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赵学军, 国果, 修江帆, 吴建伟, 陶如玉. 家蝇伴侣蛋白CCT基因序列分析与表达模式[J]. 中国公共卫生, 2015, 31(8): 1043-1046. DOI: 10.11847/zgggws2015-31-08-18
引用本文: 赵学军, 国果, 修江帆, 吴建伟, 陶如玉. 家蝇伴侣蛋白CCT基因序列分析与表达模式[J]. 中国公共卫生, 2015, 31(8): 1043-1046. DOI: 10.11847/zgggws2015-31-08-18
ZHAO Xue-jun, GUO Guo, XIU Jiang-fan.et al, . Molecular cloning, sequencing and expression pattern of chaperonin gene in housefly-Musca domesitca[J]. Chinese Journal of Public Health, 2015, 31(8): 1043-1046. DOI: 10.11847/zgggws2015-31-08-18
Citation: ZHAO Xue-jun, GUO Guo, XIU Jiang-fan.et al, . Molecular cloning, sequencing and expression pattern of chaperonin gene in housefly-Musca domesitca[J]. Chinese Journal of Public Health, 2015, 31(8): 1043-1046. DOI: 10.11847/zgggws2015-31-08-18

家蝇伴侣蛋白CCT基因序列分析与表达模式

Molecular cloning, sequencing and expression pattern of chaperonin gene in housefly-Musca domesitca

  • 摘要: 目的 对家蝇伴侣蛋白CCT η(MD-CCT η)基因进行序列分析, 克隆其cDNA序列并检测家蝇CCT η基因的表达情况。方法 采用表达序列标签(EST)测序技术从已构建的家蝇幼虫cDNA文库中筛选MD-CCTη基因, 对其进行序列测定和分析;提取家蝇3日龄幼虫RNA, 逆转录cDNA为模板, PCR扩增, 并与pMD-19T载体连接, 转化大肠杆菌DH5α中;采用实时荧光PCR技术分析MD-CCT η基因在家蝇不同生长发育阶段及3日龄幼虫组织中表达模式。结果 MD-CCT η基因开放阅读框(ORF)全长1 632 bp, 编码543个氨基酸, 理论分子量59.3 kDa, 等电点6.27;该基因推导的氨基酸序列与其他昆虫同源序列比较有较高的相似性(93%), 聚类分析结果显示, 家蝇与地中海实蝇和果蝇的亲缘关系最近(100%);MD-CCT η在发育阶段以卵期表达量最高, 其次为1日龄幼虫, 雄性成虫表达量最低;MD-CCTη基因在家蝇3日龄幼虫组织中气管表达量最高, 其次为唾液腺, 在体壁中表达量最低。结论 成功克隆出MD-CCTη基因的cDNA序列, MD-CCTη在家蝇不同发育阶段及组织中表达模式存在明显差异。

     

    Abstract: Objective To clone and analyze a novel cDNA, named as Musca domesitca chaperonin containing tailless complex polypeptide η(MD-CCTη)and to detect relative expression levels of MD-CCTη by real time quantitative reverse transcriptase PCR(QRT-PCR).Methods The total RNA was extracted from Musca domesitca larvae of 3rd day;MD-CCTη gene was cloned with PCR and then ligated into vector pMD-19T and transformed into Escherichia coli DH5α competent cells.The relative expression levels of MD-CCTη were detected in different tissues of the larvae of 3rd day and during different developmental stages by QRT-PCR.Results Sequence analysis indicated that the open reading frame was 1 632 bp,encoding a putative protein consisting of 543 amino acids,with a predicted molecular weight of 59.3 kDa and an isoelectric point(pI)of 6.27.The analysis using National Center for Biotechnology Information-Basic Local Alignment Search Tool(NCBI-BLAST)confirmed that the deduced amino acid sequence shared high identity with the reported homologous sequences from other insects(93%).Cluster analysis results showed the genetic relation of Musca domesitca was more close to that of C.capitata and Drosophila(100%).QRT-PCR showed that the expression of MD-CCTη was the highest in the egg,followed by that of in the larvae of 1st day,and lowest in male adult during development;whereas the the expression of MD-CCTη was the highest in the trachea,medium in the salivary glands,and the lowest in the body wall of 3rd day larvae.Conclusion MD-CCTη was cloned and mRNA expression levels of MD-CCTη were different during different development period and in the different tissues of Musca domesitca.

     

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