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14 Δεκ 2012 (πριν από 4 χρόνια και 9 μήνες)

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EXPRESSION
OF
AtZIP1
INCREASE OF ZINC CONCENTRATIONS IN TOBACCO

KAHYA SHUAIBU
1,2,3

, Narayanan Narayanan
1
, Eliana

Gaitan
-
Solis
1
,
Chiedozie Egesi ², Joseph Onyeka ², Emmanuel Okogbenin ², E.N.A. Mbanaso² ,

F.I.Onyenobi ³, Martin Fregene¹ and Richard Sayre
1


1
Donald
Danforth Plant Science Center, St. Louis, MO 63132,
USA

² National Root Crops Research Institute Umudike, P.M.B 7006,Umuahia, Abia state ,Nigeria

³ Abia state university Uturu,Facaulty of Agriculture and Veterinary Medicine Umudike

Umuahia Campus


ABSTRACT

METHODS

SCHEMATIC PROCEDURE FOR TOBACCO
REGENERATION




PATH

OF TRANSITION METALS AND

GENETIC
ENGINEERING TARGET

INTRODUCTION

1

.

CONCLUSIONS

Use

Use

Uptake

Phloem transport

Storage and detoxification


(d)

Storage and

detoxification



(a)

Mobilization


(b)

Uptake


(c)


Xylem


loading

Symplastic


passage

Symplastic


passage




Xylem

transport

Apoplastic


passage

unloading

PCR
AMPLIFICATION

CLONING

DIGESTION

SEQUENCING

AGRO
-
TRANSFORMATION

TOBACCO LEAF DISKS

Transition

metal

from

the

soil

to

the

sites

of

use

and

storage

in

the

leaf
.

(a)

to

enhance

mobilization

by

secretion

of

organic

acids,

(b)

to

increase

uptake

by

over

expression

or

deregulation

of

transporters,

(c)

to

stimulate

uptake

into

the

root

and

translocation

via

the

xylem

by

overproduction

of

intracellular

chelators,



(d)

to

increase

the

strength

of

metal

sinks

in

the

leaves

by

over

expression

of

storage

and

detoxification

mechanisms
.





OBJECTIVE

(Stephan et ,al;2002)

A
14
-
AtZIP
1
-
tNOS

construct

in

p
2301

was

given

as

a

gift

from

Eliana

Gaitan
-
solis
,

DDPSC
.

Primers

with

restriction

enzymes

(
EcoR
I

and

Kpn
I
)

were

designed

to

pull

out

the

construct

and

cloned

it

in

pCAMBIA
2300
.

AtZIP
1

driving

by

A
14
-

root

epidermal

promoter

was

introduced

into

cassava

(FEC)

via

Agrobacterium

-

mediated

transformation
.

A
14
-
AtZIP
1
-
tNOS

construct

was

introduced

into

tobacco

through

leaf

disk

via

Agrobacterium


mediated

transformation
.

Transgenic

tobacco

lines

were

screened

by

PCR,

RT
-
PCR

and

dot

blot

analysis

to

confirm

the

presence

of

the

gene
.

Inductively

Couple

Plasma

Mass

Spectrophotometry(ICP
-
MS)

were

carried

out

for

transgenic

and

wild

type

seeds,

leaves

and

roots
.

The

seeds

were

sterilized

and

germinated

on

150

mg/l

kanamycin

plates

(MS
0
-
media)

for

each

generation
.


S

breeding for higher Zn in cassava may not be feasible.

Current

knowledge

on

the

molecular

and

biochemical

mechanisms

of

transition

metal

homeostasis

is

commonly

used

to

modify

metal

uptake,

root

-
to
-

shoot

translocation,

and

distribution

at

the

cellular,

tissue,

and

organ

levels
.

Such

alterations

are

focus

to

enhance

crops

for

higher

mineral

levels
.

Biofortification

aims

at

an

efficient

micronutrient

uptake

mainly

from

poor

soils,

and

an

efficient

translocation

to

the

edible

parts

of

crop

plants

(Kramer

et

al
.
,
2007
;

Palmgren

et

al
.
,

2008
;

Kramer,

2009
)
.

through

genetic

engineering

as

a

promising

tool

.

Zinc(Zn)

is

essential

in

plants,

animals,

and

humans
.

However,

it

is

frequently

deficient

in

the

diet,

resulting

in

poor

health
.

Across

the

world,

there

are

many

soils

that

are

Zn
-
deficient

or

with

low

Zn

bioavailability
.

