Li A, Zhang Y, Wu X, Tang W, Wu R, Dai Z, Liu G, Zhang H, Wu C, Chen G, Pan X
Yangzhou University, Yangzhou, Jiangsu, 225009, People's Republic of China.
Plant Mol Biol. 2008 Mar;66(5):491-502. doi: 10.1007/s11103-007-9283-3. Epub 2008 Jan 6.
T-DNA tagging is a high throughput strategy for identifying and cloning functional genes in plants. In this study, we screened 4416 lab-created T(1) rice T-DNA tagged lines and identified a mutant, designated dh1 (degenerated hull1), with phenotype of degenerated hull and naked pistils and stamens. Approximately 60% florets on the dh1 panicle defected in forming normal palea and lemma. Instead, they formed degenerative velum-like or filamentous organs accompanying with the lack of lodicules, stamens and pistils at different degree. A 361 bp of genomic sequence flanking the T-DNA isolated using TAIL-PCR (Thermal asymmetric interlaced PCR) co-segregated with the mutation phenotype. Results of blastn and gene prediction revealed the T-DNA inserted into the promoter region of a function-predicted gene at 283 bp upstream of its transcription start site (TSS). The predicted gene encoded a LOB (Lateral Organ Boundaries) domain-like protein. RT-PCR analyses indicated the transcription level of target candidate gene, DH1, decreased significantly in dh1 mutant. RNAi aimed at DH1 in wild type plants could partially result in the mutation phenotype of dh1. DH1 could also rescue the mutation phenotype in the complement experiment. The result of transformation by a fused expression vector, pDH1::GFP, revealed that DH1 had the keen spatial and temporal characteristics of expressing at axillary bud, young panicle and floral organs but not at root, leaf, node and culm, and strongly expressing at young tissues but weakly at mature organs. The dh1 presented severer mutation phenotype under relatively longer daylight than under shorter daylight implied that shorter daylight induced the expression of gene(s) redundant to DH1 in function and partially compensated for the loss-of-function. It is the first time to report the LOB-domain gene participating in the development of floral organs in rice.
T-DNA标签法是一种用于鉴定和克隆植物功能基因的高通量策略。在本研究中，我们筛选了4416个实验室创建的水稻T(1)代T-DNA标签系，鉴定出一个突变体，命名为dh1（退化颖壳1），其表型为颖壳退化以及雌蕊和雄蕊裸露。dh1穗上约60%的小花在形成正常内稃和外稃时出现缺陷。相反，它们形成了退化的膜状或丝状器官，同时不同程度地缺少浆片、雄蕊和雌蕊。使用热不对称交错PCR（TAIL-PCR）分离的T-DNA侧翼361 bp基因组序列与突变表型共分离。blastn结果和基因预测显示，T-DNA插入到一个功能预测基因启动子区域，位于其转录起始位点（TSS）上游283 bp处。预测该基因编码一个类似LOB（侧生器官边界）结构域的蛋白。RT-PCR分析表明，目标候选基因DH1在dh1突变体中的转录水平显著降低。在野生型植物中针对DH1的RNA干扰可部分导致dh1的突变表型。在互补实验中，DH1也可挽救突变表型。融合表达载体pDH1::GFP转化结果显示，DH1具有敏锐的时空表达特性，在腋芽、幼穗和花器官中表达，而在根、叶、节和茎中不表达，在幼嫩组织中强烈表达，在成熟器官中弱表达。与较短日照相比，dh1在相对较长日照下表现出更严重的突变表型，这意味着较短日照诱导了功能上与DH1冗余的基因表达，并部分补偿了功能丧失。这是首次报道LOB结构域基因参与水稻花器官发育。
