鞭毛素蛋白FliCaaa诱导水稻免疫反应的活性测定

Functional analysis of the flagellin Fli Caaa from Acidovorax avenae subsp.avenae in elicitation of immune responses of rice

为阐明燕麦嗜酸菌(Acidovorax avenae subsp.avenae,Aaa)鞭毛素蛋白Fli Caaa及其结构域对水稻免疫反应的诱导作用,通过分子生物学和生物信息学方法,对Aaa菌株IPN1的鞭毛素进行基因克隆及其序列分析;在大肠杆菌中对该基因全长、N端和C端片段进行了原核表达,纯化了Fli Caaa蛋白及其截短肽段;采用水稻叶片浸润法测定了表达蛋白对水稻免疫反应的诱导活性。结果表明,通过特异性引物的PCR扩增,获得1 479 bp的鞭毛素基因fli Caaa,其编码产物含有492个氨基酸,分子量为49.4 k D,具有flagellin-N和-C两个保守结构域;原核诱导表达获得了Fli Caaa、Fli Caaa-N和Fli Caaa-C可溶性融合蛋白;3种纯化蛋白均能诱导水稻细胞死亡和H2O2产生等免疫反应,但诱导能力略有差异。

To reveal the functions of flagellin Fli Caaa from Acidovorax avenae subsp. avenae( Aaa) in induction of immune responses of rice and its domains,the fli Caaa gene cloned from the strain IPN1 of Aaa was expressed in Escherichia coli strain BL21,and the purified protein Fli Caaa and the truncated proteins Fli Caaa-N and Fli Caaa-C were assayed for induction of immune responses in rice. The results showed that three purified proteins could induce the immune response of the rice Oryza sativa L. cv.Nipponbare,including hypersensitive cell death and hydrogen peroxide( H2O2) production,when the rice leaves were inoculated by infiltration of the tested proteins. Stronger induced defense response was found in Fli Caaa-treated rice than in those treated with Fli Caaa-N and Fli Caaa-C.