荒漠昆虫小胸鳖甲Ras GTP酶激活蛋白基因 MpRasGAP 的克隆及低温表达分析

›› 2015, Vol. 58 ›› Issue (4): 367-374.

荒漠昆虫小胸鳖甲Ras GTP酶激活蛋白基因 MpRasGAP 的克隆及低温表达分析

阮梦鸽, 李洁琼, 孟闪闪, 马纪*

（新疆大学生命科学与技术学院, 新疆生物资源基因工程重点实验室, 乌鲁木齐 830046）

出版日期:2015-04-20 发布日期:2015-04-20
作者简介:阮梦鸽, 女, 1991年生, 新疆阜康人, 硕士研究生, 研究方向为昆虫低温分子生物学, E-mail: ruanmengge@sina.com

Cloning and expression profiling in response to low temperature of Ras GTPase-activating protein gene MpRasGAP in the desert beetle Microdera punctipennis (Coleoptera: Tenebrionidae)

RUAN Meng-Ge, LI Jie-Qiong,MENG Shan-Shan, MA Ji*

(Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China)

Online:2015-04-20 Published:2015-04-20

摘要/Abstract

摘要： 【目的】丝裂原活化蛋白激酶 (mitogen activated protein kinase, MAPK)级联是细胞的重要信息传递系统之一，Ras GTP酶激活蛋白（Ras GTPase-activating protein, RasGAP）基因 RasGAP 和c-Jun氨基末端激酶（c-Jun N-terminal kinase, JNK）基因 JNK 分别是MAPK信号转导途径的上、下游基因。本研究旨在确定荒漠昆虫小胸鳖甲 Microdera punctipennis RasGAP 及 JNK 基因对低温的响应情况。【方法】从荒漠甲虫小胸鳖甲中克隆获得 RasGAP 基因的cDNA序列，利用生物信息学分析软件分析其氨基酸序列并构建进化树，利用实时荧光定量PCR检测低温胁迫条件下 RasGAP 和 JNK 基因的表达情况。【结果】小胸鳖甲RasGAP cDNA的开放阅读框2 523 bp，命名为 MpRasGAP （GenBank登录号：KM677930），编码840个氨基酸，分子量96.594 kDa，编码蛋白MpRasGAP属于RasGAP超家族。MpRasGAP与赤拟谷盗 Tribolium castaneum RasGAP的氨基酸序列一致性达89%。小胸鳖甲在4℃和-4℃低温胁迫1 h后，MpRasGAP 的mRNA水平都显著高于室温对照（25℃）。小胸鳖甲在4℃处理3 h或-4℃处理1 h后, MpJNK 的mRNA水平也显著升高。【结论】本研究结果表明小胸鳖甲MpRasGAP 和 MpJNK 的mRNA水平受低温诱导。研究结果有助于深入研究荒漠昆虫在低温下MAPK信号转导途径的作用机制。

关键词: 小胸鳖甲, 低温胁迫, Ras GTP酶激活蛋白, 基因克隆, mRNA水平, 实时定量PCR

Abstract: 【Aim】 Mitogen activated protein kinase (MAPK) cascade is one of the important signal transduction pathways in cells. Ras GTPase-activating protein (RasGAP) gene RasGAP and c-Jun N-terminal kinase (JNK) gene JNK are an up-stream gene and a down-stream gene in MAPK pathway, respectively. This study aims to determine the expression profiles of RasGAP and JNK in the desert beetle Microdera punctipennis in response to low temperature. 【Methods】 A full length cDNA of RasGAP from M. punctipennis was cloned. The deduced amino acid sequence was analyzed, and the phylogynetic tree was constructed. The expression profiles of RasGAP and JNK in M. punctipennis exposed to low temperatures were detected by using real-time quantitative PCR. 【Results】 The ORF of RasGAP cDNA from M. punctipennis is 2 523 bp in length, and was named as MpRasGAP(GenBank accession no.: KM677930), encoding a polypeptide of 840 amino acids with the molecular weight of 96.594 kDa. The encoded protein MpRasGAP belongs to the RasGAP super family. Homology analysis showed that MpRasGAP shares 89% amino acid sequence identity with RasGAP from Tribolium castaneum. When M. punctipennis adults were exposed to 4℃ and -4℃ for 1 h, the mRNA levels of MpRasGAP were significantly upregulated as compared with that at room temperature (25℃). When M. punctipennis adults were exposed to 4℃ for 3 h or -4℃ for 1 h, the mRNA levels of MpJNK were also elevated significantly. 【Conclusion】 The results of this study demonstrate that the expression of both MpRasGAP and MpJNK in M. punctipennis can be induced by low temperature. Our results will help to further study the role of MAPK pathway in the desert insect under low temperature.

Key words: Microdera punctipennis, low temperature stress, Ras GTPase-activating protein (RasGAP), gene cloning, mRNA level, real-time quantitative PCR

阮梦鸽, 李洁琼, 孟闪闪, 马纪. 荒漠昆虫小胸鳖甲Ras GTP酶激活蛋白基因 MpRasGAP 的克隆及低温表达分析[J]. , 2015, 58(4): 367-374.

RUAN Meng-Ge, LI Jie-Qiong,MENG Shan-Shan, MA Ji. Cloning and expression profiling in response to low temperature of Ras GTPase-activating protein gene MpRasGAP in the desert beetle Microdera punctipennis (Coleoptera: Tenebrionidae)[J]. , 2015, 58(4): 367-374.

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