金线莲ArLBD1基因克隆、亚细胞定位及表达分析

Gene Cloning,Subcellular Localization and Expression Analyses of ArLBD1 Gene in Anoectochilus roxburghii

以金线莲(Anoectochilus roxburghii)叶片的转录组数据为基础,采用RT-PCR技术克隆到了金线莲ArLBD1转录因子基因,采用生物信息学、原核表达、Western blot、亚细胞定位及qPCR等方法对该基因功能进行初步分析,以期为进一步研究其功能提供参考依据。结果表明:ArLBD1基因CDS长度864 bp,编码287个氨基酸。ArLBD1蛋白分子量为30.62 kD,理论等电点为6.25,不稳定系数55.55,属于不稳定蛋白。ArLBD1蛋白含有LOB superfamily保守结构域,第7~28氨基酸为C基序,第36~84氨基酸为GAS基序,第89~107氨基酸为类亮氨酸拉链基序,具有完整的类亮氨酸拉链基序,属于ClassⅠ。系统进化分析表明ArLBD1与拟南芥AtLBD36的亲缘关系最近,其次是AtLBD6。SDS-PAGE电泳及Western blot结果显示,ArLBD1基因在大肠杆菌中成功诱导表达。亚细胞定位分析发现ArLBD1蛋白定位在细胞核。qPCR分析结果表明,ArLBD1基因在金线莲叶、茎和根中都能检测到表达,在叶片表达量最高,其次是根,茎的表达量最低,推测ArLBD1基因主要在金线莲叶片发育中发挥功能。

Based on the transcriptome data of Anoectochilus roxburghii leaves,an LBD transcription factor gene,named ArLBD1,was cloned by RT-PCR.The function of the gene was analyzed by bioinformatics,prokaryotic expression,Western blot,subcellular localization and expression patterns of different organs,in order to provide reference for further research on the gene′s function.The results showed that the CDS length of ArLBD1 gene was 864 bp,encoding 287 amino acids.The molecular weight of ArLBD1 protein was 30.62 kD,theoretical isoelectric point was 6.25,and instability coefficient was 55.55.ArLBD1 protein belonged to unstable protein.ArLBD1 protein contained a LOB superfamily conserved domain,its amino acid 7-28 was C-block,amino acid 36-84 was GAS-block,and amino acid 89-107 was Leucine-zipper-like block.It had a complete Leucine-zipper-like-block and belonged to Class I.Phylogenetic analysis showed that ArLBD1 was most closely related to Arabidopsis thaliana AtLBD36,followed by AtLBD6.SDS-PAGE and Western blot results showed that ArLBD1 gene was successfully induced and expressed in Escherichia coli.Subcellular localization analysis showed that ArLBD1 protein was in the nucleus.The results of qPCR analysis showed that ArLBD1 gene could be detected in leaves,stem and roots of Anoectochilus roxburghii,with the highest expression level in leaves,followed by roots,and the lowest expression level in stems.It was speculated that the ArLBD1 gene mainly played a role in the development of Anoectochilus roxburghii leaves.