Consequently,

crops

cultivated

there

contain

low

Zn

concentrations

leading

to

Zn
-
deficiency
-
based

malnutrition
.

The

mechanisms

to

enhance

Zn

accumulation

and

translocation

of

Zn

in

crop

plants

is

still

rudimentary
.

Here

in

this

study,

we

used

tobacco

(
Nicotiana

tabacum
)

as

a

model

crop

to

study

the

expression

of

a

zinc

transporter

from

Arabidopsis

(AtZIP
1
)
.

Tobacco

has

several

advantages,

including

a

high

biomass,

moderate

soil

requirements,

fast

growth

rate,

ease

of

harvesting

and

not

a

metal

hyperaccumulator
.


ICP
-
MS ANALYSIS

A

Rooting media

Selection

T


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卯楬

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浥摩m

吱†䝥湥牡瑩潮o

卥敤e

Mn Conc. (mg/kg
)

Fe Conc.(mg/kg)

Zn Conc. (mg/kg
)

Cu Conc.mg/kg

We

would

like

to

thank

Kevin

Lutke,

Tissue

Culture

Facility,

DDPSC

for

transforming

into

Tobacco
.

Funding

from

Gates

Foundation

and

support

from

Biocassava

plus

and

NRCRI

Umudike

is

greatly

appreciated


Tobacco

transgenic

lines

carrying

A
14
-
AtZIP
1
-
tNOS

shows

a

promising

phenotype

in

shoots

indicating

a

balanced

Zn

homeostasis
.

This

suggested

AtZIP
1

with

A
14

promoter

may

be

a

good

strategy

to

maintain

Zn

homoestasis
.

These

mechanism

of

Zn

distribution

and

accumulation

in

plants

will

contribute

to

biofortification

of

staple

food

crops
.



Combining

this

strategy

with

a

storage

gene

(AtMTP
1
)

and

tuber

specific

promoter

(
Patatin
)

is

under

way
.

PAT
-
AtMTP
1

is

already

known

to

accumulate

zinc

in

the

root

tubers
.

Therefore,

probably

combining

these

two

construct

will

balance

zinc

homeostasis

in

the

plant

and

maintain

high

zinc

concentration

in

the

target

root

tissue
.



Cassava

(
Manihot

esculenta

Crantz
)

is

an

important

staple

crop,

especially

for

resource

poor

populations

in

sub
-
Saharan

Africa

which

lacks

micronutrients

especially

zinc

and

iron
.

Mimicking

our

strategies

in

cassava

would

enhance

zinc

homeostasis

in

the

edible

part

of

the

crop

and

will

play

a

major

role

in

reducing

the

micronutrient

malnutriton

in

Africa
.




Preliminary

analysis

shows

that

A
14

is

expressed

in

root

epidermis

and

leaves

(Elisa

LevyaGuerrero,

unpublished

data)
.

This

should

increase

the

transport

of

zinc

into

the

root

epidermis

and

not

concentrate

in

the

cortex

there

by

preventing

the

accumulation

of

zinc

into

the

root

alone
.



ATZIP1

NOS

A14


STRATEGY

The

ICP
-
MS

analysis

shows

that

there

is

a

1
.
5

fold

increase

of

Zn

in

the

transgenic

roots

(Line

T
3

and

T
4
)

when

compared

with

the

WT
.

Also,

few

transgenic

lines

show

a

reduction

of

zinc

in

leaves

compared

to

the

wild

type
.

Interestingly,

there

was

no

significant

difference

in

zinc

concentrations

in

the

seeds

of

transgenic

and

WT

plants
.

ZIP
1

being

a

transporter

of

iron,

we

see

a

3

fold

increase

in

the

transgenic

root

when

compared

with

the

WT
.

There

is

no

significant

difference

between

Mn

and

Cu

distribution

in

the

leaves,

seeds,

root

of

transgenic

lines
.

Gene
name

Expression

A14

Epidermis, enriched
in atrichoblast



To

understand

the

molecular

mechanisms

of

zinc

accumulation,

distribution,

sequestration

in

subcellular

compartments

of

different

tissues

by

expression

of

AtZIP
1

using

a

A
14

epidermal

promoter
.



T2 Generation

T3 Generation

(Homozygous)

ACKNOWLEDGEMENTS




T1 T2 T3 T4 T5 T6 T7 T8

A14:ZIP

H

0




PCR OF TRANSGENIC LINES