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»ù´¡Ì«²î£¬¿´ÎÄÏ×ʱ²»ÄÜÀí½âÕâ¶Î»°Ôõô²Ù×÷µÄ£¬Äܲ»ÄÜÇë¶®µÄÕ½ÓѰïÎÒ½âÊÍһϣ¨²»ÊÇÒª·Òë¹þ£© Isolation of gp91phox Homologs from N. benthamiana Two cDNAs for potato rboh, StrbohA and StrbohB, were used as probes to screen an oligo(dT)-primed Uni-ZAP XR library (Stratagene, La Jolla, CA), which was constructed from poly(A)+ RNAs of N. benthamiana leaves treated with hyphal walls of Phytophthora for 6 h. From 6 105 plaques of the library, six positive clones were isolated after three rounds of plaque hybridization under low-stringency conditions by each probe (12 positive clones total). Positive phage plaques were excised directly in vivo into pBluescript SK phagemid according to the manufacturer¡¯s in-structions (Stratagene). The nucleotide sequences were determined for both strands using the Prism Dye Termination Cycle Sequencing Ready Reaction Kit and a DNA sequencing system (model 310; Applied Biosystems, Foster City, CA). The positive clones obtained by the StrbohA probe contained one NbrbohA and five NbrbohB cDNAs, whereas the StrbohB probe detected six NbrbohB cDNAs. The partial sequences of 11 NbrbohB cDNAs were identical. |
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