摘要
从简化种子生产程序考虑，今后杂交水稻育种的策略将沿三系法、两系法和一系法三个阶段发展(袁隆平，1988)。其中，所谓一系法便是借助无融合生殖途径固定杂种优势。无孢子生殖是普遍存在于草类植物的一种无融合生殖方式，其特征是：大孢子母细胞正常并发育成单倍体胚囊，其周围的二倍体珠心细胞发育成大小与大孢子母细胞相近的无孢子生殖起始细胞，这些细胞不经过减数分裂直接发育成二倍体胚囊，从而实现无孢子生殖。但在水稻中尚未发现这一生殖现象。敲除多胞囊基因(MULTIPLESPOROCYTES，MSP1)的水稻突变体msp1，在胚珠中虽能产生多个大孢子母细胞，然而其花粉囊绒毡层不发育，只形成额外小孢子母细胞，结果表现雄性不育，不能繁种。
针对这一问题，本论文研究了与无孢子生殖特性相关的水稻基因，探索了人工合成水稻无孢子生殖的材料和方法，试图为实现固定水稻杂种优势提供一条重要的途径。主要研究结果包括：
1．通过tBLASTn搜索水稻数据库，作者在日本晴(O. sativa. subsp. japonica. cv. Nipponbare)基因组中发现2个绒毡层决定基因(TAPETUM DETERMINANT1，TPD1)OsTDL1A和OsTDL1B，它们与拟南芥TPD1同源。RT-PCR结果显示，在幼穗中OsTDL1A和OsTDL1B与MSP1基因共表达。RNA原位杂交表明，这些基因的表达部位有差异。OsTDL1A和MSP1在大孢子母细胞周围的珠心细胞和小孢子母细胞周围的花粉囊囊壁细胞中均表达，而OsTDL1B仅在花粉囊囊壁细胞中表达，胚珠组织中并不表达。同时基于酵母双杂交和双分子荧光互补分析显示了这两个基因编码蛋白功能上的差异。OsTDL1A编码蛋白能与MSP1基因编码的富亮氨酸重复区域互作，而OsTDL1B却不能。通过RNA干扰技术抑制转化株OsTDL1A基因的转录水平，结果引发多个大孢子母细胞出现，而花粉囊仍然保持正常，由此推测，胚珠内的大孢子母细胞数目受MSP1与配体OsTDL1A的基因互作控制。基于上述实验，作者认为，应用RNA干扰技术产生的水稻OsTDL1A-RNA干扰系将成为人工合成水稻无孢子生殖的重要材料。
2．通过ClaustalW多序列比对，发现栽培稻和不同基因组野生稻MSP1基因非编码区序列具有高度的保守性，通过聚类分析可将供试的6个基因组野生稻分成2群。其中AA基因组3个种为一群，它们间的序列同源性高达97.7％。另一群中CC、CCDD、EE基因组间的序列同源性为98％，它们与BBCC的亲缘关系比GG更近；这为研究水稻不同基因组的进化提供了线索。通过转录分析纠正了NCBI对MSP1上游基因注释的错误，其实这是MSP1的5′非翻译区，MSP1转录起始位点应位于Nonomura等(2003)克隆的全长cDNA序列转录起始位点上游2281bp。进一步应用双向电泳和质谱分析研究其编码蛋白的分离及其表达，结果发现在水稻大孢子母细胞时期(3mm幼穗)，日本晴(野生型)和msp1突变体间，共显示9个差异蛋白。与野生型相比，突变体中P1的分了量降低，P2的等电点升高，出现了日本晴中未显示的P3蛋白，P4、P5、P6表达量下调，而P7、P8、P9上调。作者推测MSP1激酶域作为负调节物，可能导致下游冷激蛋白的磷酸化，而抑制信号传导。
3．首次发现水稻基因组中存在2个腈水解酶基因(OsNITA和OsNITB)，它们成簇分布在2号染色体上，与拟南芥NIT4基因聚为一类。OsNITA和玉米ZmNIT2的同源性(92％)高于OsNITB和ZmNIT1的同源性(81％)。通过RT-PCR显示，2个水稻腈水解酶基因能在根、茎、叶及幼穗等多种组织中表达，但在表达量上有差异，OsNITA明显高于OsNITB。利用RNA原位杂交技术检测了两基因在水稻根尖(种子萌发后10d)和幼穗(大孢子母细胞时期)中的表达部位。结果表明，OsNITA在根尖分生组织区有较强的杂交信号，而在根冠和伸长区不表达，OsNITB的表达模式与OsNITA相似，但信号很弱。OsNITA在胚珠内的珠心细胞中表达，而在大孢子母细胞内未检测到杂交信号。在花粉囊的表达主要集中于内层、中层和绒毡层，维管细胞中少量表达，小孢子母细胞中则不表达，OsNITB在3mm幼穗中未检测到任何信号。根据水稻腈水解酶基因的结构及时空表达特征，我们推测该基因可能参与催化生长素合成。
In the future, hybrid rice breeding will follow three developmental stages to facilitate seed production procedure, they are three-line hybrid, two-line hybrid and one-line hybrid (Yuan, 1988). One-line hybrid is to fix hybridity through apomixes. Although unknown in rice, apospory is a form of apomixis that is common in grasses. In apospory, the megaspore mother cell is normal and develops into a haploid embryo sac, while adjacent diploid nucellar cells give rise to aposporous initials. These resemble megaspore mother cells in their large size, but they bypass meiosis and generate diploid embryo sacs. The rice knockout mutants of the leucine-rich repeat receptor kinase MULTIPLE SPOROCYTES1 (MSP1) produce multiple megaspore mother cells in the ovule and replace the tapetum of the anther with excess meiocytes. However, msp1 homozygous mutant is sterile, it is difficult to work with.
