问题描述：
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METHODS
Plant Cultivation and Grafting
Seedlings of Cucumis sativus cv Hoffmanns Produkta,Cucurbita maxima cv Gelber Zentner,and Cucurbita ficifolia cv Clevia were grown and grafted by the technique described by Golecki et al.1998 .Cucumis sativus was used as the scion and Cucurbita maxima or Cucurbita ficifolia as rootstocks.Cucurbita maxima cv Big Max and Cucumis melo cv Charentais were used only as ungrafted control plants.
Isolation of Phloem Exudate Proteins
Phloem exudate samples were collected from scion and stock of 13-day-old Cucumis sativus and Cucurbita maxima approach grafts.In all cases,the first transverse cut across the scion hypocotyl below the cotyledons separated the Cucumis sativus scion from the Cucurbita maxima stock to avoid cross-contamination of Cucumis sativus tissue with Cucurbita maxima phloem sap.Afterward,exudate from the scion and the stock was taken immediately from the basal end of the hypocotyl below the cotyledons and from a subsequent cut below the next leaf (Figure 1,site 1).For immunoblot analysis,phloem exudates were diluted 1:4 in extraction buffer (0.1 M Tris,pH 8.2,5 mM EDTA,and 20 mM DT T).Total protein concentration of each sample was determined according to Lowry et al.1951 or by the Bradford assay (Bio-Rad),using BSA as a standard.
SDS-PAGE and Immunoblot Analysis
Reduced phloem exudate proteins were separated by SDS-PAGE in 15% acrylamide gels (Laemmli 1970 ).After electrophoresis,the gels were either stained with Coomassie Brilliant Blue R 250 or the proteins were transferred from the gel to Immobilon-P membrane (Millipore,Bedford,MA) by electroblotting in a TransBlot apparatus (Bio-Rad) by using a Tris–glycine buffer (Towbin et al.1979 ).Polyclonal antibodies against Cucurbita maxima PP1 and PP2 were raised in New Zealand white rabbits,and IgG was purified on protein A columns as described by Dannenhoffer et al.1997 and Clark et al.1997 .Blots were incubated overnight with either Cucurbita maxima PP1 (1:500,000 [v/v]) or PP2 (1:500,000 [v/v]) polyclonal antibodies.Alkaline phosphatase–conjugated goat anti–rabbit IgG (Jackson ImmunoResearch Laboratories,West Grove,PA) was diluted 1:10,000 for the secondary antibody reaction.The immunoblotting procedure and the detection with the chemiluminescent substrate were adapted from the Tropix Western-Light kit protocol (Tropix,Bedford,MA).
RNA and DNA Isolation
Total RNA and genomic DNA were extracted from various organs by using the method of Gustincich et al.1991 as modified by Clark et al.1997 .RNA was isolated from hypocotyls,stems,and petioles of individual Cucumis sativus scions and their respective Cucurbita spp rootstocks (Figure 1B,site 2),as well as from ungrafted Cucumis spp and Cucurbita spp control plants.Genomic DNA was isolated from young leaves of Cucumis spp and Cucurbita spp control plants.
METHODS
Plant Cultivation and Grafting
Seedlings of Cucumis sativus cv Hoffmanns Produkta,Cucurbita maxima cv Gelber Zentner,and Cucurbita ficifolia cv Clevia were grown and grafted by the technique described by Golecki et al.1998 .Cucumis sativus was used as the scion and Cucurbita maxima or Cucurbita ficifolia as rootstocks.Cucurbita maxima cv Big Max and Cucumis melo cv Charentais were used only as ungrafted control plants.
Isolation of Phloem Exudate Proteins
Phloem exudate samples were collected from scion and stock of 13-day-old Cucumis sativus and Cucurbita maxima approach grafts.In all cases,the first transverse cut across the scion hypocotyl below the cotyledons separated the Cucumis sativus scion from the Cucurbita maxima stock to avoid cross-contamination of Cucumis sativus tissue with Cucurbita maxima phloem sap.Afterward,exudate from the scion and the stock was taken immediately from the basal end of the hypocotyl below the cotyledons and from a subsequent cut below the next leaf (Figure 1,site 1).For immunoblot analysis,phloem exudates were diluted 1:4 in extraction buffer (0.1 M Tris,pH 8.2,5 mM EDTA,and 20 mM DT T).Total protein concentration of each sample was determined according to Lowry et al.1951 or by the Bradford assay (Bio-Rad),using BSA as a standard.
SDS-PAGE and Immunoblot Analysis
Reduced phloem exudate proteins were separated by SDS-PAGE in 15% acrylamide gels (Laemmli 1970 ).After electrophoresis,the gels were either stained with Coomassie Brilliant Blue R 250 or the proteins were transferred from the gel to Immobilon-P membrane (Millipore,Bedford,MA) by electroblotting in a TransBlot apparatus (Bio-Rad) by using a Tris–glycine buffer (Towbin et al.1979 ).Polyclonal antibodies against Cucurbita maxima PP1 and PP2 were raised in New Zealand white rabbits,and IgG was purified on protein A columns as described by Dannenhoffer et al.1997 and Clark et al.1997 .Blots were incubated overnight with either Cucurbita maxima PP1 (1:500,000 [v/v]) or PP2 (1:500,000 [v/v]) polyclonal antibodies.Alkaline phosphatase–conjugated goat anti–rabbit IgG (Jackson ImmunoResearch Laboratories,West Grove,PA) was diluted 1:10,000 for the secondary antibody reaction.The immunoblotting procedure and the detection with the chemiluminescent substrate were adapted from the Tropix Western-Light kit protocol (Tropix,Bedford,MA).
RNA and DNA Isolation
Total RNA and genomic DNA were extracted from various organs by using the method of Gustincich et al.1991 as modified by Clark et al.1997 .RNA was isolated from hypocotyls,stems,and petioles of individual Cucumis sativus scions and their respective Cucurbita spp rootstocks (Figure 1B,site 2),as well as from ungrafted Cucumis spp and Cucurbita spp control plants.Genomic DNA was isolated from young leaves of Cucumis spp and Cucurbita spp control plants.
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