藜CaMAPKK2的表达分析及盐胁迫信号通路互作组分的筛选

doi:10.3864/j.issn.0578-1752.2013.05.003

摘要/Abstract

摘要： 【目的】通过对抗逆植物藜的丝裂原活化蛋白激酶（MAPK）级联途径中MAPKK的胁迫表达模式分析及信号转导途径互作组分的筛选，探索植物藜响应外界胁迫信号诱发逆境耐受的机制。【方法】以藜叶片总RNA为模板，利用定量PCR方法对NaCl、H2O2和ABA胁迫下藜MAPKK表达规律进行了分析。利用RT-PCR结合RACE技术获得了藜MAPKK的全长cDNA序列。利用酵母双杂交技术对MAPKK盐胁迫信号通路互作组分进行了分析。【结果】获得一个藜MAPKK的全长cDNA序列，命名为CaMAPKK2，其开放阅读框为1 089 bp，编码一个由362个氨基酸组成的丝裂原活化蛋白激酶。定量PCR显示CaMAPKK2受盐胁迫诱导明显上调表达，同时受外源H2O2和ABA调控。H2O2合成抑制剂DPI与ABA合成抑制剂Na2WO4显著抑制了300 mmol•L-1 NaCl处理下CaMAPKK2的表达。以全长CaMAPKK2为诱饵蛋白，利用酵母双杂交技术筛选到5个可能与CaMAPKK2相互作用的蛋白。测序结果显示，其中1个序列可通读，该cDNA序列长794 bp，与欧洲赤杨（Alnus glutinosa）和拟南芥的噻唑合成酶（thiazole biosynthetic enzyme）基因AgTHI1和AtTHI1核酸序列相似度达79%和78%，其它4个序列没有连续的读码框。【结论】CaMAPKK2受NaCl和H2O2诱导上调表达，暗示盐胁迫可能通过诱导H2O2和ABA的积累从而导致CaMAPKK2表达增加。要进一步筛选CaMAPKK2互作组分需获得更多阳性克隆并开展相关功能验证试验。

关键词: 藜 , 盐胁迫 , 信号转导途径 , MAPKK , 酵母双杂交 , 定量PCR

Abstract: 【Objective】The aim of the present study is to understand the mechanism of stress signal transduction to induce the response of adverse tolerance with Chenopodium album, an adverse-tolerant plant species, through analysis of the expression pattern of mitogen-activated protein kinase gene (MAPKK) under different abiotic stresses and screening of interaction proteins of MAPKK in MAPK signal transduction pathways. 【Method】 Using the total RNA from the leaf of C. album as the template, the expression patterns of MAPKK under NaCl, H2O2 and ABA were analyzed by quantitative PCR technique. The full-length cDNA sequence of MAPKK was obtained by combined reverse transcription-PCR (RT-PCR) and RACE techniques. The yeast two-hybrid technique was employed to analyze the interaction components of MAPKK in salt-stress signal pathway. 【Result】 A full length cDNA fragment of MAPKK gene from C. album included a 1 089 bp ORF, which encodes with 362 putative amino acids, named as CaMAPKK2. qPCR analysis showed that CaMAPKK2 gene was significantly induced by salt and H2O2 but not by ABA stress, at least in the present study. DPI or Na2WO4 (inhibitor of H2O2 or ABA synthesis) significantly inhibited the expression of CaMAPKK2 in C. album when exposed to 300 mmol.L-1 NaCl stress. By using the CaMAPKK2 as bait protein, five potential clones which may interact with CaMAPKK2 were obtained by the first screening of yeast two hybridization. Sequencing result indicated that one 794 bp fragment out of 5 cDNA showed correct reading frame, and shared identities of 79% and 78% in nucleotide sequence to AgTHI1 and AtTHI1 (thiazole biosynthetic enzyme) from Alnus glutinosa and Arabidopsis thaliana, respectively, while other 4 cDNA sequences had no correct reading frame. 【Conclusion】CaMAPKK2 gene was significantly induced by salt and H2O2 stress, suggesting that salt stress might induce the accumulation of H2O2 and ABA, which in turn leads to expression increasing of MAPKK. More positive clones should be acquired and the relevant experiments (e.g. Pull down, CoIP) should be performed for screening and verifying the interaction component of CaMAPKK2.

Key words: Chenopodium album , salt stress , signal transduction pathway , MAPKK , yeast two-hybrid technique , quantitative PCR

陈莎莎, 贺转转, 姜生秀, 李晓荣, 邢佳佳, 吕秀云, 兰海燕. 藜CaMAPKK2的表达分析及盐胁迫信号通路互作组分的筛选[J]. 中国农业科学, 2013, 46(5): 889-897.

CHEN Sha-Sha, HE Zhuan-Zhuan, JIANG Sheng-Xiu, LI Xiao-Rong, XING Jia-Jia, 吕Xiu-Yun , LAN Hai-Yan. The Expression Analysis and Screening of Interaction Protein of Mitogen-Activated Protein Kinase (CaMAPKK2) in Salt-Stress Signal Pathways of Chenopodium album[J]. Scientia Agricultura Sinica, 2013, 46(5): 889-897.

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