Describe and explain the steps to be taken to isolate the gene responsible for r

Question

Describe and explain the steps to be taken to isolate the gene responsible for resistance to bacterial blight in petunia. You have at your disposal the Avr gene from the bacterial blight cloned into a Ti-plasmid, sensitive and resistant petunia varieties, an Agrobacterium-mediated petunia transformation system, Maize Ac and Ds elements separately available for transformation into petunia, a highly developed and a detailed molecular map of petunia.

Additional information – The petunia resistance gene has a high endogenous mutation rate of ~10-4

Explanation / Answer

Plant Materials.

Xa1 donor rice cultivars (cv.) IR-BB1 and Kogyoku (Xa1/Xa1) and a susceptible cv. IR24 (xa1/xa1) were used. For high-resolution linkage analysis, F3 populations (4,225 plants) derived from IR24/IR-BB1 and IR24/Kogyoku crosses were tested by bacterial inoculation. For complementation tests of Xa1, susceptible cv. Nipponbare and Kitaake were used for transformation.

cDNA Libraries.

To obtain pathogen-induced as well as constitutive mRNA, 2-month-old IR-BB1 was inoculated with T7174, a representative strain of Japanese Xoo race 1 (19). One gram of 5-cm leaf tips were harvested at 1, 2, 3, 4, and 5 days after inoculation (DAI) and pooled for RNA isolation. Poly(A)+ RNA was isolated by using oligotex-dT30 (TaKaRa). cDNA was synthesized from 5 g of poly(A)+ RNA and cloned into vector lambda gt10 (Amersham). A total of 2.5 × 105 plaque-forming units (pfu) were screened with Y5212 DNA. To separate the YAC DNA from yeast chromosomes, DNAs in the YAC clone Y5212 were electrophoresed with CHEF (contour-clamped homogeneous electric field) in 0.5× TBE (89 mM Tris base/89 mM boric acid/2 mM EDTA, pH 8.0) at 14°C by using 1% gel for 60 hr at 4 V/cm by switching from 20 to 60 sec. YAC DNA was extracted from the gel with the Prep-A-gene DNA purification kit (Bio-Rad). For plaque hybridization, 30 ng of 32P-labeled YAC DNA was used as a probe.

Another cDNA library was constructed with the same cDNA described above in vector Lambda ZAPII. A total of 3.9 × 105 pfu were screened to obtain the full-length cDNA corresponding to Xa1 gene. Labeling of the probes and signal detection were done with the ECL system (Amersham) following the manufacturer’s instructions.

Similarity Search.

Computer searches for similar sequences were carried out with the data registered in nonredundant protein sequence databases GenPept, PDB, SwissProt, SPupdate, and PIR by using the blast algorithm (20).

Rapid Amplification of cDNA Ends (RACE)-PCR.

RACE-PCR was performed with the Marathon cDNA amplification kit (CLONTECH) according to the manufacturer’s instructions. Template mRNA was extracted as described above. RACE products were cloned into the PCRII vector (Invitrogen) and sequenced.

Cosmid Library.

Sau3AI partially digested DNA from the cv. IR-BB1 was ligated with BamHI-digested cosmid vector SuperCosI (Stratagene). The clones were packaged in vitro with GigapackIII Gold (Stratagene) and transfected into competent Escherichia coli XL1-BlueMR. Colony filters including 5 × 104 colony-forming units were screened with restriction fragment length polymorphism markers with the ECL system (Amersham).

Transformation.

Protoplasts were isolated from suspension culture cells of rice (cv. Nipponbare or Kitaake) and purified according to ref. 21. Protoplast density was adjusted to about 2 × 106 with transformation buffer (22). Cosmid 3-2 and pCH (23), the plasmid harboring the 35S promoter-hygromicin resistance gene, or CLD04541XA and pCH were added to the protoplast suspension and mixed gently and well. The polyethylene glycol (PEG) treatment was done according to the method of Datta (24) with modification to transform large DNA; PEG solution was added to tubes containing the protoplast–DNA mixture and mixed by shaking. Tubes were incubated at room temperature for 10 min. Washing solution (20 ml of 0.4 M mannitol and 0.01 M CaCl2) was added and the tubes were incubated for another 15 min. After incubation, the layer of protoplasts and that of washing solution were mixed well by gently inverting the tube. Protoplasts were collected by centrifugation and washed 4 times by repeating the above steps. After PEG treatment, protoplasts were cultured according to ref. 25.

Southern Hybridization.

Genomic DNA was isolated from green leaves by the CTAB method (26). Genomic DNA was digested with the restriction enzymes BamHI, BglII, DraI, EcoRI, EcoRV, or HindIII; separated by electrophoresis on 0.6% agarose gel; and transferred onto positively charged nylon membrane (Boehringer Mannheim). Labeling of probes and signal detection were done with the ECL system

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