毛果杨中与PtMKK4互作的PtMPKs的筛选及验证

doi:10.11707/j.1001-7488.20151008

林业科学 ›› 2015, Vol. 51 ›› Issue (10): 60-66.doi: 10.11707/j.1001-7488.20151008

毛果杨中与PtMKK4互作的PtMPKs的筛选及验证

王磊¹, 宿红艳², 顾亮³, 常志远², 陈娜²

1. 鲁东大学生命科学学院烟台 264025;
2. 鲁东大学农学院烟台 264025;
3. 烟台市农业科学研究院果树研究所烟台 265500

收稿日期:2015-01-03 修回日期:2015-03-17 出版日期:2015-10-25 发布日期:2015-11-10
通讯作者: 宿红艳
基金资助:
国家自然科学基金项目"MAPK级联途径在调控毛果杨干旱、高盐胁迫信号转导中的作用及其机制的研究"(31470661);"干旱、高盐胁迫耐受性相关的胡杨PeNAC基因的功能解析"(31570649);山东省自然科学基金项目"高盐、干旱胁迫诱导的两个杨树PtMKK基因的功能解析"(ZR2013CM018)。

Screening and Verification of PtMPKs Interacting with PtMKK4 of Populus trichocarpa

Wang Lei¹, Su Hongyan², Gu Liang³, Chang Zhiyuan², Chen Na²

1. College of Life Sciences, Ludong University Yantai 264025;
2. College of Agriculture, Ludong University Yantai 264025;
3. Fruit Institute, Yantai Academy of Agricultural Sciences Yantai 265500

Received:2015-01-03 Revised:2015-03-17 Online:2015-10-25 Published:2015-11-10

摘要/Abstract

摘要： [目的] 在外界刺激传递到胞内引起细胞响应的信号转导网络中,促分裂原活化蛋白激酶(MAPK)级联途径处于中心环节。该途径由 MAPKKK,MAPKK和MAPK 3种蛋白激酶组成,各组分之间以逐级磷酸化的方式构成信号放大途径,特异感受上游刺激,将信号放大并向下传递给靶蛋白。迄今尚未在杨树中确定一条完整的 MAPK信号通路。毛果杨MAPKK成员PtMKK4在干旱信号转导中发挥重要作用,筛选、鉴定其进一步激活的靶标MAPKs将为深入理解MAPK级联途径调控植物逆境响应机制提供重要信息。[方法] 利用酵母双杂交和双分子荧光互补技术筛选、鉴定与PtMKK4互作的MAPKs,并采用半定量PCR方法对筛选到的PtMPKs在干旱和高盐胁迫处理下表达水平的变化进行分析。[结果] 分别构建pGBKT7-PtMKK4诱饵表达载体和pGADT7-PtMPKs猎物表达载体,共转化酵母Y₂H感受态细胞。仅共转化pGBKT7-PtMKK4和pGADT7-PtMPK6-1的酵母菌可在营养缺陷型筛选培养基SD/-Ade/-Trp/-Leu/-His和SD/-Ade/-Trp/-Leu/-His/X-α-gal中生长,且在SD/-Ade/-Trp/-Leu/-His/X-α-gal中生长的菌落显示蓝色。双分子荧光互补试验进一步证实PtMKK4和PtMPK6-1存在蛋白互作。此外,与PtMKK4相似,PtMPK6-1可被20% PEG和200 mmol·L^-1 NaCl诱导表达。[结论] 在毛果杨MAPK级联途径中,PtMPK6-1可能为PtMKK4激活的靶标。

关键词: 毛果杨, MAPK级联途径, 酵母双杂交, 双分子荧光互补

Abstract: [Objective] Mitogen-activated protein kinase (MAPK) cascades play central roles in the signal transduction networks from external stimuli to the intracellular responses. A typical MAPK cascade consists of three subsequently acting protein kinases, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK) and finally, the MAP kinase (MAPK). Poplar is not only the model organism of forest research, but also the important economic tree species of China. Thus, it is of great significance to carry out research on the MAPK signal pathways of poplar under various stress conditions. To date, a complete MAPK signal pathway has not yet been established in poplar. PtMKK4 has been shown to be involved in mediating the drought signal in Populus trichocarpa. It will provide insights into the molecular mechanism of MAPK cascades regulating the plant stress responses to identify the downstream PtMPKs target of PtMKK4. [Method] In this study, yeast two-hybrid system and bimolecular fluorescence complementation(BiFC)were performed to screen and verify of the PtMPKs, which interacted with PtMKK4. Semi-quantitative RT-PCR was used to analyze the expression patterns of the identified PtMPKs under drought and high salinity conditions. [Result] pGBKT7-PtMKK4 bait plasmid and pGADT7-PtMPKs prey plasmids were constructed, respectively, and co-transformed into the yeast Y₂H competent cells. The results showed that only the Y₂H cells transformed with pGBKT7-PtMKK4 and pGADT7-PtMPK6-1 plasmids could grow in the nutrient deficient media SD/-Ade/-Trp/-Leu/-His and SD/-Ade/-Trp/-Leu/-His/X-α-gal, and furthermore the yeast colonies in the SD/-Ade/-Trp/-Leu/-His/X-α-gal medium were blue. The BiFC results also confirmed that PtMKK4 interacted with PtMPK6-1. Besides, the identified PtMPK6-1 could be induced by 20% PEG and 200 mmol·L^-1 NaCl, which was similar to PtMKK4. [Conclusion] These results above indicated that PtMPK6-1 may be the downstream target of PtMKK4 in P. trichocarpa.

Key words: Populus trichocarpa, MAPK cascades, yeast two-hybrid system, bimolecular fluorescence complementation

中图分类号:

S718.46

王磊, 宿红艳, 顾亮, 常志远, 陈娜. 毛果杨中与PtMKK4互作的PtMPKs的筛选及验证[J]. 林业科学, 2015, 51(10): 60-66.

Wang Lei, Su Hongyan, Gu Liang, Chang Zhiyuan, Chen Na. Screening and Verification of PtMPKs Interacting with PtMKK4 of Populus trichocarpa[J]. Scientia Silvae Sinicae, 2015, 51(10): 60-66.

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毛果杨中与PtMKK4互作的PtMPKs的筛选及验证

Screening and Verification of PtMPKs Interacting with PtMKK4 of Populus trichocarpa

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