State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou, 310006, China; College of Life Sciences, Shanxi Agricultural University, Taigu, 030801, China.
J Integr Plant Biol. 2013 Nov;55(11):1016-25. doi: 10.1111/jipb.12098. Epub 2013 Sep 10.
A narrow leaf mutant was isolated from transgenic rice (Oryza sativa L.) lines carrying a T-DNA insertion. The mutant is characterized by narrow leaves during its whole growth period, and was named nal9 (narrow leaf 9). The mutant also has other phenotypes, such as light green leaves at the seedling stage, reduced plant height, a small panicle and increased tillering. Genetic analysis revealed that the mutation is controlled by a single recessive gene. A hygromycin resistance assay showed that the mutation was not caused by T-DNA insertion, so a map-based cloning strategy was employed to isolate the nal9 gene. The mutant individuals from the F₂ generations of a cross between the nal9 mutant and Longtepu were used for mapping. With 24 F₂ mutants, the nal9 gene was preliminarily mapped near the marker RM156 on the chromosome 3. New INDEL markers were then designed based on the sequence differences between japonica and indica at the region near RM156. The nal9 gene was finally located in a 69.3 kb region between the markers V239B and V239G within BAC OJ1212_C05 by chromosome walking. Sequence and expression analysis showed that an ATP-dependent Clp protease proteolytic subunit gene (ClpP) was most likely to be the nal9 gene. Furthermore, the nal9 mutation was rescued by transformation of the ClpP cDNA driven by the 35S promoter. Accordingly, the ClpP gene was identified as the NAL9 gene. Our results provide a basis for functional studies of NAL9 in future work.
一个窄叶突变体是从携带 T-DNA 插入的转基因水稻（Oryza sativa L.）品系中分离得到的。该突变体的特征是在整个生长过程中叶片狭窄，被命名为 nal9（窄叶 9）。该突变体还有其他表型，如幼苗期叶片浅绿色、植株高度降低、小穗和分蘖增加。遗传分析表明，该突变由单个隐性基因控制。潮霉素抗性测定表明，该突变不是由 T-DNA 插入引起的，因此采用基于图谱的克隆策略来分离 nal9 基因。nal9 突变体与 Longtepu 杂交的 F₂代突变个体用于作图。利用 24 个 F₂突变体，nal9 基因初步定位在 3 号染色体上标记 RM156 附近。然后根据 RM156 附近粳稻和籼稻序列差异设计新的 INDEL 标记。最终，nal9 基因位于BAC OJ1212_C05 上标记 V239B 和 V239G 之间的 69.3kb 区域内，通过染色体步移定位。序列和表达分析表明，一个 ATP 依赖的 Clp 蛋白酶解亚基基因（ClpP）很可能是 nal9 基因。此外，ClpP cDNA 由 35S 启动子驱动转化可以拯救 nal9 突变。因此，ClpP 基因被鉴定为 NAL9 基因。我们的研究结果为未来 NAL9 功能研究提供了依据。
