (SIGMA Chemicals, USA) as described earlier

[24]. The quality and quantity of the isolated total RNA

was assessed based on O.D. at 260/280 nm. First strand

cDNA was synthesized from the isolated RNA (1.0 lg)

using QuantiTect� Reverse Transcription Kit (Qiagen,

USA) in the reaction volume of 20 lL at 42 �C for 30 min

in a thermal cycler (Bio-Rad S1000, Singapore).

Primers specific to b Actin (as an internal control) and

curcin precursor gene were designed (Table 2) using Pri-

mer3 software [25]. The reaction mixture was prepared

using QuantiFastTM SYBR�Green PCR master mix con-

taining pre-optimized ROX as passive reference dye (Qia-

gen, USA), gene specific primers (10 lM), cDNA as a

template and nuclease free molecular biology grade water.

Thermal cycling program consisted initial denaturation

(95 �C, 10 min), followed by 30 cycles of denaturation

(95 �C, 30 s), primer annealing (55 �C, 30 s) and primer

extension (72 �C, 20 s). The qRT PCR reactions were

carried out in a real time thermal cycler-Mx3005P (Strata-

gene, Germany). Fold expression of the target genes was

quantified based on CT values using DDCT method [26].

The qRT PCR products were further separated on aga-

rose gel (2 %) and the fluorescence emitted was detected

using a multipurpose phosphor-imager (Typhoon 9410, GE

Table 1 Summary of the in

planta genetic transformationSr.

no.

Transformation

experiments

O.D.

600 nm

Infection time

(min)

Vacuum

(min)

Survival

(%)

Positive plants

(%)

1 I 2.0 30 10 Nil NA

2 II 2.0 60 20 Nil NA

3 III 2.0 30 – Nil NA

4 IV 1.0 40 10 Nil NA

5 V 1.0 40 – Nil NA

6 VI 1.0 20 – 70 12

7 VII 1.0 20 10 50 15

Mol Biol Rep

123

Page 4: RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.)

Healthcare, USA) with emission filter: 610 BP30 (Deep

Purple, SYPRO, Ruby, EtBR) at 488 nm, with focal plane

adjusted ?3 mm above the surface. The captured 1D gel

images were further analyzed using ImageQuantTM TL

software (GE Healthcare, USA).

Results

Establishment of in planta transformation

and confirmation

Infection with Agrobacterium to germinating seeds was

carried out at 5 days after sowing. Seeds infected at an

earlier stage resulted in complete mortality whereas,

infection at later stages resulted in failure in genetic

transformation. The seeds were then transferred to the

sterilized sand medium in the contained laboratory and

were allowed to grow for 90 days. Transgenic confirma-

tion was carried out with real time PCR analysis using

NPT II gene specific primers with genomic DNA as a

template from the putative transgenic seedlings, subjected

to in planta transformation. Ct values were obtained only

in positive samples (Fig. 1). Possibilities of false positives

were eliminated as no Ct was obtained in case of negative

(no template controls) and wild type seedlings. Further,

the genomic DNA samples were PCR amplified with

primers specific to b Actin as an internal control. The

results revealed amplification in all the test samples, thus,

eliminating the possibility of false negatives. Ct was

obtained in positive reaction with pRI101 AN plasmid

DNA as a template. Higher bacterial cell density used for

transformation (as measured by O.D. at 600 nm of 2.0) or

longer infection (30 min or higher) resulted in zero sur-

vival of the seedlings. The O.D. of 1.0 and infection time

of 20 min resulted in higher survival (70 %) as well as

successful transformation in 12 % of the transformed

seedlings. Infection of the germinating seeds without

Acetosyringone treatment resulted in lower transformation

efficiency. Application of vacuum for 10 min after the

infection slightly reduced the survival (by 20 %), however

improved the rate of success of transformation (Table 1).

The transgenic confirmation was repeated twice, with the

same results. The transgenic plants are being grown in

containment for analysis of stability of gene integration

and expression in subsequent generations.

Cloning and sequence analysis of curcin precursor

gene

Curcin precursor gene was amplified and cloned in pGEM-

T vector. The gene sequence was determined by DNA

sequencing, followed by sequence analysis using BLASTn

at NCBI server. The processed sequence (1,075 bp),

bearing variations from previously known sequences, was

submitted to NCBI GenBank database (Accession No.

