Arumugam Thangavelu U, Davies Eric, Morita Eugene Hayato, Abe Shunnosuke
Laboratory of Molecular Cell Physiology, Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan.
Plant Physiol Biochem. 2007 Oct-Nov;45(10-11):767-80. doi: 10.1016/j.plaphy.2007.07.006. Epub 2007 Jul 24.
The makorin (MKRN) RING finger protein gene family encodes proteins (makorins) with a characteristic array of zinc-finger motifs and which are present in a wide array of eukaryotes. In the present study, we analyzed the structure and expression of a putative makorin RING finger protein gene in rice (Oryza sativa L. ssp. Japonica cv. Nipponbare). From the analysis of the genomic (AP003543), mRNA (AK120250) and deduced protein (BAD61603) sequences of the putative MKRN gene of rice, obtained from GenBank, we found that it was indeed a bona fide member of the MKRN gene family. The rice MKRN cDNA encoded a protein with four C3H zinc-finger-motifs, one putative Cys-His zinc-finger motif, and one RING zinc-finger motif. The presence of this distinct motif organization and overall amino acid identity clearly indicate that this gene is indeed a true MKRN ortholog. We isolated RNA from embryonic axes of rice seeds at various stages of imbibition and germination and studied the temporal expression profile of MKRN by RT-PCR. This analysis revealed that MKRN transcripts were present at all the time points studied. It was at very low levels in dry seeds, increased slowly during imbibition and germination, and slightly declined in the seedling growth stage. After 6days of germination, an organ-dependent expression pattern of MKRN was observed: highest in roots and moderate in leaves. Similarly to MKRN transcripts, transcripts of cytoskeletal actin and tubulin were also detected in dry embryos, steadily increased during imbibition and germination and leveled off after 24h of germination. We studied the spatial expression profile of MKRN in rice tissues, by using a relatively fast, simple and effective non-radioactive mRNA in situ hybridization (NRISH) technique, which provided the first spatial experimental data that hints at the function of a plant makorin. This analysis revealed that MKRN transcripts were expressed in young plumules, lateral root primordia, leaf primordia, leaves and root tissues at many different stages of germination. The presence of MKRN transcripts in dry seeds, its early induction during germination and its continued spatiotemporal expression during early vegetative growth suggest that MKRN has an important role in germination, leaf and lateral root morphogenesis and overall development in rice.
马克罗林（MKRN）泛素连接酶蛋白基因家族编码的蛋白（马克罗林）具有一系列特征性的锌指基序，存在于多种真核生物中。在本研究中，我们分析了水稻（日本晴）中一个假定的马克罗林泛素连接酶蛋白基因的结构和表达。通过对从GenBank获得的水稻假定MKRN基因的基因组序列（AP003543）、mRNA序列（AK120250）和推导的蛋白质序列（BAD61603）进行分析，我们发现它确实是MKRN基因家族的一个真正成员。水稻MKRN cDNA编码一种含有四个C3H锌指基序、一个假定的Cys-His锌指基序和一个泛素连接酶锌指基序的蛋白质。这种独特的基序组织和整体氨基酸一致性表明该基因确实是一个真正的MKRN直系同源基因。我们从不同吸胀和萌发阶段的水稻种子胚轴中分离RNA，并通过RT-PCR研究MKRN的时间表达谱。该分析表明，在所有研究的时间点都存在MKRN转录本。在干种子中其水平非常低，在吸胀和萌发过程中缓慢增加，在幼苗生长阶段略有下降。萌发6天后，观察到MKRN的器官依赖性表达模式：在根中最高，在叶中中等。与MKRN转录本类似，细胞骨架肌动蛋白和微管蛋白的转录本在干胚中也被检测到，在吸胀和萌发过程中稳定增加，并在萌发24小时后趋于平稳。我们通过使用一种相对快速、简单且有效的非放射性mRNA原位杂交（NRISH）技术研究了MKRN在水稻组织中的空间表达谱，该技术提供了首个暗示植物马克罗林功能的空间实验数据。该分析表明，MKRN转录本在萌发的许多不同阶段的幼芽、侧根原基、叶原基、叶和根组织中表达。MKRN转录本在干种子中的存在、其在萌发过程中的早期诱导以及在早期营养生长过程中的持续时空表达表明，MKRN在水稻的萌发、叶和侧根形态发生以及整体发育中起重要作用。
