文心兰‘南茜’谷氧还蛋白基因克隆定位及表达

Cloning,subcellular localization,and expression of a glutaredoxin(OnROXY1) in Oncidium ‘Gower Ramsey’

为研究文心兰谷氧还蛋白超家族基因ROXY在花药发育中的功能,以文心兰‘南茜’（Oncidium Gower Ramsey）为试验材料,从‘南茜’的转录组网站选取一条与拟南芥At ROXY1/2相似度最高的基因序列（命名为OnROXY1）,以该序列的cDNA为模版设计引物,用RT-PCR从‘南茜’花器官中扩增该基因,并对扩增产物进行测序和保守结构域分析.结果表明,OnROXY1基因编码区402 bp,编码133个氨基酸,与‘南茜’转录组网站中的预测序列只有一个碱基差异,但与该预测序列的氨基酸序列和保守结构域完全一致（GenBank登录号：MF696154）.OnROXY1基因具有典型的谷氧还蛋白（Glutaredoxin,GRX）基因家族的结构域和CMCC活性位点,因此该基因属于GRX基因家族,并且属于亚类CC型的GRX基因.进化树分析表明,OnROXY1与水稻的Os ROXY1、Os ROXY2和拟南芥的At ROXY1、At ROXY2蛋白序列的一致性最高,进化距离最近.亚细胞定位结果显示,OnROXY1主要定位于细胞核中,少量定位在细胞质中.RT-PCR检测发现,在不同组织部位中,OnROXY1在假鳞茎中的表达量最高,唇瓣中的表达量最低;不同花期的定量结果显示,OnROXY1在脱落干枯期时的表达量最高,盛开期时的表达量最低.本研究推测谷氧还蛋白基因OnROXY1可能在文心兰假鳞茎与根的发育、消除植物体内过多的活性氧以及延缓文心兰花器官的衰老等方面具有重要作用.

In order to investigate the function of the glutathione superfamily ROXY gene during anther development in Oncidium（Orchidaceae）, sequences in the glutaredoxin gene family from the Oncidium ‘Gower Ramsey＇ transcriptomic library were selected and aligned with AtROXY1 and AtROXY2 of Arabidopsis thaliana for genetic homology analysis. Next, OnROXY1（Gen Bank accession No. MF696154） was screened, and using RACE-PCR, several conserved amino acid sequence domains were revealed. The results showed that the complete cDNA sequence of OnROXY1 was 402 bp and encoded 133 amino acids, which was only one base different from the predicted sequence selected from the Oncidium ‘Gower Ramsey＇ transcriptomic library and completely consistent with the amino acid sequence and conserved domain of the predicted sequence. In addition, OnROXY1 belonged to the glutaredoxin（GRX） superfamily and had a c-type cytochrome in the CMCC superfamily domain. Moreover, protein sequence and phylogenetic tree analysis showed that OnROXY1 had the closest amino acid identity with OsROXY1, OsROXY2, AtROXY1, and AtROXY2. Subcellular localization using a GFP-fusion tag of OnROXY1 revealed nucleic and cytosolic localization. Differential expression profiles of OnROXY1 via quantitative real-time PCR produced a significantly greater amount of m RNA transcript in pseudobulb tissues and lesser in the labellum. Moreover, OnROXY1 exhibited the greatest gene expression in the withered and abscised period and lowest during the blooming periods. Here, the results showed the importance of OnROXY1, specifically in pseudobulbs and roots, via its function of scavenging excessive reactive oxygen species and delaying aging in the floral organs of Oncidium.