| 温度胁迫下沙葱萤叶甲小热激蛋白基因GdHsp20.6的克隆及表达分析 |
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| 引用本文：霍志家,周晓榕,谭瑶,庞保平,单艳敏,张卓然.温度胁迫下沙葱萤叶甲小热激蛋白基因GdHsp20.6的克隆及表达分析.植物保护学报,2019,46(4):874-884 |
| DOI：10.13802/j.cnki.zwbhxb.2019.2018155 |
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| 中文摘要:为明确小热激蛋白在沙葱萤叶甲Galeruca daurica应对温度胁迫中的作用，采用PCR方法克隆沙葱萤叶甲小热激蛋白基因Hsp20（GdHsp20.6）完整的开放阅读框（open reading frame，ORF）序列，应用在线软件对GdHsp20.6基因进行生物信息学分析，通过原核表达技术诱导表达及纯化其编码蛋白，并通过实时荧光定量PCR（real-time quantitative PCR，RT-qPCR）技术分析不同温度胁迫下GdHsp20.6基因的表达量。结果显示，GdHsp20.6基因ORF序列长度为543 bp，编码180个氨基酸，预测分子量为20.6 kD，无跨膜区和信号肽。GdHsp20.6氨基酸序列有高度保守的α-结构域。GdHsp20.6氨基酸序列与其它鞘翅目昆虫的Hsp20氨基酸序列有较高的一致性，其中与花绒寄甲Dastarcus helophoroides的Hsp20.99氨基酸序列的一致性最高，为63%。GdHsp20.6基因在大肠杆菌Escherichia coli BL21（DE3）细胞系中成功表达，经异丙基-β-D-硫代吡喃半乳糖苷（isopropyl-β-d-thiogalactoside，IPTG）诱导后GdHsp20.6蛋白成功表达，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis，SDS-PAGE）和Western-blot分析表明融合蛋白大小与预测大小一致，并纯化获得了纯度较高的目的蛋白GdHsp20.6。低温（-10~5℃）和高温（35~40℃）处理1 h以及处理后25℃恢复30 min均能诱导GdHsp20.6基因表达上调，并且0℃处理30~120 min也能诱导GdHsp20.6基因表达上调。表明GdHsp20.6基因在沙葱萤叶甲应对低温和高温胁迫中可能起着重要作用。 |
| 中文关键词:沙葱萤叶甲 小热激蛋白 温度胁迫 表达谱 Western-blot分析 |
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| Molecular cloning and expression analysis of small heat shock protein gene GdHsp20.6 under temperature stress in Galeruca daurica (Celeoptera: Chrysomelidae) |
| Author Name | Affiliation | E-mail | | Huo Zhijia | Research Center for Grassland Entomology, Inner Mongolia Agricultural University, Hohhot 010020, Inner Mongolia Autonomous Region, China | | | Zhou Xiaorong | Research Center for Grassland Entomology, Inner Mongolia Agricultural University, Hohhot 010020, Inner Mongolia Autonomous Region, China | | | Tan Yao | Research Center for Grassland Entomology, Inner Mongolia Agricultural University, Hohhot 010020, Inner Mongolia Autonomous Region, China | | | Pang Baoping | Research Center for Grassland Entomology, Inner Mongolia Agricultural University, Hohhot 010020, Inner Mongolia Autonomous Region, China | pangbp@imau.edu.cn | | Shan Yanmin | Grassland Station of Inner Mongolia Autonomous Region, Hohhot 010020, Inner Mongolia Autonomous Region, China | | | Zhang Zhuoran | Grassland Station of Inner Mongolia Autonomous Region, Hohhot 010020, Inner Mongolia Autonomous Region, China | |
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| Abstract:In order to clarify the function of small heat shock proteins (sHsps) of Galeruca daurica under tempersture stress, the complete open reading frame (ORF) sequence of GdHsp20.6 (Hsp20 gene in G. daurica) was cloned by PCR, and bioinformatics analysis was performed using on-line software. The gene expression was induced by prokaryotic expression technology, and the expressed protein was purified. The expression level of GdHsp20.6 gene under temperature stress was analyzed using the real-time quantitative PCR (RT-qPCR). The results showed that the ORF sequence of GdHsp20.6 gene was 543 bp in length, encoding 180 amino acids; the predicted molecular weight was 20.6 kD without a transmembrane region or signal peptide. The amino acid sequence of GdHsp20.6 had a highly conserved α-crystallin domain, a higher identity with those of the Hsp20s in other coleopteran insects and the highest identity with the Hsp20.99 from Dastarcus helophoroides (63%). GdHsp20.6 gene was successfully expressed in Escherichia coli BL21 (DE3) cells, and then GdHsp20.6 protein was induced to express by isopropyl-β-d-thiogalactoside (IPTG). The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot analysis showed that the size of the fusion protein was consistent with the predicted value, and the target protein with high purity was obtained. The results showed that the relative expression level of GdHsp20.6 gene was significantly up-regulated by low (-10-5℃) and high (35-40℃) temperature treatments for one hour with recovery or no recovery at 25℃ for 30 min. Additionally, GdHsp20.6 gene was also up-regulated when the G. daurica larvae were treated at 0℃ for 30-120 min. These results suggested that GdHsp20.6 gene might play an important role in response to cold and hot stress in G. daurica. |
| keywords:Galeruca daurica small heat shock protein temperature stress expression profiling Western-blot analysis |
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