小胸鳖甲热激蛋白基因(Mphsp70)的克隆、序列分析及温度对其表达的影响

Cloning and sequence analysis of a heat shock protein gene (Mphsp70) from Microdera punctipennis and its expression in relation to high temperatures

采用RACE-PCR技术,从荒漠甲虫小胸鳖甲Microdera punctipennis Kaszab克隆hsp70基因全长cDNA序列,命名为Mphsp70。测序结果表明,序列全长2207bp,该序列覆盖了完整编码区,编码647个氨基酸,分子量大小为70.69ku,理论等电点为5.57(GenBank登录号JF421286.1)。此序列包含142bp的5'端非翻译区和124bp的含有多聚腺苷酸信号序列AATAAA和poly A尾的3'端非翻译区以及1941bp的开放阅读框。该基因无内含子,符合诱导型Hsp70的特征。经BLAST检索分析,由Mphsp70的核苷酸序列推定的氨基酸序列与已知的光滑鳖甲Hsp70高度同源,同源性高达97.22%。通过荧光定量RT-PCR技术研究昆虫受到高温胁迫时该基因的表达,结果表明:经37℃和42℃处理昆虫1h后诱导昆虫体内hsp70的表达,其表达量分别为对照组(25℃)的21.57倍和389.3倍,随着处理时间的延长,表达量降低。该研究结果为深入研究小胸鳖甲的抗逆机理提供了新的思路。

The full length cDNA sequence of the heat shock protein 70 gene from Microdera punctipennis Kaszab （a desert beetle found in Xinjiang） was isolated using the RACE-PCR technique,and designated as Mphsp70.Sequence analysis indicated that Mphsp70 is 2 207 bp in length and contains a 5＇-terminal untranslated region （5＇-UTR） of 142 bp,a 3＇-UTR of 124 bp with a polyadenylation signal sequence AATAAA and a poly （A） tail,and an open reading frame of 1 941 bp encoding 647 amino acid residues with estimated molecular weight of 70.69 ku.The theoretical isoelectric point is 5.57 （GenBank No.JF421286.1）.Mphsp70 contains no intron,a distinctive feature of inducible hsp70 genes.A sequence homology search using BLAST revealed that MpHsp70 had a high similarity （97.22%） to ApHsp70 from the desert beetle Anatolica polita.The results of real-time quantitative PCR indicate that the mRNA level of Mphsp70 at 37℃ and 42℃ was respectively 21.57 and 389.3 times that of the control at 25℃,and that these levels decreased with time at high temperatures.These results will facilitate further research on the environmental adaptability of this desert beetle.