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Cloning and Expression of MRP1-RNAi in Arabidopsis plants. Plants produce and / or absorb toxic chemicals from the environment. Transgenic plants will help the plants and other organisms to: prevent certain diseases survive longer improve productivity.
Cloning and Expression of MRP1-RNAi in Arabidopsis plants. Plants produce and / or absorb toxic chemicals from the environment. Transgenic plants will help the plants and other organisms to: prevent certain diseases survive longer improve productivity.
Cloning and Expression of MRP1-RNAi in Arabidopsis plants. Plants produce and / or absorb toxic chemicals from the environment. Transgenic plants will help the plants and other organisms to: prevent certain diseases survive longer improve productivity.
Cellular and Molecular Biology Introduction Plants produce and/ or absorb toxic chemicals form the environment that can be harmful toward plant life
These chemicals include: lead, arsenic, cadmium, and herbicides
With the enhancement of transgenic plants to the capacity to remove toxic
chemicals form the environment, that will in turn help the plants and other organisms to: prevent certain diseases survive longer improve productivity Why I Chose Arabidopsis Plants? • Arabidopsis Plants ABC Transporters have ABC transporters • ABC (ATP binding cassette) transporters have been identified to detoxify heavy metals in plants • Produces large quantities of seeds Example of Transgenic Plants The best known example of a transgenic plant is the use of B.t. genes in corn, commonly called B.T. Corn.
B.t., or Bacillus thuringiensis, is a naturally occurring bacterium
that produces crystal proteins that are lethal to insect larvae.
Crystal protein genes have been transferred into corn, enabling
the corn to produce its own pesticides against insects. Objective/ Purpose The objective is: • To clone the MRP1-RNAi gene • To determine if the knock out gene (MRP1-RNAi) will be transformed in Arabidopsis • To select the Arabidopsis plants containing gene MRP1-RNAi on selection medium containing spectinomycin • To determine if the T1 seeds contained heavy metal resistance Hypothesis • I hypothesize that the MRP1-RNAi gene will be transformed in Arabidopsis plant. • Also that it will be confirmed by PCR using gene specific primers. Health and Nutrition Benefits of Transgenic Plants with Metal Resistance Plants oxygenate the atmosphere and reduce atmospheric pollutants Plants eliminate chemical fertilizers, produce food, restore ecosystems, and clean air Plants that recreate healthy ecosystems in residential areas They reduce some of the adverse effects of residential buildings on ecosystems Plants that improve air quality and human productivity They create residential composting systems for efficient waste removal. Materials • Media: Half strength MS (Murashige and Skoog, 1962) medium (MSI, MSII, MSIII, MSIV, MSV, MSVI), which are the nutrient containing solutions, 100mL pipette, paper measuring plate, small lab spatulas, flask, magnetic tube,3 bottles, ph meter, rubber gloves, lab coat, foil, beaker, Mettler Toledo (weighing balance), Stirrer/Hot plate, Mili-Q water dispenser, Sucrose, Phytael, HCl, and an Autoclave. • Surface sterilization of Arabidopsis seeds: Mol. Bio. Grade ethanol (100%), Whatman filter paper (90mm diameter circles), 1.5-2mL eppendorf tubes, a sterile hood, a steady hand, 70% ethanol, a votex machine, • Gel Electrophoresis: Agarose (Sigma, St. Louis), weighing balance, 1x TAE, Erlenmeyer flask, weighing paper, microwave, gel cast with comb, UV-illuminator, Gel Documentation, pipettes • RT-PCR: RT buffer (5X), dNTP mix (10mM), Rnase inhibitor (4U/mL), Reverse Transcriptase, Rnase- free water, Template RNA, PCR buffer, OligodT Primer, PCR enzyme • Transformation: PCR product, Donor vector, TE buffer, Vortex, Proteinase K solution, BP reaction, competent cells, liquid LB medium, Petri plate, • T0 and T1 seed generation analysis: liquid LB