作者姓名：李学勇   
  论文题目：水稻分蘖控制基因MOC1的克隆与研究   
  作者简介：李学勇，男，1972年5月出生，1998年9月师从于中国科学院遗传与发育生物学研究所李家洋院士，于2003年3月获博士学位。   
     
  中 文 摘 要   
  　

分蘖是决定水稻产量的重要农艺性状，在生产实践中主要通过栽培措施和育种手段来增加有效分蘖，减少无效分蘖。同时，分蘖又是水稻生长发育过程中的一种特殊的分枝现象。根据形态解剖学的研究，水稻分蘖的生长过程分为两个阶段：（1）在每个叶子的叶腋里分化形成一个分蘖芽；（2）分蘖芽进一步生长发育成分蘖。但是，对于分蘖芽形成和伸长的分子机理却是一无所知。分离鉴定各种分蘖突变体并且克隆相应的基因，将有助于揭示控制水稻分蘖乃至高等植物分枝的分子机理；而分蘖机理的研究结果对于水稻的生产具有重要的指导意义。

1990年，在粳稻品种H89025中发现了一株丧失分蘖能力的自发突变体，只有一个茎秆，而没有分蘖，我们将其命名为mono culm1 (moc1)。组织解剖学的观察表明，在moc1突变体的叶腋里没有腋生分生组织和分蘖芽，因此推测MOC1基因是腋生分生组织的起始和分蘖芽的形成所必需的。遗传分析表明，moc1突变体是由单个细胞核基因的隐性突变造成的，并且初步定位在第6染色体的长臂上，位于分子标记R1559和S1437之间。我们采用图位克隆的方法分离了MOC1基因，它与番茄中控制侧枝形成的Lateral Suppressor基因有比较高的同源性，在氨基酸水平上的一致性为44％，相似性为59％。序列分析表明，在moc1突变体中，有一个1.9 kb的逆转座子插在MOC1基因的编码区内，导致蛋白质的翻译提前终止。将野生型MOC1基因的基因组片段转入moc1突变体内，又恢复了分蘖能力，这说明我们克隆的MOC1基因是正确的。而且，MOC1转基因后代的分蘖数目是原始野生型品种H89025的3-5倍，分蘖数目的增加有两个原因：（1）在一些叶腋内形成了2个分蘖，而野生型中1个叶腋只能形成1个分蘖；（2）高节位上的分蘖以及三级、四级、五级等高级别分蘖的伸长，而野生型中这些分蘖芽一般保持休眠状态。这说明MOC1基因除了控制腋生分生组织的起始和分蘖芽的形成以外，还具有促进分蘖芽的生长发育的功能。

MOC1蛋白属于GRAS家族的转录因子，具有2个亮氨酸七聚体重复（leucine heptad repeats），1个VHIID结构域和1个SH2-like结构域。虽然没有发现典型的核定位信号序列，但是MOC1:GFP的融合蛋白却定位在了细胞核内。MOC1基因的组织原位杂交实验表明，在腋生分生组织形成之前，MOC1基因就已经在叶腋里的少数表皮或亚表皮细胞里表达，并且MOC1基因的表达伴随着腋生分生组织形态建成的整个过程；在腋生分生组织分化形成分蘖芽以后，MOC1仍然在整个分蘖芽中表达。这种表达模式与MOC1基因促进腋生分生组织的起始、分蘖芽的形成、和分蘖芽的生长发育等功能相符合。另外，我们发现在moc1突变体中由于MOC1基因的功能缺失突变，导致了分生组织特异性的OSH1基因和控制侧枝伸长的OSTB1基因的表达水平降低。因此，我们推测MOC1可能通过调控OSH1等分生组织特异性的基因促进腋生分生组织的起始，通过调控OSTB1等基因调节分蘖芽的生长发育。

我们对MOC1基因的图位克隆、表达模式以及作用机理的初步研究，将有助于阐明控制水稻分蘖的分子机理。而且，通过MOC1基因的正义或者反义转基因技术，有望创造出具有最佳分蘖数目和理想株型的水稻育种材料。

  关键词：水稻、分蘖、分枝、腋生分生组织、分蘖芽的形成和伸长、mono culm1 (moc1)突变体、图位克隆、MOC1基因、GRAS家族转录因子、Lateral Suppressor基因、细胞核定位、组织原位杂交、OSH1基因、OSTB1基因、逆转座子

