烟草赤霉素2-氧化酶基因NtGA2ox1的克隆、表达及亚细胞定位分析

Cloning,Expression and Subcellular Localization of Gibberellin 2-oxidase Gene NtGA2ox1 from Nicotiana tobacum

赤霉素2-氧化酶(GA2ox)通过2-β-羟基化作用产生失活的赤霉素,进而调节植物体内的赤霉素的活性水平。前期,本研究在烟草侧枝发育突变体转录组数据中,发现一个赤霉素2-氧化酶基因,其表达水平与野生型相比存在显著差异,命名为NtGA2ox1。为了更好地研究该基因在烟草侧枝发育中的作用,本研究从普通烟草中分离克隆了NtGA2ox1基因。通过测序分析该基因的编码及全长序列,发现NtGA2ox1基因含有2个外显子和1个内含子,编码一条长度为379个氨基酸的序列。同源进化分析表明,该基因在多种植物中存在同源序列,特别是茄科植物。组织特异性表达分析发现,NtGA2ox1基因在烟草的各个生长阶段均有表达,其中,在花和根中表达量较高。同时,激光共聚焦显微镜结果表明,YFP-NtGA2ox1融合蛋白在细胞质和细胞核中有很强的荧光信号,表明NtGA2ox1蛋白很可能定位于细胞核和细胞质中。本研究为进一步研究赤霉素调控烟草侧枝发育提供了理论依据。

GA2-oxidase(GA2 ox) can produce biologically inactive GAs by 2-β-hydroxylated process,subsequently,regulate the endogenous levels of bioactive GAs in plants.We isolated a GA2-oxidase gene,termed as NtGA2 ox1,of which the expression level showed significantly different in axillary-development mutant compared to wild type during screening the genes related to axillary-development in common tobacco.To further investigate the role of NtGA2 ox1 gene in common tobacco,the CDS and full-length sequence of NtGA2 ox1 were isolated from common tobacco.Sequence blast showed that NtGA2 ox1 gene contains two exons and one intron,which encoded a protein with 379 amino acids in length.BlastP and phylogenetical tree analyzed with homologs from the sequence from several species,suggested that NtGA2 ox1 was highly homologous with these plants,especially with the species from Solanum.Tissue-specific expression indicated that the NtGA2 ox1 m RNA can be detected in all selected tissues,and showed higher expression in flower and root.Meanwhile,strong fluorescence signaling was detected in cytosol and nucleus by laser confocal microscope,suggesting that YFP-NtGA2 ox1 fusion protein is probably located in cytosol and nucleus.This study provides a theoretical basis for the further study on the regulation of gibberellin on the development of lateral branching of tobacco.