Cloning and

Expression of
MRP1-RNAi in
Arabidopsis Plants

By: Victoria Dawson

Cellular and Molecular Biology
 Introduction
 Plants produce and/ or absorb toxic chemicals form the environment that can be
harmful toward plant life

 These chemicals include: lead, arsenic, cadmium, and herbicides

 With the enhancement of transgenic plants to the capacity to remove toxic

chemicals form the environment, that will in turn help the plants and other
organisms to:
prevent certain diseases
survive longer
improve productivity
 Why I Chose Arabidopsis Plants?
• Arabidopsis Plants ABC Transporters
have ABC transporters
• ABC (ATP binding
cassette) transporters
have been identified to
detoxify heavy metals in
plants
• Produces large
quantities of seeds
 Example of Transgenic Plants
 The best known example of a transgenic plant is the use of B.t.
genes in corn, commonly called B.T. Corn.

 B.t., or Bacillus thuringiensis, is a naturally occurring bacterium

that produces crystal proteins that are lethal to insect larvae.

 Crystal protein genes have been transferred into corn, enabling

the corn to produce its own pesticides against insects.
 Objective/ Purpose
The objective is:
• To clone the MRP1-RNAi gene
• To determine if the knock out gene (MRP1-RNAi)
will be transformed in Arabidopsis
• To select the Arabidopsis plants containing gene
MRP1-RNAi on selection medium containing
spectinomycin
• To determine if the T1 seeds contained heavy metal
resistance
 Hypothesis
• I hypothesize that the
MRP1-RNAi gene will
be transformed in
Arabidopsis plant.
• Also that it will be
confirmed by PCR using
gene specific primers.
 Health and Nutrition Benefits of Transgenic
Plants with Metal Resistance
 Plants oxygenate the atmosphere and reduce atmospheric
pollutants
 Plants eliminate chemical fertilizers, produce food, restore
ecosystems, and clean air
 Plants that recreate healthy ecosystems in residential areas
 They reduce some of the adverse effects of residential
buildings on ecosystems
 Plants that improve air quality and human productivity
 They create residential composting systems for efficient
waste removal.
 Materials
• Media: Half strength MS (Murashige and Skoog, 1962) medium (MSI, MSII, MSIII, MSIV, MSV, MSVI),
which are the nutrient containing solutions, 100mL pipette, paper measuring plate, small lab spatulas,
flask, magnetic tube,3 bottles, ph meter, rubber gloves, lab coat, foil, beaker, Mettler Toledo (weighing
balance), Stirrer/Hot plate, Mili-Q water dispenser, Sucrose, Phytael, HCl, and an Autoclave.
• Surface sterilization of Arabidopsis seeds: Mol. Bio. Grade ethanol (100%), Whatman filter paper
(90mm diameter circles), 1.5-2mL eppendorf tubes, a sterile hood, a steady hand, 70% ethanol, a votex
machine,
• Gel Electrophoresis: Agarose (Sigma, St. Louis), weighing balance, 1x TAE, Erlenmeyer flask, weighing
paper, microwave, gel cast with comb, UV-illuminator, Gel Documentation, pipettes
• RT-PCR: RT buffer (5X), dNTP mix (10mM), Rnase inhibitor (4U/mL), Reverse Transcriptase, Rnase-
free water, Template RNA, PCR buffer, OligodT Primer, PCR enzyme
• Transformation: PCR product, Donor vector, TE buffer, Vortex, Proteinase K solution, BP reaction,
competent cells, liquid LB medium, Petri plate,
• T0 and T1 seed generation analysis: liquid LB medium, Petri plate, spectinomycin, 2uM methyl mercury,
2uM lead, 2uM cadmium
 Procedures
The first step in determining the gene expression of MRP1-RNAi in
Arabidopsis plants is growing the Arabidopsis plants. In order to grow the plants
half strength MS Media was used and the seeds were sterilized and grew in a
growth chamber. Once the plants developed leaves the next step is to perform RNA
isolation. RNA was checked on gel using gel electrophoresis, which helps to view
the RNA. The next step was to perform RT-PCR. After that was the transformation
of E-coli which would allow the E-coli to enter the PCR DNA fragment. The
transformed colonies grew after overnight incubation of Petri plates in an
incubator at 37°C. In addition the plasmid was isolated. Then the plasmid was
checked using PCR. The PCR fragment was cloned into the binary vector
(pK7GWIWG2 (I) and checked with gene specific primers using PCR. Afterwards,
the Agro bacterium tumefaciens or soil bacteria were transformed with a binary
vector, which was used to transform it into the Arabidopsis plant using the floral-
dip method (Clough and Bent, 1998). The plants formed seeds were harvested and
inoculated in a medium containing spectinomycin to determine if plants were
transformed with MRP1-RNAi. Once the seed grew, the T1 generation seeds were
harvested and inoculated into three selection medium’s each containing 2uM
mercury, 2uM lead, and 2uM cadmium.
 Results/Discussion
Using NCBI (National Center for Biotechnology Information) Genbank
analysis, the MRP1-RNAi primers were synthesized to isolate the gene that encodes
ABC transporter protein MRP1 for Arabidopsis thaliana. Total RNA was isolated
using RNeasy plant kit (Qiagen). The 400bp gene fragment was isolated by RT-
PCR using gene specific primers. The 400bp PCR product was cloned in
pDONR221 vector, sequenced and subsequently cloned into binary vector
pK7WIWG2 (I). The plasmid harboring MRP1-RNAi gene was transformed into
Agro bacterium tumefaciens. Plants were inoculated by submerging inflorescences
in the bacteria suspension using floral-dip method (Clough and Bent, 1998). The
transformed T0 seeds were collected and selected on selection medium containing
spectinomycin. The transformed T0 seeds grew and formed seeds (T1 generation).
T1 seeds were placed in three medium’s containing 2uM mercury, 2uM lead, and
2uM cadmium, which were used to check for heavy metal resistance. The three
medium’s containing T1 seeds and the heavy metals were compared to Arabidopsis
seeds growing in selection medium’s containing no heavy metals. The results
showed more growth in the selection medium containing no heavy metals, but the
T1 seeds did show growth in selection medium’s containing heavy metals.
Transformation
Metal Resistance Comparisons
 Chart Data

