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根据川牛膝基因组数据提供的基因序列合成特异性引物,利用常规PCR方法和cDNA末端快速扩增(RACE)技术克隆川牛膝Obg C(Gene Bank登录号KU847910)全长cDNA序列,克隆获得2 226 bp全长CoObgC序列,开放阅读框为1 818 bp,编码605个氨基酸序列,预测CoObgC蛋白相对分子质量为66.39 k Da,等电点p I 5.35,为稳定蛋白,并进行多重序列比对和构建系统进化树分析。以川牛膝actin为内参,采用实时荧光定量PCR(qRT-PCR)分析CoObgC基因在川牛膝根、茎、叶、花4种组织中表达特征,结果显示在叶片中表达丰度最高,其次为根、花、茎;构建p CABIA2300-CoObgC重组载体,利用农杆菌介导法在烟草中进行瞬时表达,激光扫描共聚焦显微镜观察显示川牛膝CoObgC定位于叶绿体。该研究为进一步解析Obg基因的结构和功能,开展川牛膝分子生物学研究奠定基础。
The full length cDNA of Obg C (Gene Bank accession number KU847910) was cloned by using conventional PCR and rapid amplification of cDNA ends (RACE) according to the sequence of the genomic DNA provided by Achyranthes bidentata. 226 bp full-length CoObgC sequence with an open reading frame of 1818 bp encoding a 605 amino acid sequence predicted the relative molecular mass of CoObgC protein was 66.39 k Da and isoelectric point p I 5.35 was a stable protein and multiple sequence alignment Build phylogenetic tree analysis. The expression of CoObgC gene in four tissues of roots, stems, leaves and flowers of Achyranthisura were analyzed by real-time quantitative PCR (qRT-PCR). The results showed that the expression of CoObgC gene was the highest in leaves, followed by As the root, flower and stem. The recombinant vector pCABIA2300-CoObgC was constructed and transiently expressed in tobacco by Agrobacterium tumefaciens. Laser scanning confocal microscopy showed CoObgC located in the chloroplast. This study laid the foundation for the further study of the structure and function of Obg gene and the development of molecular biology of Achyranthis shrub.