JF357910). Gene structure prediction using FGENESH 2.6

tool indicated an exon in the plus chain (score 30.280566).

Coding sequence and ORF (11–940) with a length of

930 bp was predicted (score 36.15) along with poly A

starting at 973 bp (score 1.06).

Motif scanning of the predicted amino acid sequence

(FGENESH tool) using MyHits program [23] indicated

confirmed status of the Shiga/ricin ribosomal inactivating

toxins active site signature (204–220) as well as ribosome

inactivating protein motif (48–293 aa; N-score = 66.096;

E-value = 1.7e-59).

Construction of RNAi vector and genetic

transformation

A 334 bp long gene fragment was cloned in sense and

antisense orientation separated by intron sequence (192 bp)

in plant expression binary vector pRI101 AN to produce

double stranded RNA as a trigger for the sequence specific

silencing, upon genomic integration and expression of the

cassette (Fig. 2). Construction of the vector confirmed by

double digestion with restriction enzymes (Eco R1 and Sac

1), produced digestion product of expected size (860 bp).

Further, sequencing also confirmed the cloning of correct

gene sequence in sense and antisense orientation with

intron. The minimum free energy secondary structure

prediction of the cloned sequence with RNAfold program

suggested formation of the hairpin RNA with loop [27].

The binary silencing vector was then transferred to chem-

ically competent Agrobacterium tumefaciens strain GV

3101 cells by heat shock method and the recombinant

Table 2 Primers designed for

quantitative transcript

expression analysis using

Primer3 tool

Sr. no. Primer Length (50–30) Tm (oC) Amplicon (bp)

1 Curcin F TTCCAGAGGCAGCAAGATTC 60 111

2 Curcin R GGTCTCCCCAGTTGTTCTCA 60

3 Actin F CAGAGGAACACCCAGTGCTT 60 148

4 Actin R CGACCACTGGCATACAAAGA 60

Mol Biol Rep

123

Page 5: RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.)

colonies appeared on Kanamycin selection medium were

confirmed by real-time PCR with primers and fluorescent

dual labeled probe specific to the marker gene NPT II.

Germinating seeds after in planta genetic transforma-

tion, carried out as described above, were grown in labo-

ratory and transgenic confirmation carried out using real

time PCR with the primers specific to NPT II. Genomic

DNA isolated from ten out 30 tested seedlings produced Ct

(Fig. 3) confirming transgenic nature of the seedlings for

the construct. However, Cts were not obtained in negative

control or wild type sample.

Transcript expression of curcin precursor gene

Under non-stress condition, the transcript expression of the

curcin precursor gene was not detected in the leaves of the

wild type or the lines transformed with the silencing con-

struct (Data not shown). However, the transcript expression

was increased on mechanical wounding of the leaves in the

wild types. In one transgenic line, Ct was not obtained in

qRT PCR with the curcin precursor gene, indicating

reduction in transcript expression below the detectable

level (Fig. 4). In other four transgenic lines tested, lower

Transgenic

Wild types, NTC, non-transgenic

Fig. 1 Amplification plot depicting transgenic confirmation in plants

transformed with binary vector pRI101 AN. Genomic DNA isolated

from leaves of the putative transgenic and wild types was used as a

template in quantitative PCR for transgenic confirmation with NPT II

gene specific primers. Cts were obtained in five of the tested (34)

samples but not in the rest of the putative transgenic plants or the wild

types

Sense Intron Anti-sense

pRI101-AN-CurSil-1

(11,277 bp)

Fig. 2 Custom designed RNAi construct (pRI101 AN–CurSil-1). Sequence of the custom designed precursor silencing fragment (860 nt) cloned

in the binary vector (pRI101 AN). Sequence (334 nt) in sense and antisense orientation is separated by intronic sequence (192 nt)

Mol Biol Rep

123

Page 6: RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.)

