PDF《酵母双杂交筛选OIPK相互作用蛋白*》

应用与环境生物学报 2010, 16(4): 474~477 Chin J Appl Environ Biol=ISSN 1006-687X 2010-08-25 DOI: 10.

3724/SP.J.1145.2010.00474 壳寡糖激发子能够激发植物的免疫反应, 在农业生产 上可有效控制植物病害发生和危害,对绿色农业生产起到 了积极的作用[1~3] . 据报道, 壳寡糖可以诱导苯丙氨酸解氨酶 和酪氨酸解氨酶的活性提高, 并且诱导叶片中木质素含量提 高[4] , 诱导过氧化氢和过氧化物酶的活性[5] , 以及许多抗性相 关基因的转录水平提高[6] , 从而提高植物的抗性水平. 作者所 在实验室研究壳寡糖的作用机理时发现壳寡糖处理可以诱 导烟草中一个Ser/Thr蛋白激酶OIPK(Oligochitosan induced protein kinase, OIPK, GenBank accession No. AY319760)的转 录水平提高, 并且OIPK与壳寡糖诱导烟草对烟草花叶病毒 (TMV)的抗性有密切的关系, OIPK可能为MAPK级联途径 的一个关键酶[7~8] . 后来又在烟草中发现两个OIPK基因的选 择性剪接体: OIPKL(EU359698)和OIPKS(EU359699) , 预 示着OIPK基因可能通过选择性剪接, 在植物中执行更为复 杂的生物学功能. 植物体中的蛋白激酶在植物对病原菌以及 一些生物和非生物刺激的抗性中起着重要作用,蛋白激酶 通过对其它蛋白的磷酸化,对许多抗性基因起着直接和间 接的调控[9~10] . 高盐、病菌刺激可以诱导植物中的Ser/Thr激 酶如SOS2和Pto;

而一类特殊的Ser/Thr蛋白激酶MAPKs, 如AtMPK1/

2、BWMK

1、 GhMAPK、 WIPK、SIPK等, 都参与了 生物或非生物刺激引起的植物抗性反应[11~14] . 壳寡糖诱导的 OIPK激酶在植物中的作用机理还有待进行深入研究. 研究蛋白-蛋白间的相互作用是揭示蛋白质的作用机 制、发现新的蛋白质功能的重要途径, 而酵母双杂交是寻 找蛋白相互作用的常用方法, 能在体内测定蛋白质的结合 作用, 具有高度敏感性[15~16] . 在植物中, 应用酵母双杂交, 发 现了许多的蛋白相互作用, 如AtMPK12和IBR5,ATMPK4和MEK1等[17~19] . 而关于OIPK激酶的相互作用蛋白还未见报道. 为探索OIPK在壳寡糖诱导的信号转导中的作用,本研究通 酵母双杂交筛选OIPK相互作用蛋白* 杨金丽1,

2 赵小明1** 尹恒1 张洪艳1,

2 杜昱光1** (1 中国科学院大连化学物理研究所生物技术部 大连 116023) (2 中国科学院研究生院 北京 100049) Analysis of Proteins Interacted with OIPK by Yeast Two-hybrid Method* YANG Jinli1,

2 , ZHAO Xiaoming1** , YIN Heng1 , ZHANG Hongyan1,

2 &

DU Yuguang1** (1 Biotechnology Department, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China) (2 Graduate School of the Chinese Academy of Sciences, Beijing 100049, China) Abstract In our previous study, an oligochitosan-induced protein kinase (OIPK) was identified to be involved in oligochitosan-induced defense reactions in tobacco. Two alternatively spliced variants of OIPK gene, referred to as OIPKL and OIPKS, were found in tobacco and OIPKL was the longest one. To further investigate the function of OIPK in the signal transduction pathway of oligochitosan induced plant resistance, yeast two-hybrid screening was performed to find out its interaction partners. OIPKL cDNA was inserted downstream of the DNA-binding domain of the bait vector pGBKT7 and the recombinant pGBKT7-OIPKL plasmid was used as a bait plasmid to screen a tobacco By-2 cell suspension library constructed in pACT2 vector. Four proteins were identified to be interacted with OIPKL protein in the yeast two-hybrid system, including S-adenosyl-L-methionine synthetase, ubiquitin-activating enzyme E1, pyruvate kinase and peroxidase. The results provide evidence for the mechanism of oligochitosan induced plant resistance. Fig 3, Ref