川牛膝CoObgC基因克隆、亚细胞定位及表达分析

Cloning,subcellular lacalization and expression analysis of chloroplast-targeted Obg gene in Cyathula officinalis

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。

According to ObgC gene sequences from Cyathula officinalis genomic data, the specific primers were designed, and a full-length CoObgC cDNA （2 226 bp） was obtained by polymerase chain reaction （PCR） and rapid amplification of cDNA ends （RACE） methord. Sequence alignment showed that CoObgC gene contained a 1 818 bp open reading frame （ORF） encoding 605 ami- no acids. Sequence analysis predicted that molecular weight of CoObgC protein was about 66. 39 kDa, the academic isoelectric point was 5.35, and the protein was stable protein. Then multiple sequence alignment was applied to construct phylogenetic tree. The real- time fluorescence quantification PCR （RT-qPCR） demonstrated that a high expression level in leaf, followed by root and flower, the low transcription was in stem. The recombinant vector pCABIA2300-CoObgC was constructed and introduced into tobacco epidermal cells by agrobacterium-mediated transformation, green fluorescence was tested and targeted to chloroplast under a laser scanning confo- cal microscope. These findings will be helpful to lay a foundation for studying the structure and function of CoObgC gene, and elucida- ting C. officinalis molecular biology experiment.