levels of transcripts of curcin precursor gene were detected

as indicated by higher Ct values than the wild type. The

transcripts of the normalizer gene (b Actin) were detected

in all the cDNA samples. The normalized quantitative

transcript expression analysis also indicated reduced tran-

script abundance in these lines by more than 98 % as that

of the wild type (Fig. 4). Further, the qRT PCR products

were separated on agarose gel without ethidium bromide

and SYBR specific fluorescence was detected with multi-

purpose phosphor-imager (Fig. 5). Fluorescence was

detected for all the cDNA samples in the normalizer

reactions but not in no template control. Whereas, fluo-

rescence was detected only with wild type cDNA in reac-

tion with curcin precursor gene, but not in cDNAs from the

five transgenic samples or no template control. Thus, the

results confirmed the qRT PCR data on considerable

reduction in transcript expression of the curcin precursor

gene. Thus, the results confirmed the development of

transgenic Jatropha with considerably reduced transcript

expression of the curcin precursor gene through RNAi

technology. Morphologically transgenic plants were simi-

lar to that of wild types up to seedling stage. Further, the

transgenic plants with low curcin precursor gene transcript

expression are being grown for analysis on curcin content

in seeds and other plant parts.

Discussion

Jatropha curcas, is a potential source of biodiesel, espe-

cially in third world countries. Jatropha cake, which con-

stitutes up to 70 % by weight of the oil extracted seed, is a

protein-rich by-product, and a potential source of livestock

feed. However, higher levels of curcin along with phorbol

esters impart toxic nature to Jatropha cake [8]. Besides, the

composition analysis also revealed presence of other toxic/

antimetabolic constituents including trypsin inhibitors,

Transgenic

Wild types, NTC, non-transgenic

Fig. 3 Amplification plot

depicting transgenic

confirmation of the plants

transformed with RNAi vector

for curcin silencing. Genomic

DNA isolated from leaves of the

putative transgenic and wild

types was used as a template in

real time PCR for transgenic

confirmation with NPT II gene

specific primers. Cts were

obtained in ten of the tested (30)

samples but not in the rest of the

putative transgenic plants, NTC

or the wild types

-0.200

0.000

0.200

0.400

0.600

0.800

1.000

1.200

WT 93A 88A 71 93 100Fo

ld E

xpre

ssio

n O

ver

WT

Transgenic Lines

Fig. 4 Quantitative transcript expression analysis of curcin precursor

gene in leaves. The transcript expression was normalized with

expression of house keeping gene b Actin. Fold transcript expression

in five transgenic lines over the wild type (WT) is represented in

graph

Fig. 5 Gel images showing transcript abundance of curcin precurosr

gene in leaves of WT and transgenic Jatropha lines. Fluorescence of

SYBR Green 1 dye bind with real time PCR products was

documented in multi-purpose phosphor-imager (Typhoon Scanner,

Model Typhoon TRIO?, GE HealthCare, USA) with 610 BP30

emission filter and blue laser. PCR products obtained for the internal

control (b Actin) were also visualized for data normalization

Mol Biol Rep

123

Page 7: RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.)

phytates, saponins and lectins in the Jatropha seed meals.

Though, the seeds of non-toxic edible Jatropha genotypes

from Mexico have considerably low phorbol esters, con-

tents of curcin and other antimetabolic constituents are

similar to that of toxic genotypes [8, 28]. Currently, acid

and alkali treatments are used to reduce the toxicity of

Jatropha cake [29] however, no treatment has been suc-

cessful in completely eliminating the antimetabolic factors

and toxic principles of defatted Jatropha kernel meal of

non-toxic and toxic varieties. Biotechnological interven-

tions, like RNAi mediated gene silencing coupled with

genetic transformation techniques may be utilized to

develop the curcin deficient transgenic Jatropha lines.

