中华蜜蜂促分裂原活化蛋白激酶ERK-A基因的克隆与低温表达分析  

Molecular cloning and cryogenic expression of ERK-A gene from Apis cerana cerana

作　　者：陈文凤 张卫星[1] 王红芳[1] 胥保华[1] Chen Wenfeng, Zhang Weixing, Wang Hongfang, Xu Baohua(College of Animal Science and Technology, Shandong Agricultural University, Tai＇ an 271018, China)

机构地区：[1]山东农业大学动物科技学院,泰安271018

出　　处：《中国蜂业》2018年第2期68-72,共5页Apiculture of China

基　　金：国家蜂产业技术体系建设专项资金(CARS-45);国家自然科学基金(31572470);山东省"双一流"学科建设经费;山东省农业良种工程项目"优质高产蜜蜂及蚕桑新品种培育"(2014-2016)

摘　　要：昆虫属变温生物,冷胁迫是其最常见的环境刺激。丝裂原活化蛋白激酶(MAPK)信号通路与细胞应对外界刺激密切相关,为了解MAPK信号通路中ERK基因在中华蜜蜂耐寒机制中的作用,从NCBI上面查找意大利蜜蜂的ERK-A的序列,设计引物,以中华蜜蜂为模板,扩增cDNA;利用多种生物信息学软件预测分析其编码蛋白的理化特性和结构特征;采用MEGA 5.2软件中的邻接法(neighbor-joining,NJ)构建ERK-A与不同物种之间的进化树;通过实时定量PCR分析蜜蜂在4℃环境下ERK-A基因的表达情况。生物信息学分析表明,ERK-A全长1098 bp,编码365个氨基酸。ERK-A的理论分子量是42 kDa,含有ERK-A激酶所共有的磷酸化位点,属于MAPK超家族。实时定量PCR检测ERK-A基因在低温下的表达谱发现,4℃处理1 h后,ERK-A基因的表达上调与对照组差异显著(P<0.05),2 h时达到高峰,为对照组的4倍。研究结果表明ERK-A基因是冷响应的,可能在中华蜜蜂应对低温环境时发挥信号转导作用。Cold stress is one of the most common environmental stimuli to insects. MAPK signaling pathway is closely related to cellular response to external stimuli. To study the function of ERK in MAPK signaling pathway in the cold hardy mechanism of Apis cerana cerana, A eDNA of an extracellular signal-regulated protein kinase （ERK-A） gen~ was selected from the NCBI. Physiochemical properties and structure characteristics of the deduced amino acid sequence were analyzed by multiple bioinformatics methods. Phylogenetic tree between ERK-A and different species was constructed using neighbor-joining of MEGA 5.2. The expression levels of ERK-A were detected by real-time PCR when the 15-d workers were placed at extreme temperatures （4＂C）. Bioinformatic analysis showed that the full length of ERK-A is 1098 bp, encoding 365 amino acids. The predicted molecular mass of ERK-A is 42 kDa, containing the highly conserved kinase phosphorylation site. ERK-A belongs to MAPK supeffamily. Real-time quantitative PCR analysis showed that after treated the insect was at 4 E for 1 h, the expression of ERK-A is significantly different with the control, and 4 fold of the control after 2 h treatment. Our results indicate that ERK-A is cold responsive, and may play a role in signal transduction in Apis cerana cerana when it is exposed to low temperatures.

关 键 词：中华蜜蜂 MAPK通路 克隆 低温 ERK—A 

分 类 号：S891[农业科学—特种经济动物饲养]