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

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

【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,Ras GAP)基因Ras GAP和c-Jun氨基末端激酶(c-Jun Nterminal 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 Ras GAP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Ras GAP基因的c DNA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Ras GAP和JNK基因的表达情况。【结果】小胸鳖甲Ras GAP c DNA的开放阅读框2 523 bp,命名为MpRas GAP(Gen Bank登录号:KM677930),编码840个氨基酸,分子量96.594 k Da,编码蛋白MpRas GAP属于Ras GAP超家族。MpRas GAP与赤拟谷盗Tribolium castaneum Ras GAP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,MpRas GAP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,Mp JNK的mRNA水平也显著升高。【结论】本研究结果表明小胸鳖甲MpRas GAP和Mp JNK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。

【Aim 】Mitogen activated protein kinase（ MAPK） cascade is one of the important signal transduction pathways in cells. Ras GTPase-activating protein（ Ras GAP） gene Ras GAP and c-Jun Nterminal 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 Ras GAP and JNK in the desert beetle Microdera punctipennis in response to low temperature. 【Methods】A full length c DNA of Ras GAP from M. punctipennis was cloned. The deduced amino acid sequence was analyzed,and the phylogynetic tree was constructed. The expression profiles of Ras GAP and JNK in M. punctipennis exposed to low temperatures were detected by using real-time quantitative PCR. 【Results】The ORF of Ras GAP c DNA from M. punctipennis is 2 523 bp in length,and was named as MpRas GAP（ Gen Bank accession no. ：KM677930）,encoding a polypeptide of 840 amino acids with the molecular weight of 96. 594 k Da. The encoded protein MpRas GAP belongs to the Ras GAP super family. Homology analysis showed that MpRas GAP shares 89% amino acid sequence identity with Ras GAP from Tribolium castaneum. When M.punctipennis adults were exposed to 4℃ and- 4℃ for 1 h,the mRNA levels of MpRas GAP 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 Mp JNK were also elevated significantly. 【Conclusion】The results of this study demonstrate that the expression of both MpRas GAPand Mp JNK 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.