Though, tissue culture in vitro regeneration from various

explants have been reported in Jatropha [16–19], its appli-

cation to establish efficient and reproducible genetic trans-

formation method for genetic engineering towards the

improvement in desired traits have failed. Tissue culture

dependent genetic transformation methods with marker

genes have been reported in Jatropha with transformation

efficiency up to 13 %, however the methods are laborious,

time consuming and genotype specific [19]. Therefore, a

number of tissue-culture independent techniques have thus

been developed in other plants, wherein the transformation

of plant cells is carried out in planta. Such methods, though

produce chimeric individuals up on transformation, in the

next generation stably transformed lines can be easily

selected based on the activity of the selectable marker gene

with successful transformation rate often higher than 1 %

[21]. In the present study, we have achieved up to 15 %

transformation efficiency in T0 generation. Selection will be

applied in the next generation to isolate stably transformed

lines. Thus, the need for tissue culture plant regeneration has

been successfully avoided. The established in planta trans-

formation protocol is simpler, rapid and less resource

intensive than the reported tissue culture dependent proto-

cols [30]. To the best of our knowledge, this is the first report

on standardization of in planta transformation in Jatropha.

Interestingly, optimal bacterial load for transformation was

found at culture O.D. of 1.0. Higher bacterial load resulted in

complete mortality of the germinating seeds, while lower

cell density resulted in poor transformation efficiency. Fur-

ther, addition of Acetosyringone to the Agrobacterium sus-

pension resulted in higher transformation efficiency.

Curcin being ribosomal inhibiting protein (RIP; molec-

ular weight 28.2 kDa) is essentially a vital component of

plant’s defence system, improving its chances of survival

under stress and unfavourable conditions [31]. Molecular

evidences indicate length polymorphism of curcin ORF

isolated from different accessions. While, finally processed

protein has a length of 251 amino acid residues, nascent

polypeptide was found spanning lengths between 293 and

309 amino acids [6, 7]. In the line of previous studies [6],

the curcin gene, in this study was found intronless, based

on computational predictions.

As curcin imparts toxicity to Jatropha seeds, it hinders

use of Jatropha cake as animal feed after extraction of oil.

RNA interference (RNAi) pathway to silence curcin pre-

cursor gene holds huge promise for industrial applications

of Jatropha. RNAi, a post-transcriptional mode of gene

silencing, has been successfully used for reducing toxicity

in a number of plant species including Jatropha [30, 32]. It

holds various benefits over other methods of gene

silencing. It is highly specific, highly potent, systemically

spread across tissues, and significantly down-regulates the

target gene. However, such efforts to knock down the

curcin gene expression in Indian Jatropha genotypes have

not been reported so far. In the present study, we report

more than 98 % reduction in the accumulation of curcin

transcript in the leaf tissues. This has been achieved by

construction of binary silencing vector by cloning part of

curcin precursor gene in sense and antisense orientations,

separated by intron. This vector was used for in planta

Agrobacterium mediated genetic transformation in Jatro-

pha strain DARL-2. Silencing efficacy varies with RNAi

construct types. Efficient silencing in a wide range of

plant species has been reported using hpRNA (hairpin

RNA) constructs containing sense/anti-sense arms ranging

from 98 to 853 nt [33]. Further, inclusion of an intron in

these constructs (ihpRNA) had a consistently enhancing

silencing effect [33]. Use of ihpRNA type of RNAi con-

struct containing sense/antisense arm of 334 nt length with

intron could be the reason for efficient curcin precursor

gene silencing in the present study. At the molecular level,

target gene silencing is brought about by systematic

degradation of specific mRNA by small interfering

(20–25 nt.) double stranded RNA (siRNA/dsRNA). RNA

mediated silencing also involves various cellular mecha-

nisms apparently triggered by double stranded RNA

(dsRNA) that results in suppression of target gene

expression [10].

As involvement of curcin in plant defence has been

established [31], constitutive silencing of the gene may

render Jatropha plants susceptible to the stresses. There-

fore, we are further focusing on achieving seed specific

silencing of the curcin precursor gene through the tissue

specific instead of constitutive promoter. In Jatropha it

takes minimum 3 years to flower and fruit. Hence, con-

stitutive gene silencing was followed in the present work

for early analysis of reduced transcript expression of the

gene in the leaf tissues. Otherwise, with seed specific

silencing construct, the analysis could be delayed up to

seed development. Thus, the curcin precursor gene

silencing in Jatropha may further enhance its economic

value and development of a strategic resource for bio-

diesel and various by-products.

Mol Biol Rep

123

Page 8: RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.)

Acknowledgments Research fellowship from DRDO to Deepti

Khatri and Kamal Kumar is gratefully acknowledged.

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