  
  Study on the Isolation and Characterization of the MOC1 Gene Required for Tillering Control in Rice (Oryza Sativa)
Li Xueyong
ABSTRACT   
        Rice tillering is an important agronomic trait that determines the grain yield. In agriculture practice, rice tillering is mainly regulated through cultivation and breeding techniques to increase the productive tillers and reduce non-productive ones. Meanwhile, tillering is a special branching character during rice growth and development. Morphological and anatomical studies have demonstrated that tillering process in rice can be divided into two stages: the formation of a tiller bud at each leaf axil and the outgrowth of tiller bud into tiller. However, the molecular mechanism of tiller bud formation and outgrowth is nearly totally unknown in rice. Characterization of various tillering mutants and cloning related genes will be conducive to reveal the molecular mechanism controlling rice tillering and plant branching. And the discovery of tillering mechanism will be very instructive to rice production.

In 1990, a spontaneous rice mutant losing tillering ability was found in a Japonica rice variety H89025. This mutant is designated as mono culm 1 (moc1) because it has only the main culm without tillers. Morphological and anatomical studies revealed that there are no axillary meristem and tiller bud in the leaf axil of moc1 mutant plants. Therefore, we predict that MOC1 gene is required for axillary meristem initiation and tiller bud formation. Genetic analysis revealed that moc1 mutant is caused by a recessive mutation in a single nuclear gene. MOC1 is primarily mapped onto the long arm of chromosome 6 flanked by two RFLP markers, R1559 and S1437. We then isolated the MOC1 gene through a map-based cloning approach. MOC1 shows high homology to the Lateral Suppressor protein which controls lateral branching in tomato, with 44% identity and 59% similarity. Sequencing analysis showed that there is a 1.9 kb retrotransposon inserted into the coding region of MOC1 gene in the moc1 mutant, which predicts a premature translation stop. The genomic fragment of wild type MOC1 gene was introduced into the moc1 mutant, and the tillering ability was restored in the MOC1 transgenic plants. This complementation test confirmed the identity of MOC1 gene. Moreover, the tiller number of MOC1 transgenic plants was increased to 3-5 times of that of original wild-type plants H89025. Two reasons explain the increase in tiller number: (1) the formation of two tillers in some leaf axils, in contrast, there is only one tiller formed in a leaf axil in wild-type plants; (2) the outgrowth of tiller buds at higher nodes and higher orders, in contrast, these tiller buds are usually arrested in wild-type plants. The latter also suggests that MOC1 promotes the development and outgrowth of tiller buds in addition to its promotive effect on axillary meristem initiation and tiller bud formation.

MOC1 protein belongs to the GRAS transcription factor family, with two leucine heptad repeats, a VHIID domain, and an SH2-like domain. Although no typical nuclear localization signals were found in MOC1, the MOC1::GFP fusion protein was localized into the nuclear. The RNA in situ hybridization experiments revealed that MOC1 was initially expressed in a few epidermal or subepidermal cells in the leaf axil prior to the formation of axillary meristem. The expression of MOC1 was continued during the formation process of axillary meristem. MOC1 was still expressed after axillary meristem has differentiated into tiller buds. The expression pattern of MOC1 is consistent with its promotive roles on axillary meristem initiation, tiller bud formation and tiller bud outgrowth. Moreover, the loss of function mutation of MOC1 gene caused the reduced transcript level of meristem specific OSH1 gene and axillary branch specific OSTB1 gene in the moc1 mutant. Therefore, we predict that MOC1 promotes axillary meristem initiation and tiller bud outgrowth through the activation of OSH1 and OSTB1 gene respectively.

The isolation of MOC1 gene and the primary studies on its expression pattern and function mechanism will be conducive to the elucidation of rice tillering mechanism. In addition, it is hopeful to generate new breeding materials with optimal tiller number and ideal shoot architecture through transformation of sense or anti-sense MOC1 genes.

Key Words: Rice, Tiller, Branch, Axillary meristem, Tiller bud, Mono culm1 mutant, Map-based cloning, MOC1, GRAS family, Lateral Suppressor, OSH1, OSTB1, Retrotransposon