Our purpose is to study the function of some rice genes which are related to apospory developmental characteristics and explore relative materials and methods toward synthetic apospory in rice to fix hybridity. Herein the main results were summarized as follows.
1. When conducting tBLASTn analysis of TPD1 (TAPETUM DETERMINANT1) against the rice genomes (O. sativa. subsp. japonica. cv. Nipponbare), we discovered two homologues in rice, they were tentatively named as OsTDL1A and OsTDL1B. RT-PCR results showed that OsTDL1A and OsTDL1B were co-expressed with MSP1 in 1-3 mm spikelets, at the peak of meiosis. RNA in situ hybridization establishes that OsTDL1A, like MSP1, is expressed in both ovule and anther of rice, whereas OsTDL1B is expressed in the anther but not the ovule. Only OsTDL1A displays an ability to bind to the LRR domain of MSP1, while OsTDL1B doesn't have this ability. RNA interference of OsTDL1A phenocopies the msp1 mutant in the ovule but not in the anther. We discuss these results in relation to the development of synthetic apospory for hybrid rice, producing multiple MeMCs without causing male sterility. Our data suggest that OsTDL1A-RNAi lines will be a suitable material for developing synthetic apospory for hybrid rice.
2. We found that the un-translated region of MSP1 gene were highly conserved between cultivar and different genome wild rice by multiple sequence alignment analysis (ClustalW). Unrooted dendrogram based on sequencing results showed that six different genome wild rice was divided into two groups. Three AA genome belong to one group with 97.7% similarity. While in another group, the similarity between CC, CCDD and EE genome is 98%, and BBCC is less relative to GG genome comparing to CC, CCDD and EE genome. The results provide very valuable clues for rice evolution between different genome. Through detailed transcription analysis, we corrected the annotation of NCBI database on MSP1 upstream gene, the annotated upstream gene is only part of MSP1 un-translated region, and the real MSP1 transcription site is located on 2281bp upstream of the cloned full-length cDNA by Nonomura (Nonomura et al., 2003). Furthermore, using two dimensional electrophoresis method and mass spectrum analysis, at least 2000 protein spots were clearly separated from 3mm spikelets protein (Megaspore Mother Cell Stage) of wild type and msp1 homozygous mutant. Among those proteins, we found nine protein spots which showed difference between wild type and mutant. The molecular weight of P1 is lower in mutant than wild type, while the p1 of P2 is increased in mutant, and P3 was only appeared in mutant. In addition, P4, P5 and P6 were down-regulated, P7, P8 and P9 were up-regulated in mutant. According to the previous report, we hypothesize that the kinase domain of MSP1 encoded protein has the ability to phosphorylate the cold stress protein.
3. We found that rice genome contains two nitrilase genes: OsNITA and OsNITB, they are tandemly arranged on chromosome2 and belong to Arabidopsis NIT4 group. The similarity (92%) between OsNITA and maize ZmNIT2 is higher than that of the similarity (81%) between OsNITB and ZmNIT1. RT-PCR results indicated that two rice nitrilase genes can express in a wide range of tissues including root, shoot leaf and spikelets, but in each tissue the expression level of OsNITA was several times higher than OsNITB. In addition, the cellular location of two rice nitrilase genes was also checked with 10DAG root tips and spikelets at the megaspore mother cell stage by RNA in situ hybridization. The data showed that strong signal was detected in meristem cells in root tips, while no signal in root cap cells and exlongation zone with OsNITA, OsNITB has the similar expression pattern, but the signal was very weak. In ovule, OsNITA can express in the nucellus cells, however, no signal was appeared in the megaspore mother cell. In anther, the signal was mainly concentrated in endothecium, middle layer and tepatum cells, with weak staining in vascular cells, but no signal in microspore mother cell. OsNITB was not expressed in 3mm spikelets.These dates will help us establish the relationship between nitrilase expression and IAA biosynthesis in rice.
引文
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