Transcript Development of a Rapid qPCR Assay for On
Cloning of the Salt Tolerant Gene 9-Cis-Epoxycarotenoid Dioxygenase in Gingko biloba.
Abreeotta J. Williams M.S.
Dr. K. Soliman REU-AAMU Advisor Dr. Lian Xu REU-NFU Advisor
Gingko biloba
• • • Ginkgo biloba is a deciduous tree of the Gymnosperm group in the family Ginkgoaceae and order Ginkgoales. G. biloba is a living fossil dating back over 200 million years, with an individual living more than 3000 years. Ginkgo is native to the Guizhou Province in south China.
• • The standard leaf contains about 24% flavonoid and 6% isoprenoids (ginkgolides and bilobalide).
It is medically used to alleviate: – Mountain sickness – Depression – Pre-Menstrual Syndrome – Glaucoma – Tinnitus – Cochlear Deafness (Cupp, 1999)
• • China exported $510 million worth of Gingko extract between January and August of 2010.
The gingko nut is used for food. Gingko nuts cost $1.50/500 grams
Salinity
• • Soil salinity is the salt content in the soil.
Results of Soil salinity: – detrimental effects on plant growth and yield – weakens the plant's ability to absorb water – plant nutrients absorption strained
• • • Saline soils cannot be reclaimed by chemical amendments, conditioners or fertilizers. A field can only be reclaimed by removing salts from the plant root zone.
There are three ways to manage saline soils – leaching requirement – artificial drainage – managed accumulation It is generally estimated to cost between 25-50% yield loss for agricultural crops in China annually (Cheng-Nan, 2006).
• • • Through the years these Gingko has had to adapt to the factors of their environment, one being high salinity.
China, the Gingkos native land, has historically had and continues to have high salt content in the soil.
The gingko, however, has shown much Tolerance to soil salinity.
Objective
• The goal of this experiment is to clone the gene to obtain the 5’ end.
Materials and Methods
• • Collect young leaves.
Freeze with liquid nitrogen for later use.
Materials and Methods continued… RNA
Materials and Methods continued…
RNA Evaluation Methods
Materials and Methods continued…
RNA Evaluation
• Quality and quantity of RNA was measured with the NanoDrop.
• • Spectrophotometer 260/280 – Should be ~2 for quality RNA
4500 bp 1200bp 200 bp Materials and Methods continued…
RNA Evaluation
• Principle – – Nucleic acids are negative.
Larger particles move slower.
• Measures quality of nucleic acid.
• Measures size of nucleic acid.
Materials and Methods continued… • • RNA can not be cloned directly.
cDNA synthesis
Materials and Methods continued… • Polymerase Chain Reaction (PCR) – In-vitro DNA Replication.
– Chemicals • DNase free water • Primer Set • Target DNA • dNTP • • MgCl 2 Taq Polymerase – Steps • Denaturation • • Annealing Extension
Materials and Methods continued…
Materials and Methods continued… • Nested PCR
Materials and Methods continued… • Nested PCR evaluation and size selection 4500 bp 1200 bp 200 bp
Materials and Methods continued… • Gel Extraction
Materials and Methods continued… • Cloning
Materials and Methods continued… • • • • • • • Vector with sticky ends was purchased from TaiGen Biotechnology Company.
DNA was recombined to vector.
Solution was heat shocked.
Immediately placed on ice.
Grown on LB media containing ampicilian.
Clone selection.
Grown in LB broth.
Materials and Methods continued… • Bacterial PCR
Materials and Methods continued… • • • DNA sequencing is the process of determining the nucleotide order of a given DNA fragment Sequencing was contracted out to BGI LifeTech Company.
Sequences were analyzed using Basic Local Alignment Search Tool (BLAST).
Results and Discussion
• • Quality RNA exhibits a quality score (260/280 ratio) of ~2. RNA from this study exhibited a score of at least 2.005. From 2 grams of tissue, 20 µL of RNA was extracted with a quantity of 1,915 ng/ µL . 4500 bp 1200 bp 200 bp
Results and
Discussion
continued…
• Nested PCR successfully amplified the target. This is shown below as a band with the approximate size of 1200 bp. 4500 bp 1200 bp 200 bp
Results and
Discussion
continued…
• Several colonies developed on LB media containing ampicilian.
Results and
Discussion
continued…
• Bacterial PCR was successful for 3 of the 5 samples. They exhibit a band at approximately 1360 bp.
Successful bands 1200 bp 200 bp
Conclusion
• • Although the product recovered was of expected size, BLAST results of these sequences did not provided the gene of interest. Thus, targeted salt tolerance gene was not obtained.
We believe the possible reason for not removing target gene may be due to: – Error in Primer design – Nested PCR conditions
Acknowledgments
• • • • • • • • • • • Dr. Lian Xu Dr. K. Soliman Wang Na (Cherry) Dr. E. Moss Dr. Y. Wang Ni Zhou Xian (Larry) Tao Yuan Yuan (Rebecca) Xuan Lei (Leo) Alabama A&M University Nanjing Forestry University National Institute of Food and Agriculture
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