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Rashidah H. Farid, AAMU M.S. Student Dr. Tomgming Yin, Host Professor, NFU Danial Hassani, PhD Candidate Mentor, NFU.

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Presentation on theme: "Rashidah H. Farid, AAMU M.S. Student Dr. Tomgming Yin, Host Professor, NFU Danial Hassani, PhD Candidate Mentor, NFU."— Presentation transcript:

1 Rashidah H. Farid, AAMU M.S. Student Dr. Tomgming Yin, Host Professor, NFU Danial Hassani, PhD Candidate Mentor, NFU

2 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

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4 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

5 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.

6 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 , 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 ,China. AFLP Analysis on Dual Color Flower Gladiolus Mutants Induced by Radiation[J];Acta Botanica Boreali- Occidentalia Sinica; 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.

7 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.

8 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

9 3RD PCR JUN 5TH RED FLOWER Primer Annealing Temperature Taq Enzyme Specificity Figure 2. Amplification failed. M

10 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

11 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'

12 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 H 2 O Changed to: 34 µl of H 2 O 5 µl of 10x Ex Taq Buffer Changed to: La Taq Buffer 4 µl of dNTP Mix 4 µl of MgCl 2 (25mM) 0.3 µl Ex Taq Changed to: 0.3 µl La Taq Annealing Temp 58°C

13 WHITE JUN 7TH NEW cDNA EXACT TM FOR PRIMERS RED JUN 7TH NEW cDNA EXACT TM FOR PRIMERS M M

14 Figure? M

15 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.

16 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.

17 Sample IDUser name Date and Time Nucleic Acid Conc.UnitA260A280260/280260/230Sample Type RNA-CTAB #1Guest :15: ng/µl RNA sample 2Guest :20: ng/µl RNA PLGuest :48: ng/µl RNA PL 2Guest :50:3825.4ng/µl RNA PL 3Guest :51:4582.7ng/µl RNA PL 4Guest :52:53126ng/µl RNA PL 5Guest :54:1074ng/µl RNA

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19 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


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