Figure 4
Recommended thermal cycling conditions used for PCR.
*Calculated annealing temperature for LTP 5 primers
determined by {Ta=81.5+0.41(%GC)-(675/primer length)=67}
Gradient PCR of annealing temperatures ranged from 62-72°C.
**Extension time is 1 min./ kb of PCR product.
ACCTTCTACTTTCATGCATTTGCACTCATAAGCTCTCTCTATATAAACCCAACCTTCATTACCATTCTTCATTA
TCCAATCCAATCCATTACTTACTAAGTAAAAAGAGAAAAAAAAAAAATTAAGATATGGAGGGACTCTTGAA
GTTGTCAACTTTGGTGATTGTGTGCATGTTAGTGACCGCTCCAATGGCGTCCGAGGCAGCAATCTCGTGC
GGCGCAGTCACCGGCAGCTTAGGTCAATGCTATAACTACTTGACCCGAGGCGGTTTCATTCCTAGAGGGT
GTTGCTCTGGCGTTCAGAGGCTCAACAGCTTGGCTCGTACCACCCGTGACCGCCAACAAGCTTGTCGTT
GTATCCAGGGAGCAGCGAGAGCCTTGGGTTCTCGACTTAACGCTGGTCGTGCTGCTCGTCTCCCTGGTG
CTTGCCGTGTTAGGATCTCTTACCCCATCAGTGCCAGAACCAACTGTAACACGTACGTTATCTTCTATCTCA
CTCTATATTTATTACATTTTTACTTGAGGTCCTAATATTTAATGGAGTGGTAGTAACCATATGATTGATGTATTT
GGTTGGTTGCAGCGTCAGGTGATCAAAAGAAGATAGACGTTGAAGGGTTTTCATATAATCGATGAGAGAG
TATTAAAAATAATTAAGATGTCACACTATATACACACTAGTGTATCCTTTTACTGTTATTATTGTTGTGTTTCTA
GATTTTGTGTTTTCATGTCTTTCTTTGTATATGACGGTCCATTTAATTGGTCGTCTGTGTTATGTACTCCTCTA
ATGATATATTGAATTTACGATTATGTATTATCTTCAAACTCTTGCCATTAACTTTGGTCACGTTCTGCTT
`
The goal of this project was to successfully isolate
and amplify specific lipid transfer protein gene
regions (LTP 5 and LTP 12) from Arabidopsis thaliana.
DNA was extracted from a wild type and primers were
designed in order to amplify the LTP sequence of
interest. Studies undergoing plant senescence have
shown that suppressed LTP3 and LTP4 genes have a
role in regulating plant senescence and regenerate
plant growth after apparent death. These LTP genes
were chosen in order to determine the roles other
members of the LTP family might have in plant
development. (Supported by NIH grant 5 R25 GM
49001-10)
ABSTRACT
BACKGROUND
• Polymerase chain reaction (PCR) is a biochemical
technique used to amplify a specific fragment of
DNA many thousands of fold. The technique uses a
thermal cycler that undergoes a programmed cycle
of heating and cooling in order to establish
conditions for denaturation, annealing, and
extension by enzymatic activity.
• PCR requires primers that are short complimentary
sequences of nucleotides that are designed to attach
to a target sequence, which begins DNA
amplification by DNA polymerase.
• Lipid transfer proteins (LTPs) are small proteins (9
kDa) found in the epidermal tissues of plants. In-vitro studies suggest that non-specific lipid transfer
proteins are responsible for the transfer of
phospholipids, glycolipids, fatty acids and steroids
between membranes [5]. LTPs may play a role in the
development of plant organs but their relationship to
mechanisms underpinning plant physiology are still
unknown.
• Research shows that LTPs function as signal
carriers, in plant antimicrobial defense, and
correlate with cell death in other plants. In the model
plant Arabidopsis thaliana, LTPs were found to be
part of a multigene family encompassing over 15
identified genes [1].
