Rashidah H. Farid, AAMU M.S. Student
Dr. Tomgming Yin, Host Professor, NFU
Danial Hassani, PhD Candidate Mentor, NFU

Research Topic
 Importance of Peach Flower
 Anthocyanin Pathway
 Literature Review
 Research Purpose
Identified Problems

Methods

Results & Discussion
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Personal Goal: CTAB Protocol
 Methods
 Results
China Experience
Thank You
Peach blossoms are highly
prized in Chinese culture
 More vitality than any other tree
 Peach rods to protect them from
evils
 Identifiable by their bright pink
flowers increase pollination

Anthocyanins are a group
of reddish-blue, watersoluble pigments common
in many flowers, fruits and
vegetables.
Possible health attributes,
such as reduction of
coronary heart diseases,
antioxidant properties, and
anti-cancer activities.
Bright coloration essential
to pollination.
Figure 1. Genes involved in color expression
in flowers.


Stanciu, Gabriela al et., 2010, Spectrophotometric study on
stability of anthocyanins extracts from black grapes skins.,
Ovidius University Annals of Chemistry Volume 21, Number
1, pp.101-104, 2010.
 Varying factors effect the concentration of anthocyanins:
pH, light exposure, temperature and oxygen.
LIU Chang-ming,ZHANG Xian,XU Qing,XIAN
Feng(College of Horticulture,Northwest A&F
University,Yangling,Shaanxi 712100,China. AFLP
Analysis on Dual Color Flower Gladiolus Mutants
Induced by Radiation[J];Acta Botanica BorealiOccidentalia Sinica; 2009-01.

Dual color flower mutant and the control were
related to the changes of genetic material, some of
them may be related to the genes involved in
flower color formation.

James T. Midcap, Extension Horticulturist,
University of Georgia, Athens. Hydrangea Flower
Color,
In acidic soils, aluminum more available- binds to anthocyanin
pigments producing blues
 In alkaline soils, erratic aluminum uptake so pigments are pink.



To determine whether there is a difference in
anthocyanin pathway genes expression that
distinguish red color from white color in peach
flowers.
Focused on six genes: ANS, CHS, CHI, DFR,
F3H, UFGT
M
3RD PCR JUN 5TH RED FLOWER
Figure 2. Amplification failedPrimer
.
Annealing Temperature
Taq Enzyme Specificity
Re-configure the grouping of
primers based on annealing
temperatures
 Use a higher Specificity Taq
Enzyme vs Master Mix
 Optimized the total cDNA to 50
ng per reaction

ANS-1F
5'
GTGCTGTCACTTGGGTTGG
3'
ANS-1R
5'
TCTTCTCCTTTGGTGGCTC
3'
ANS-2F
5'
GCGTGCTGTCACTTGGGTT
3'
ANS-2R
5'
TCTTCTCCTTTGGTGGCTC
3'
ANS-3F
5' GTTGAAGAAGGCAGCAGTG 3'
ANS-3R
5' ACGCTTGTCCTCAGGGTAT 3'
ANS-4F
5'
AGCGACATTACCAATACAC
3'
ANS-4R
5'
CTGCTGCCTTCTTCAACTC
3'
CHI-1F
5'
CGTTTCCACCGTCAGTCAA
3'
CHI-1R
5'
GATCACCACGTTCCCAGCT
3'
CHI-2F
5'
AACGATACTGCCACTAACC
3'
CHI-2R
5'
TTCCCATTTCCTGCCTCAT
3'
CHI-3F
5'
AACGATACTGCCACTAACC
3'
CHI-3R
5'
CTACACTCTGCCTTGCTCC
3'
CHI-4F
5'
AACGATACTGCCACTAACC
3'
CHI-4R
5'
AAACGCCGTGCTTTCCTAT
3'
CHS-1F
5'
CCCAGTGACACCCACCTTG
3'
CHS-1R
5'
TTCTTCGTAGCCCTCTTCC
3'
CHS-2F
5'
CACCCACCTTGACAGTTTA
3'
CHS-2R
5'
CTTTCTTCGTAGCCCTCTT
3'
DFR-1F
5'
CGAAGAGCACCAGAAGTCA
3'
DFR-1R
5'
GGGAAGAACAAATGTAGCG
3'
DFR-2F
5'
TGACGAAACCGACTGGAGC
3'
DFR-2R
5'
ATCGAACTTTGTGGGAATA
3'
DFR-3F
5'
GAACCGTGAATGTCGAAGA
3'
DFR-3R
5'
GGGAAGAACAAATGTAGCG
3'
F3'5'H-1F
5'
TTCTCCAACCGTCCACCTA
3'
F3'5'H-1R
5'
CAACATCTCCGCCACCACA
3'
F3'5'H-2F
5'
TTCTCCAACCGTCCACCTA
3'
F3'5'H-2R
5'
ATCCCTTGTAAATCCATCC
3'
F3'5'H-3F
5' CTCCAACCGTCCACCTAAT 3'
F3'5'H-3R
5' ATCCCTTGTAAATCCATCC 3'
F3H-1F
5'
TCATCGTCTCCAGCCATTT
3'
F3H-1R
5'
CAGCATTCTTGAACCTCCC
3'
F3H-2F
5'
GTTCTTCGCTCTGCCCTCG
3'
F3H-2R
5'
CGCCTTTGTCAATGCCTCT
3'
F3'H-1F
5'
TCCCAGTTCCTGAAGACCC
3'
F3'H-1R
5'
CACTCCTGCCAACACCATC
3'
F3'H-2F
5'
CCAGTTCCTGAAGACCCAC
3'
F3'H-2R
5'
AACACTCCTGCCAACACCA
3'
UFGT-1F
5'
TATCATTCGGCAGTTTCGC
3'
UFGT-1R
5'
GGAGCTGTGAGCCCTATTT
3'

