沙葱萤叶甲表皮蛋白基因GdAbd的克隆及对温度胁迫的响应

Molecular cloning of a cuticular protein gene GdAbd from Galeruca daurica(Coleoptera: Chrysomelidae) and its response to temperature stress

为揭示沙葱萤叶甲Galeruca daurica表皮蛋白基因GdAbd在其应对温度胁迫中的作用,采用RACE技术克隆表皮蛋白基因GdAbd的cDNA全长序列,并进行生物信息学分析;利用实时荧光定量(qPCR)技术比较GdAbd经不同温度处理1 h及25℃恢复30 min后在沙葱萤叶甲2龄幼虫体内的表达水平。结果表明,GdAbd基因全长708 bp(GenBank登录号:MG874710),开放阅读框为477 bp,编码158个氨基酸;蛋白预测分子量为16.98 kD,等电点为4.26;编码蛋白具有典型的表皮蛋白R&R保守结构域,属于RR-2亚族;具有1个跨膜结构和1个信号肽。同源序列比对和系统发育分析表明,GdAbd与马铃薯甲虫Leptinotarsa decemlineata SgAbd-4的同源性最高,氨基酸序列一致性达50.63%。qPCR检测结果表明,与25℃对照相比,GdAbd基因表达水平在-10~5℃低温胁迫时未发生显著变化,而在35℃高温胁迫时发生显著上调;-10、-5和0℃低温胁迫后25℃恢复30 min可诱导GdAbd显著上调表达,但5℃低温和35℃高温胁迫处理与对照差异不显著。说明短时低温胁迫不能显著影响GdAbd表达,但胁迫后回温可诱导其上调表达。

To investigate the functions of the cuticular protein gene (GdAbd) in leaf beetle Galeruca daurica in response to temperature stress, the full-length cDNA encoding GdAbd was cloned from G. daurica larvae by RCAE method, and bioinformatics analysis was performed. The expression levels of GdAbd were compared in the 2nd larvae exposed to different temperatures (-10,-5, 0, 5, 25, and 35℃) for one hour and those undergoing the recovery at 25℃ for 30 min after different temperature stresses by using qPCR. The results showed that the length of GdAbd gene was 708 bp (GenBank accession no. MG874710), with an open reading frame (ORF) of 477 bp, encoding 158 amino acids with a predicted molecular weight of 16.98 kD and pI of 4.26. The encoded protein shared a typical consensus region of R & R consensus, with a transmembrane domain and a signal peptide. Homologous sequence alignment and phylogenetic analysis showed that GdAbd shared the highest amino acid sequence identity (50.63%) with Leptinotarsa decemlineata SgAbd-4. Compared to the control (25℃), the expression levels of GdAbd did not change under the cold stresses from -10℃ to 5℃, but significantly up-regulated under the hot stress only at 35℃. The significantly up-regulated expression of GdAbd was found after recovery at 25℃ for 30 min from the stresses at -10℃,-5℃ or 0℃, whereas there was no significant difference between 5 or 35 and 25℃. The results indicated that rapid cold stress had no significant effects on the expression of GdAbd, while the recovery from cold shock elicited the up-regulated expression.