稻瘟菌诱导性稻叶脂氧合酶的进一步纯化及抗体制备

PURIFICATION AND ANTIBODY PREPARATION OF LIPOXYGENASES FROM RICE INDUCED BY INCOMPATIBLE INFECTION OF MAGNAPORTHE GRISEA

采用ＤＥＡＥ－Ｔｏｙｏｐｅａｒｌ离子交换、Ｂｕｔｙｌ－Ｔｏｙｏｐｅａｒｌ疏水层析、ＣＭ－Ｔｏｙｏｐｅａｒｌ离子交换、ＳｅｐｈａｃｒｙｌＳ１００凝胶过滤和ＦＰＬＣＭｏｎｏＱ等步骤，从非亲和性稻瘟菌侵染稻叶中纯化了两种诱导性脂氧合酶，即ＣＭ－Ｌｏｘｌ和ＣＭ－Ｌｏｘ２。ＳＤＳ－ＰＡＧＥ检测结果表明：ＣＭ－Ｌｏｘ１和ＣＭ－Ｌｏｘ２为单链多肽，它们的分子量分别为９８ｋｄ和１０２ｋｄ。利用微量免疫法制备了ＣＭ－Ｌｏｘ１和ＣＭ－Ｌｏｘ２的抗体，制备的抗体效价达到１∶１０００倍，能检测０．５～２．０ｎｇ的抗原。免疫印迹杂交证明ＣＭ－Ｌｏｘ１和ＣＭ－Ｌｏｘ２之间存在密切的血清学关系。此外，Ａｎｔｉ－ＣＭ－Ｌｏｘ１和Ａｎｔｉ－ＣＭ－Ｌｏｘ２与水稻中另一稻瘟菌诱导性脂氧合酶ＲＬＬ以及发芽种子出现的ＲＬ－２也存在交叉反应。

Two lipoxygenases, CM Lox1 and CM Lox2, which are induced in rice leaves in response to incompatible infection of Magnaporthe grisea , were purified to electrophoresis homogeneity by combination of DEAE Toyopearl, Butyl Toyopearl, CM Toyopearl, Sephacryl S100 gel filtration and Mono Q chromatography. SDS PAGE showed that CM Lox1 and CM Lox2 were a single plypeptide and their molecular weights were 98 kd and 102 kd, respectively.Polyclonal antibodies were raised against the purified CM Lox1 and CM Lox2 by the micro immunological method. Titeration of Anti CM Lox1 and Anti CM Lox2 was about 1∶1000 and 0 5～2 0 ng of the antigens was detectable by the antibidies. Immunological blot showed that CM Lox1 and CM Lox2 were closely related. Besides, their antibodies could cross react with RL 2, a lox occurred in germinating rice seeds and RLL, a lox induced in rice leaves also by rice blast fungus.