Studies On Related Genes Toward Rice Apospory

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.