medium, Petri plate, spectinomycin, 2uM methyl mercury, 2uM lead, 2uM cadmium Procedures The first step in determining the gene expression of MRP1-RNAi in Arabidopsis plants is growing the Arabidopsis plants. In order to grow the plants half strength MS Media was used and the seeds were sterilized and grew in a growth chamber. Once the plants developed leaves the next step is to perform RNA isolation. RNA was checked on gel using gel electrophoresis, which helps to view the RNA. The next step was to perform RT-PCR. After that was the transformation of E-coli which would allow the E-coli to enter the PCR DNA fragment. The transformed colonies grew after overnight incubation of Petri plates in an incubator at 37°C. In addition the plasmid was isolated. Then the plasmid was checked using PCR. The PCR fragment was cloned into the binary vector (pK7GWIWG2 (I) and checked with gene specific primers using PCR. Afterwards, the Agro bacterium tumefaciens or soil bacteria were transformed with a binary vector, which was used to transform it into the Arabidopsis plant using the floral- dip method (Clough and Bent, 1998). The plants formed seeds were harvested and inoculated in a medium containing spectinomycin to determine if plants were transformed with MRP1-RNAi. Once the seed grew, the T1 generation seeds were harvested and inoculated into three selection medium’s each containing 2uM mercury, 2uM lead, and 2uM cadmium. Results/Discussion Using NCBI (National Center for Biotechnology Information) Genbank analysis, the MRP1-RNAi primers were synthesized to isolate the gene that encodes ABC transporter protein MRP1 for Arabidopsis thaliana. Total RNA was isolated using RNeasy plant kit (Qiagen). The 400bp gene fragment was isolated by RT- PCR using gene specific primers. The 400bp PCR product was cloned in pDONR221 vector, sequenced and subsequently cloned into binary vector pK7WIWG2 (I). The plasmid harboring MRP1-RNAi gene was transformed into Agro bacterium tumefaciens. Plants were inoculated by submerging inflorescences in the bacteria suspension using floral-dip method (Clough and Bent, 1998). The transformed T0 seeds were collected and selected on selection medium containing spectinomycin. The transformed T0 seeds grew and formed seeds (T1 generation). T1 seeds were placed in three medium’s containing 2uM mercury, 2uM lead, and 2uM cadmium, which were used to check for heavy metal resistance. The three medium’s containing T1 seeds and the heavy metals were compared to Arabidopsis seeds growing in selection medium’s containing no heavy metals. The results showed more growth in the selection medium containing no heavy metals, but the T1 seeds did show growth in selection medium’s containing heavy metals. Transformation Metal Resistance Comparisons Chart Data
Parent Generation
T seeds and plants T0 seeds and plants T1 seeds and plants
Two genetically altered piglets stand with a normal piglet (L) in this undated photograph taken at the University of Missouri-Columbia in Columbia, Missouri Other Transgenic Organisms Websites • http://www.ncbi.nlm.nih.gov • Kolukisaoglu H.U. Klein M., Eggmann • http://blast.ncbi.nlm.nih.gov/Blast.cgi T., Geisler M., Wanke D., Marttiona E., • https://tools.invitrogen.com/content.cfm Schulz B. (2002). Family business: the ?pageid=9716 multidrug-resistance related protein Lu Clough S.J. and Bent A.F. (1998), Floral Y.U., Li Z.S., Drozdowicz Y.M., • dip: a simplified method for Hortensteiner S., Martinoia E., Rea P.A. Agrobacterium-mediated transformation (1998), AtMRP2, an Arabidopsis ATP of Arabidopsis thaliana. Plant J. 16: 735- binding cassette transporter able to 743, Fernandez R.S. Davies T.G.E., transport glutathione S-conjugates and Coleman O.D., Rea P.A. (201). The chlorophyll catabolites: functional Arabidopsis thaliana ABC Protein comparisons with AtMRP1. The Plant Superfamily, a complete Inventory. The Cell, 10: 267-282 J. Biol Chem. 276 (32): 30231-30244.
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