文心兰铁氧还蛋白基因克隆定位及表达分析

Cloning,Subcellular Localization and Expression Analysis of Ferredoxin Gene OnFdin Oncidium hybridum

采用RACE-PCR法,从‘小樱桃’文心兰中克隆到一个全长989bp的铁氧还蛋白基因cDNA序列,命名为OnFd(登录号KX461907)。OnFd基因开放阅读框长为465bp,预测可编码154个氨基酸;gDNA和cDNA序列比对结果显示OnFd基因不含内含子。生物信息学分析表明,OnFd具有1个典型的[2Fe-2S]结构域;同源分析显示,OnFd与玉米Fd3的相似度最高(64.29%)。蛋白亚细胞定位结果显示OnFd定位于叶绿体。实时荧光定量PCR检测发现:OnFd基因在花中表达量最高,其次是叶与根,在假鳞茎中表达量最低;接种病原菌研究显示,文心兰感染软腐病后,各个部位的OnFd基因表达量均呈上升趋势,尤其是接种部位假鳞茎在接种病原菌1d后表达量便表现出极显著上调,并且在5个感病阶段的表达量是健康植株的2.83~3.98倍。研究表明,OnFd基因可能在文心兰响应抗软腐病过程中具有重要作用。

The RT PCR combined with RACE method was used to clone the complete cDNA sequence of ferredoxin gene （named as OnFd,GenBank accession No. KX461907） from Oncidium Kutoo ‘Litter Cherry’ （O.ornithorynchun × T. variegata）. The complete cDNA sequence of OnFd was 989 bp,encoding 154 amino acids. Comparisons between the cDNA and genomic DNA sequences showed that intron was absent in OnFd. Bioinformatic analysis revealed that OnFd owns a typical [2Fe 2S] domain. Blastp analysis showed that OnFd shares the highest similarity with Zea mays Fd3 （64.29%）. Subcellular localization analysis indicated that the OnFd was located in the chloroplast. qRT PCR results showed that the expression level of OnFd was significantly higher in the flower than that in leaf and root,the lowest expression was found in pseudobulb. Moreover,we investigated the expression of OnFd in response to root rot pathogen infection in different tissues or organs. Result showed that the expression of OnFd accumulated quickly after pathogen infection,and very significant up regulation was found in pseudobulb at only 1 day post pathogen inoculation （dpi）（P〈0.01）,The gene expression in plants from these five infected stages is 2.83-3.98 times than that of the healthy plants. The result suggested that the OnFd may play important role in Oncidium soft root pathogen interaction.