ANTHOCYANIN PATHWAY GENE EXPRESSION IN

PEACH (PRUNUS PERSICA) FLOWERS

Rashidah H. Farid, AAMU M.S. StudentDr. Tomgming Yin, Host Professor, NFUDanial Hassani, PhD Candidate Mentor,

NFU

Outline Research Topic

Importance of Peach Flower Anthocyanin Pathway Literature Review Research Purpose

Identified Problems

Methods

Results & Discussion

Personal Goal: CTAB Protocol Methods Results

China Experience Thank You

Importance of Peach Flower

Importance of Peach Flower (delete slide)

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

Introduction: Anthocyanin Pathway

Anthocyanins are a group of reddish-blue, water-soluble 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.

Literature Review 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 Boreali-Occidentalia 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.

Literature Review 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.

Research Purpose 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

Research: Identified Problem

3RD PCR JUN 5TH RED FLOWERPrimer Annealing Temperature

Taq Enzyme SpecificityFigure 2. Amplification failed .

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Methods

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

Study Primers

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'

Methods: Final Modifications

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 TaqAnnealing Temp 58°C

Results

WHITE JUN 7TH NEW cDNA EXACT TM FOR PRIMERS

RED JUN 7TH NEW cDNA EXACT TM FOR PRIMERS

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Clone Genes

Figure?

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Personal Goal: RNA Extraction CTAB

• 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.

Methods: CTAB Protocol Overview

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.

Results

Sample ID User nameDate and Time

Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type

RNA-CTAB #1 Guest2012-6-8 15:15:24 440.1ng/µl 11.002 5.101 2.16 2.11 RNA

sample 2 Guest2012-6-8 15:20:23 419.7ng/µl 10.492 4.83 2.17 2.13 RNA

PL Guest2012-6-15 10:48:50 200.6ng/µl 5.016 3.274 1.53 0.63 RNA

PL 2 Guest2012-6-15 10:50:38 25.4 ng/µl 0.635 0.342 1.86 0.6 RNA

PL 3 Guest2012-6-15 10:51:45 82.7 ng/µl 2.068 0.988 2.09 1.38 RNA

PL 4 Guest2012-6-15 10:52:53 126 ng/µl 3.149 1.605 1.96 1.01 RNA

PL 5 Guest2012-6-15 10:54:10 74ng/µl 1.849 0.897 2.06 1.11 RNA

Results

Acknowledgements 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