Parent Generation

T seeds and plants T0 seeds and plants T1 seeds and plants

Two genetically
altered piglets stand
with a normal piglet
(L) in this undated
photograph taken at
the University of
Missouri-Columbia in
Columbia, Missouri
Other Transgenic Organisms
 Websites
• http://www.ncbi.nlm.nih.gov
• http://blast.ncbi.nlm.nih.gov/Blast.cgi
• https://tools.invitrogen.com/content.cfm
?pageid=9716
Clough S.J. and Bent A.F. (1998), Floral
•
dip: a simplified method for
Agrobacterium-mediated transformation
of Arabidopsis thaliana. Plant J. 16: 735-
743, Fernandez R.S. Davies T.G.E.,
Coleman O.D., Rea P.A. (201). The
Arabidopsis thaliana ABC Protein
Superfamily, a complete Inventory. The
J. Biol Chem. 276 (32): 30231-30244.
• Kolukisaoglu H.U. Klein M., Eggmann
T., Geisler M., Wanke D., Marttiona E.,
Schulz B. (2002). Family business: the
multidrug-resistance related protein Lu
Y.U., Li Z.S., Drozdowicz Y.M.,
Hortensteiner S., Martinoia E., Rea P.A.
(1998), AtMRP2, an Arabidopsis ATP
binding cassette transporter able to
transport glutathione S-conjugates and
chlorophyll catabolites: functional
comparisons with AtMRP1. The Plant
Cell, 10: 267-282