水稻白叶枯病菌受寄主诱导基因的鉴定

Identification of Plant-Induced Genes of Xanthomonas Oryzae pv.oryzae

用一个具链霉素（Ｓｍ）抗性并含无启动子ＣＡＴ基因的广宿主启动子探针质粒ＰＩＪ３１００，用鸟枪法在ＣＡＴ基因上游的ＢａｍＨ１克隆位，或插入水稻白叶枯病菌（Ｘａｎｔｈｏｍｏｎａｓｏｒｙｚａｅｐｖ．ｏｒｙｚａｅ），用ＢａｍＨ１完全酶切的染色体ＤＮＡ片段，将重组质粒转化大肠杆菌ＥＤ８７６７在含链霉素的ＬＢ单板上筛选转化子。得到容量为６０００个转化子的克隆群体，其中２％的克隆含有水稻白叶枯病菌启动子活性片段，在平板上表现氯霉素（Ｃｍ）抗性。在帮助质粒ＰＲＫ２０１３的帮助下，通过三亲支配，将含有水稻白叶枯病菌启动子片段的重组质粒转移进野生型的水稻白叶枯病菌中去，在含链霉素的ＰＳＡ平板上筛选１６００个接合子，其中一个在平板上表现氯霉素抗性及含有组成表达的水稻白叶枯病菌启动子。随机选取２００个平板上对氯霉素敏感的接合子，接种用氯霉素处理的水稻感病品种金南风，得到１５个比对照明显致病的克隆。用其中一个含受水稻特异诱导启动子的重组质粒为探针，在水稻白叶枯病菌野生菌基因文库中筛选到２７个阳性克隆。

A broad host range promoter-probe plasmid PIJ3100 containing a promoterless chiorampenicoi actrytransferase gene ( CAT gene)and harbouring Sm-resistence character was used to “shotgun ” clone DNAfragments of the bacterial pathogen of leaf blight of rice Xanthomonas oryzae pv. oryzae into the BamH1restriction site in front of CAT gene of the plasmid. Reconstructed plasmids were transferred an mase intoE.coli ED8767. 6000 transformants were screened on LB plates containing Sm. Helped by PRK2013 , thereconstructed plasmids containing Xoo promoters were conjuagted in to wild type of Xoo,1600transconjugants were obtained from PSA plates con taining Sm and Fif.Among them one that renders Cm-resistance on plate is considered to have Xoo' s promoter expressing constitutionally. Two hundrend of thetransconjugants senstive to Cm on plate were selected and inoculated to Cm-treated compatibie rice cultivarby leafcut methed. Fifteen clones which are more pathogenic to rice than the control were obtained. Onereconstructed plasm id of the fifteen clones contain ing Xoo.'s promoter induced speciall y by host was usedas probe. Twenty seven postive clones were screened by colony in situ hybridization between the probe andXoo cosmid genomic libarary.