金线莲ArCRC基因的克隆、亚细胞定位和表达分析

Cloning, Subcellular Localization and Expression Analysis of ArCRC Gene in Anoectochilus roxburghii

摘要:

为了解金线莲(Anoectochilus roxburghii) YABBY基因家族的CRABS CLAW (CRC)亚族基因在花和叶片发育中的功能，基于金线莲全长转录组数据RT-PCR克隆ArCRC基因，对其编码蛋白进行生物信息学、原核表达、亚细胞定位分析，采用qPCR技术对基因的表达模式进行分析。结果表明，金线莲ArCRC基因CDS长度为576 bp (GenBank登录号: OR394646), 编码191个氨基酸，含有YABBY superfamily和HMG-box_SF superfamily保守结构域，分子量为21.514 kD，理论等电点为9.16，不稳定系数41.12，属于不稳定蛋白。系统进化分析表明，ArCRC与水稻(Oryza sativa)的OsDL和拟南芥(Arabidopsis thaliana)的AtCRC聚为一类，属于CRC亚家族，且定位在细胞核。SDS-PAGE电泳和Western blot结果表明，ArCRC基因可在大肠杆菌中成功诱导表达。qRT-PCR分析表明，ArCRC基因在花的表达量最高，其次是叶，且在叶片中脉的表达量显著高于叶片外缘，因此，推测ArCRC基因主要在花器官发育中发挥功能，同时还参与调控叶片中脉的发育。

Abstract:

To understand the function of CRABS CLAW (CRC) subgene of the YABBY gene family of Anoectochilus roxburghii in flower and leaf development, the ArCRC gene was cloned by RT-PCR based on the full-length transcriptome data of A. roxburghii. The bioinformatics, prokaryotic expression and subcellular localization of encoded ArCRC protein were analyzed, and the expression pattern of ArCRC gene was analyzed by qPCR. The results showed that the CDS length of ArCRC gene was 576 bp (GenBank accession No.: OR394646), coding 191 amino acids. ArCRC contains YABBY superfamily and HMG-box_SF superfamily conserved domain, with molecular weight 21.514 kD, theoretical isoelectric point 9.16, instability coefficient 41.12, belonging to the unstable protein. Phylogenetic analysis showed that ArCRC was clustered with OsDL of rice (Oryza sativa) and AtCRC of Arabidopsis thaliana, belonging to the CRC subfamily and localized in the nucleus. SDS-PAGE electrophoresis and Western blot results showed that ArCRC gene could be successfully induced in Escherichia coli. qRT-PCR analysis showed that the expression of ArCRC gene was the highest in flowers, followed by leaves, and the expression in the midrib of leaves was significantly higher than that in the margin of leaves. Therefore, it was speculated that ArCRC gene mainly played a role in the development of flower organs and was also involved in regulating the development of the midrib of leaves.