沙葱萤叶甲海藻糖磷酸酶基因GdTPP的克隆、原核表达及对温度胁迫的响应
Molecular cloning and prokaryotic expression of the trehalose-6-phosphate phosphatase gene GdTPP in Galeruca daurica during temperature stress
路 标1** 周晓榕1 庞保平1*** 董瑞文2 娜布其亚2 那仁满都呼2
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DOI：10.7679/j.issn.2095-1353.2019.032
作者单位：1. 内蒙古农业大学草原昆虫研究中心，呼和浩特 010020；2. 四子王旗草原工作站，四子王旗 011800
中文关键词： 沙葱萤叶甲；海藻糖磷酸酶；基因克隆；温度胁迫；原核表达；表达分析
英文关键词：Galeruca daurica; trealose-6-phosphate phosphatase; gene cloning; temperature stress; prokaryotic expression; expression profiling
中文摘要：【目的】 海藻糖磷酸酶（Trealose-6-phosphate phosphatase，TPP）是参与昆虫海藻糖合成的关键酶之一。本研究旨在克隆和表达沙葱萤叶甲Galeruca daurica海藻糖磷酸酶基因，分析其对不同温度胁迫的响应，以期为进一步揭示其在沙葱萤叶甲生长发育及抗寒、耐热中的作用奠定基础。【方法】 根据沙葱萤叶甲转录组数据，通过cDNA 末端快速扩增（Rapid-amplification of cDNA ends，RACE）技术克隆沙葱萤叶甲TPP的全长cDNA序列，并对该基因进行生物信息学分析；将该基因与pET28a载体链接构建表达载体，导入大肠杆菌Escherichia coli BL21(DE3)使其表达；利用实时荧光定量PCR（qPCR）检测TPP基因在沙葱萤叶甲2龄幼虫不同温度下的表达格局。【结果】 克隆获得1条沙葱萤叶甲TPP基因cDNA全长序列（GdTPP，GenBank登录号：MG431210），该基因全长1 372 bp，开放阅读框（ORF）864 bp，编码287个氨基酸；蛋白预测分子量为32.32 ku，等电点（pI）为6.19；无信号肽和跨膜结构；包含2个N-糖基化位点；蛋白质亚细胞定位预测该蛋白位于细胞质中。同源性分析表明，GdTPP与马铃薯甲虫Leptinotarsa decemlineata TPP亲缘关系最近。成功构建原核表达载体pET28a-GdTPP，经IPTG诱导，GdTPP在大肠杆菌Escherichia coli BL21(DE3)中高效表达。qPCR结果表明，低于25 ℃对照时，GdTPP表达量随着温度下降而上升，﹣10 ℃时达到最高值，为对照的1.82倍；高于25 ℃对照时，GdTPP表达量随着温度上升而上升，40 ℃时达到最高值，为对照的1.68倍。【结论】 沙葱萤叶甲幼虫通过上调GdTPP的表达来应对高温和低温胁迫，该结果为进一步揭示TPP在昆虫应对温度胁迫过程中的作用奠定了基础。
英文摘要：
[Objectives] To clone the Trealose-6-phosphate phosphatase (TPP), a key enzyme in the trehalose synthesis pathway, in Galeruca daurica, quantify its expression during temperature stress, and lay a foundation for further investigation on the role of this gene in the growth and development of G. daurica. [Methods] Based on transcriptomic data, the full-length cDNA sequence of the G. daurica TPP gene was cloned using the RACE method, and analyzed with bioinformatic software. The gene was then inserted into the prokaryotic expression vector pET-28a (+) to create a recombinant plasmid pET-GdTPP, which was then transformed into Escherichia coli BL21 (DE3) to express TPP. qPCR was performed to profile the expression of the gene in 2nd-instar larvae under different temperatures. [Results] The full-length cDNA sequence of the G. daurica TPP gene (GdTPP, GenBanka accession No: MG431210) was obtained from G. daurica larvae. GdTPP is 1 372 bp in length with an open reading frame (ORF) of 864 bp, encoding 287 amino acids and has a predicted molecular weight of 32.32 ku and pI of 6.19. The encoded protein contains two potential N-glycosylation sites. Subcellular localization prediction indicates that the encoded protein is located in the cytoplasm and lacks signal peptide and transmembrane domains. GdTPP has one conserved domain, and is most similar to Leptinotarsa decemlineata TPP in amino acid sequence identity (68%). A recombinant plasmid, pET-GdTPP, was successfully constructed, and GdTPP was efficiently expressed in E. coli. The qPCR results show that, at temperatures lower than 25 ℃ (control), the expression level of GdTPP increases with decreasing temperature to a maximum of 1.86 times that of the control at ﹣10 ℃. However, at temperatures higher than 25 ℃, the expression increased with temperature to a maximum of 1.68 times that of the control at 40 ℃. [Conclusion] The expression of GdTPP in G. daurica larvae was up-regulated in response to cold- and heat-stress. These results provide a basis for further investigation on the role of TPP in temperature stress in insects.