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EXPRESSION OF AtZIP1 INCREASE OF ZINC CONCENTRATIONS IN TOBACCO
KAHYA SHUAIBU1,2,3 , Narayanan Narayanan1, Eliana Gaitan-Solis1, Chiedozie Egesi ², Joseph Onyeka ², Emmanuel Okogbenin ², E.N.A. Mbanaso² ,
F.I.Onyenobi ³, Martin Fregene¹ and Richard Sayre1
1Donald
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
PCR OF TRANSGENIC LINES
METHODS
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 Zndeficiency-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 (AtZIP1). Tobacco has several advantages,
including a high biomass, moderate soil requirements, fast growth rate, ease of harvesting and not a metal
hyperaccumulator.
A14-AtZIP1-tNOS construct in p2301 was given as a gift from Eliana Gaitan-solis, DDPSC.
Primers with restriction enzymes (EcoRI and KpnI) were designed to pull out the construct
and cloned it in pCAMBIA2300. AtZIP1 driving by A14- root epidermal promoter was
introduced into cassava (FEC) via Agrobacterium - mediated transformation. A14-AtZIP1tNOS 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 (MS0media) for each generation.
T1
T2
T3
T4
T5
T6
T7
T8
A14:ZIP
INTRODUCTION
WT H₂0
ABSTRACT
ICP-MS ANALYSIS
PCR AMPLIFICATION
DIGESTION
PATH OF TRANSITION METALS AND GENETIC
ENGINEERING TARGET
Fe Conc.(mg/kg)
CLONING
To understand the molecular mechanisms of zinc accumulation, distribution, sequestration in
subcellular compartments of different tissues by expression of AtZIP1 using a A14 epidermal
promoter.
Zn Conc. (mg/kg)
OBJECTIVE
AGRO-TRANSFORMATION
TOBACCO LEAF DISKS
Use
Cu Conc.mg/kg
(d)
Storage and
detoxification
Mn Conc. (mg/kg)
SEQUENCING
Apoplastic
passage
Symplastic
passage
Use
Uptake
unloading
Xylem
transport
1
Phloem transport
(c)
Xylem
loading
SCHEMATIC PROCEDURE FOR TOBACCO
REGENERATION
The ICP-MS analysis shows that there is a 1.5 fold increase of Zn in the
transgenic roots (Line T3 and T4) 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. ZIP1 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.
Symplastic
passage
CONCLUSIONS
(a)
Mobilization
(b)
Uptake
Storage and detoxification
(Stephan et ,al;2002)
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.
Selection
STRATEGY
A14
ATZIP1
Preliminary analysis shows that A14 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.
Gene
name
A14
Germination
media
Rooting media
Combining this strategy with a storage gene (AtMTP1) and tuber
specific promoter (Patatin) is under way. PAT-AtMTP1 is already known to
breeding
for higher
cassava
not beTherefore,
feasible.
accumulate
zincZnininthe
rootmay
tubers.
probably combining these
two construct will balance zinc homeostasis in the plant and maintain
high zinc concentration in the target root tissue.
T2 Generation
A
NOS
T3 Generation
(Homozygous)
Tobacco transgenic lines carrying A14-AtZIP1-tNOS
shows a
promising phenotype in shoots indicating a balanced Zn homeostasis.
This suggested AtZIP1 with A14 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.
Soil
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.
T1 Generation
ACKNOWLEDGEMENTS
Expression
Epidermis, enriched
in atrichoblast
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
S
Seeds
T₀ Generation