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1、Extraction and PCR of plastid DNA from a plant Charles Hill, Wymondham College, Norwich, UKLevel of difficulty Although this is not an easy experiment to be performed at school, it can be organised with help from scientists or other experienced teachers. If you need support in identifying a scientis
2、t or an experienced teacher to contact in your area, please ask us, sending an email to scisocembo.org.Preface The D1 protein in the Photosystem II Reaction Centre in plant chloroplasts plays a key role in electron transport, and the structure and function of Photosystem II has been reviewed (Hankam
3、er, Barber and Boekma, 1997, and Raghavendra, 2000). It has been known for some time that the site of action of certain herbicides is this D1 protein (see De Prado, and Jorrn and Garca-Torres, 1997). Hirschberg and MacIntosh (1983) analysed the sequence of the plastid psbA gene in Amaranthus hybridu
4、s and found that resistance was due to a point mutation in codon 264 causing the amino acid serine to be replaced by glycine. This was due to the substitution of the base A by G at position 790. This mutation has also been found in Solanum nigrum (Goloubinoff, Edelman, and Hallick, 1984) and Senecio
5、 vulgaris (Frey, 1999), and has been associated with resistance to the triazine herbicides. DCMU (Diuron) is also known to inhibit electron transport by binding to the D1 protein. This point mutation (SNP) creates a restriction site for the restriction enzyme BstX 1.Introduction This protocol provid
6、es a means of identifying either the resistant or the herbicide susceptible genotype of Rapid Cycling Brassicas by amplifying a 963 basepair (bp) fragment of the psbA gene (from the coding region) and subsequent incubation with the restriction enzyme BstX 1, which cleaves the gene for resistance int
7、o 764bp and 199bp fragments but has no effect on the non-resistance gene. The protocol is divided into 4 stages: extraction, PCR, restriction digest, and gel electrophoresis. Certain aspects of this protocol have been designed to match a protocol for the amplification of human mitochondrial DNA (Sch
8、ollar and Harrison, 2002), and readers may find reference to this protocol helpful. If a restriction digest has been carried out, it should be possible to see a difference between the uncut fragment (963bp) and the larger fragment resulting from the digestion of the resistant gene product (764bp). I
9、t may not be possible to see the smaller fragment due to the sensitivity of the staining procedure. If bromophenol blue is used as the loading dye then its position on the gel after electrophoresis and the small amount of diffusion which occurs, may mask the smaller fragment.Apparatus needed (for re
10、agents, see experimental protocol that follows) micropipettes to measure volumes from 1 to 50 l, and tips (e.g. Gilson or Eppendorf pipettes) plastic reaction tubes, 500 l and 1,5 ml sizes microcentrifuge suitable for these tubes PCR thermal cycler (heating block with variable programme) or constant
11、 temperature water baths agarose gel apparatus (tank) high voltage power supply capable of delivering up to 300 volts D.C. (a lower voltage supply can be used with longer running times)Sources of Biological Material Seeds for Rapid Cycling Brassicas can be obtained from the Crucifer Genetics Coopera
12、tive at the University of Wisconsin in the USA http:/www.fastplants.org/research/phenotype.html. Seeds can also be obtained in the UK from Blades Biological http:/www.blades-bio.co.uk although they may not hold the atrazine resistant biotype in stock.A convenient source of leaf tissue for PCR only i
13、s salad rape (Brassica napus). Senecio vulgaris and lettuce (Lactuca) have also given positive results but the primers do not appear to work with barley (Hordeum spp).Analyses of the psbA gene sequences from other plants suggest that Nicotiana tabacum, Solanum nigrum, Arabidopsis thaliana, Chenopodi
14、um rubrum and Amaranthus hybridus should all give a PCR product using the same primer sequences.1st stage: High pH extraction method for Plastid DNA Material: Forceps Cocktail stick or toothpick at least 6cm long 1.5 ml microcentrifuge tube Plastic drinking straw cut into approx 5cm lengths Micropes
15、tle* or glass rod to fit tube Micropipette to measure 50l with tips 70% ethanol for sterilisation Plastic gloves Leaf material the cotyledon leaves of rape (Brassica napus) or other related Brassicas work well Solution A 50l 0.9M KCl 0.1M KOH10mM EDTA Solution B50l 0.9M KCl0.1M HCl 10mM EDTA 0.2M Tr
16、is pH 8.8If in doubt about the cleanliness of forceps or micropestle, rinse with 70% ethanol and dry. The micropestles and cocktail sticks/toothpicks could be autoclaved.Procedure: 1.Pick a healthy leaf with the forceps. Use the straw to punch a small disk about 2mm2 (= 1.4 x 1.4 mm or 1.6 mm diameter). It is helpful to support the disk on a soft surface e.g. a plastic glove on a finger tip. Use the stick to push the leaf disk into the bottom o
