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条斑紫菜丝裂原活化蛋白激酶PyMAPK3基因的克隆及胁迫条件下的功能分析
李娜, 孔凡娜, 李超, 孙佩佩, 董道英, 茅云翔
中国海洋大学海洋生命学院海洋生物遗传学与育种教育部重点实验室 青岛 266003
摘要:
本研究从条斑紫菜中克隆了一个丝裂原活化蛋白激酶(mitogen activated protein kinase)基因,命名为PyMAPK3。该基因全长2155bp,开放阅读框(ORF)1401bp,编码466个氨基酸,含有两个内含子。氨基酸序列分析表明,PyMAPK3磷酸化位点的氨基酸基序为TDY,其等电点为7.17,蛋白分子量为51.6KDa,亚细胞定位于叶绿体。荧光定量PCR技术对该基因在不同非生物胁迫条件下的转录差异分析结果显示,极端胁迫条件下基因表达量显著增加。蛋白免疫印迹技术对蛋白在不同胁迫条件下的表达量分析结果显示,在不同的胁迫条件下其翻译水平的表达模式表现出与转录水平的不一致性。本研究为深入了解条斑紫菜在逆境适应机制中MAPK蛋白的作用奠定基础,同时为条斑紫菜抗逆品系的选育提供理论指导。
关键词:  条斑紫菜  丝裂原活化蛋白激酶  非生物胁迫  荧光定量PCR  蛋白质免疫印迹
DOI:10.11693/hyhz20180400094
分类号:Q71
基金项目:国家自然科学基金项目,31672641号;国家高技术研究发展计划项目,2012AA10A401号。
CLONING AND EXPRESSION OF PYMAPK3 IN PYROPIA YEZOENSIS UNDER DIFFERENT STRESSES
LI Na, KONG Fan-Na, LI Chao, SUN Pei-Pei, DONG Dao-Ying, MAO Yun-Xiang
The Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, China
Abstract:
Mitogen-activated protein kinase (MAPK) cascades are ubiquitous signaling models in eukaryotes. Many studies have indicated that MAPK cascading could play a crucial role in plant growth and development, and participate in biotic and abiotic stress responses. The red macroalgae Pyropia yezoensis, classified as Bangiophyceae, is a suitable species for the elucidation of molecular mechanisms against the environmental stress in marine red algae. A PyMAPK3 gene was isolated through a silico clone based on the transcriptomic data in this research. The full length of PyMAPK3 contains two introns and an open reading frame of 1401bp encoding 466 amino acids. The isoelectric points (PI) and molecular weights (MW) of the PyMAPK3 amino acid sequence are 7.17 and 51.6KDa, respectively. To better understand the expression patterns of PyMAPK3 in responses to an abiotic stress, qRT-PCR, and the Western-blot were used to investigate the expression patterns of PyMAPK3 in different treatment conditions (salinity, dehydration, and temperature). At mRNA levels, the gene expression increased under extremely stress conditions, while the expression pattern at the translation level was inconsistent with the transcription levels. The isolation and expression characterization of the PyMAPK3 gene in P. yezoensis may provide insights for understanding the mechanisms of PyMAPK3 responding against abiotic stresses.
Key words:  Pyropia yezoensis  MAPK  abiotic stress  real time-PCR  Western-blot
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