• As an annual plant, Arabidopsis thaliana undergoes
senescence soon after seeding. The accumulation of
oxidative radicals such as hydrogen peroxide begins
to degrade what remains of its tissues.
• Research suggests that the suppression of LTP 3 and
LTP 4 genes allow the plant to regenerate from the
remaining meristem, essentially converting the
annuals into hybrid-perennials. Figure 3 shows a
comparison of a wild type and a mutant LTP3
Arabidopsis thaliana during rosette growth (R.
Vellanoweth).
MATERIALS AND METHODS
RESULTS
• LTP5 was successfully amplified. Optimal annealing
temperature was 62.1°C. Gel electrophoresis
showed positive bands at the expected product size
of 753 bp.
• LTP12 gradient PCR was not successful; no bands
were noticeable at the LTP12 expected product size
of 682 bp.
References
[1] Arondel, Vincent, Chantal Vergnolle, Catherine Cantrel, and Jean-Claude Kader.
"Lipid Transfer Proteins Are Encoded by a Small Multigene Family in Arabidopsis
Thaliana." Plant Science 157 (2000): 1-12. Print.
[2] Banuelos, G., R. Argumedo, K. Patel, V. Ng, F. Zhou, and R. Vellanoweth. "The
Developmental Transition to Flowering in Arabidopsis Is Associated with an
Increase in Leaf Chloroplastic Lipoxygenase Activity." Plant Science 174.3 (2008):
366-73. Print.
[3] Cheng, Chao-Sheng, Yaw-Jen Liu, and Ping-Chiang Lyu. "Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, 2 June
2004. Web. 14 Aug. 2012. <http://www.ncbi.nlm.nih.gov/pubmed/15504025>.
[4] Eklund, D. M. "Localization of Nonspecific Lipid Transfer Proteins Correlate with
Programmed Cell Death Responses during Endosperm Degradation in Euphorbia
Lagascae Seedlings." Plant Physiology 132.3 (2003): 1249-259. Print.
[5] Marion, Didier. "Structure, Biological and Technological Functions of Lipid Transfer
Proteins." INRA Angers-Nantes. N.p., 22 Feb. 2006. Web. 12 Aug. 2012.
<http://www.angers-
nantes.inra.fr/angers_nantes_eng/unites_de_recherche_unites_experimentales/bio
polymeres_interactions_assemblages_bia/edifices_lipoproteiques_et_proteo_polys
accharidiques/proteines_de_transfert_de_lipides>.
[6] "National Center for Biotechnology Information." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 14 Aug. 2012.
<http://www.ncbi.nlm.nih.gov/>.
[7] "Primer3 Input (version 0.4.0)." Primer3 Input (version 0.4.0). N.p., n.d. Web. 14
Aug. 2012. <http://frodo.wi.mit.edu/>.
[8] Saiki, R., D. Gelfand, S. Stoffel, S. Scharf, R. Higuchi, G. Horn, K. Mullis, and H.
Erlich. "Primer-directed Enzymatic Amplification of DNA with a Thermos table DNA
Polymerase." Science 239.4839 (1988): 487-91. Print
[9] "TAIR - Home Page." TAIR - Home Page. N.p., n.d. Web. 14 Aug. 2012.
<http://www.arabidopsis.org/>.
[10] Thomas, S., Y. Kaneko, and C. Somerville. "A Non-specific Lipid Transfer Protein
from Arabidopsis Is a Cell Wall Protein." The Plant Journal 3.3 (1993): 427-36. Print.
Acknowledgments
This project was possible through the support of Dr.
Robert L. Vellanoweth, Jessica Ortiz, and all of Dr.
Vellanoweth’s biochemistry lab. Special thanks to the
California State University Los Angeles M.O.R.E.
programs and the (NIH) National Institute of Health.