RT-PCR total volume of 50 µl consist of the
following:







3 µl of cDNA (50 ng)
Changed to: 1 µl of cDNA
3 µl of primer (1.5 µl F/R) Changed to: 2 µl of primer
30.7 µl of H2O
Changed to: 34 µl of H2O
5 µl of 10x Ex Taq Buffer Changed to: La Taq Buffer
4 µl of dNTP Mix
4 µl of MgCl2 (25mM)
0.3 µl Ex Taq
Changed to: 0.3 µl La Taq
Annealing Temp 58°C

M
M
WHITE JUN 7TH NEW cDNA
EXACT TM FOR PRIMERS
RED JUN 7TH NEW
cDNA EXACT TM FOR
PRIMERS
M
Figure?
• Objective: To extract total RNA
from the Hydrangea using the
RNA CTAB Protocol.
• Sample Collection: Collected
hydrangea flowers ; transfer to 80°C until RNA extraction. Petals
were ground in liquid nitrogen
in preparation for extraction.
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0.5 g of Material add to 5ml CTAB extraction buffer water bath, plus 2% 2 mercaptoethanol, heated two 65 ° C placed in 10ml centrifuge tubes; then
incubated at 65 ° water bath 15min. Centrifuged at room
temperature 12000rpm 10min.
The supernatant is conserved and treated twice, with chloroform: isoamyl
alcohol (24:1) solution . Centrifuged at room temperature 12000rpm 10min.
Add 1/4 volume of 10M LiCl; 4 ° overnight- next day, 4 °
12000rpm centrifuge 20min
Discard supernatant, add 500ul SSTE dissolved about 10
minutes, transferred to 1.5ml tubes
Add an equal volume of chloroform: isoamyl alcohol, vortex, mix, 4 °
12000rpm centrifuge 10min; conserve the supernatant
Adding twice the volume of ethanol in the supernatant, placed in -70 °,
30min to 2 hours
4 ° C, 12000rpm centrifuge 15-20min, the supernatant drained, dried at room
temperature10min, adding appropriate amount of DEPC
water (60ul) dissolved RNA, spare.
Date and
Time
Nucleic Acid
Conc.
Unit
RNA-CTAB #1 Guest
2012-6-8
15:15:24
440.1 ng/µl
11.002
5.101
2.16
2.11 RNA
sample 2
Guest
2012-6-8
15:20:23
419.7 ng/µl
10.492
4.83
2.17
2.13 RNA
PL
Guest
2012-6-15
10:48:50
200.6 ng/µl
5.016
3.274
1.53
0.63 RNA
PL 2
Guest
2012-6-15
10:50:38
25.4 ng/µl
0.635
0.342
1.86
0.6 RNA
PL 3
Guest
2012-6-15
10:51:45
82.7 ng/µl
2.068
0.988
2.09
1.38 RNA
PL 4
Guest
2012-6-15
10:52:53
126 ng/µl
3.149
1.605
1.96
1.01 RNA
PL 5
Guest
2012-6-15
10:54:10
74 ng/µl
1.849
0.897
2.06
1.11 RNA
Sample ID
User name
A260
A280
260/280
260/230
Sample Type
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National Science Foundation
Alabama A&M University/CREST
Nanjing Forestry University
Dr. K. Soliman, Advisor, AAMU
Dr. Y. Wang, Co-Advisor, AAMU
Ms. Lisa Gardner and Dr. Elica Moss