Future work
• In order to amplify LTP12 we must expand our
temperature gradient to find the optimal annealing
conditions for our primer. In addition concentrations
of PCR master mix (MgCl2, Go taq enzyme , DNA
template) must be reassessed.
• Additional care must also be taken to extract pure DNA
samples due to contamination which could inhibit
PCR.
Figure 2
DNA sequence for LTP 5 gene includes primers (green), intron
(underlined), and protein-coding sequence (yellow). Primers
where chosen to be 15-30 nucleotides long with 40-60% GC
content.
Figure: 1
The plants were transferred into a growing chamber
which maintained a 16hr photoperiod in 23ºC. Twice a
week plants were fed a nutrient solution containing
0.0005 M KNO3, 0.0025M KH2PO4, 0.004 M MgSO4_7
H2O, and 0.002 M Ca(NO3)2_4H2O.
Figure: 3
LTP 3,4 mutant (top) and Wild type
Arabidopsis thaliana (bottom).
Picture taking at 8-10 growth period.
Department of Chemistry and Biochemistry
California State University, Los Angeles
Israel Santana, Michelle Reid, Jessica Ortiz, and Robert L Vellanoweth
DNA AMPLIFICATION OF SPECIFIC LIPID TRANSFER
PROTEINS (LTPs) FROM ARABIDOPSIS THALIANA
PLANT GROWTH
The soil used was composed of a 4:3:2 potting soil: vermiculite:
perlite mixture which was autoclaved and treated with
0.05% fungicide and 0.4% larvacide. The pots were filled ¾
full; 1-4 seeds were placed in each pot only slightly below the surface. The seeds were placed in an incubating room in 4ºC for
two days to stimulate and synchronize germination.
DNA Extraction
A CTAB: mercapto ethanol: chloroform protocol was used
to extract DNA from plant tissue. A small amount (0.25g) of leaf
tissue was frozen in liquid nitrogen and ground into a fine
powder using a mortar and pestle.
PCR
OPTIMIZATION
A specific PCR protocol was
designed for GoTaq Flexi DNA
polymerase. A temperature gradient (+-5˚C) PCR was used to determine optimal
amplification conditions(Figure 4).
ELECTROPHORESIS
A 1.0% agarose gel containing 0.5g of agarose, 50mL of 1x TAE buffer and 0.5uL of ethidium bromide
was used to verify amplification of gene
regions. Expected product size of LTP 5 gene region is
753 bp and 682 bp for LTP12 (Figure 5).
Initial
denaturation
95°C 2 min.
Denaturation 95°C 30 sec.
Annealing 67°C* 30 sec. 30X
Extension 72°C** 45 sec.
Final extension 72°C 5 min.
Soak 4°C Indefinite
Lane
1 2 3 4 5 6 7 8 9 10
Figure 5
PCR run (with temperature gradient ) showed positive results at 750bp (left). Lane 1 shows the optimal
amplification settings with annealing temperature at 62.1°C. Lanes 2-5 show a decline in amplification
(right) shows the LTP12 PCR reaction with an unsuccessful amplification
Gel Electrophoresis
LTP5 LTP12
Lane
1 2 3 4 5 6 7 8 9 10
ANNEALING TEMPS. °C 62.1 62.5 63.2 64.2 65.5 66.8 68.2 69.5 70.7
Lane
1 2 3 4 5 6 7 8 9 10
CONCLUSION
The functions of the various LTPs can be investigated
through generating transgenic or mutant plants that
express antisense RNA. But before any of that is
possible, the isolation and amplification of specific LTP
regions must be performed. Our data shows that LTP 5
was successfully amplified but LTP 12 was not. There are
many possible contributing factors (i.e. wrong primers,
inhibitors in reaction, too much dNTPs etc.) in the
methods that may have affected the amplification of
LTP12 (Figure 5).
250 bp----
500----
750----
1,000----
10,000----
3,000----
250 bp----
500---- 750----
1,000----
10,000----
3,000----