Functional Analysis On Rice Telomeric And Centromeric Sequence Using Transformation Techniques

Centromere, telomere and origin of replication are the three basic functional elements of eukaryotic chromosomes. Among them, centromeres are the specialized chromosome regions at which kinetochore complex is assembled and hence serve as the attachment site for spindle microtubules. Centromeres have an essential role in correct transmission of chromosomes in mitosis or meiosis. Telomeres are basic structures at the ends of linear eukaryotic chromosomes. Telomeres prevent truncation of chromosome ends during DNA replication. Telomeres have a closed DNA structure at their extreme termini bound by various proteins, preventing them from being wrongly recognized as a broken chromosome end. Nevertheless, both of centromere and telomere are the essential component of the artificial chromosome construction.As a good monocot model system, rice has been extensively studied for decades. Its 430Mb genome has been completely sequenced. It is relatively easy to prepare well-spreading pachytene chromosomes in rice, which makes it a good system for molecular cytogenetic study as well. Both molecular and cytogenetic characterizations about its centromere and telomere have been well documented in recent years.In the present study, three expression constructs were constructed containing rice centromeric or telomeric DNA sequences, which were further transformed into different rice cultivars by agrobacterium-mediated transformation system. As a result, more than one thousand transgenic rice plants were obtained. Morphological and genetic characterizations were conducted on those transgenic plants. The main results were as followings:1. There are two subspecies in the cultivated rice species Oryza sativa: japonica and indica. The japonica rice can be more easily transformed than indica rice for using Agrobacterium-mediated transformation system. To improve the techniques for indica rice transformation, an indica rice variety, Zhongxian 3037, was used for optimizing the Agrobacterium-mediated transformation system. Several factors affected the transformation efficiency were studied and a suitable transformation system was created. In this transformation system, immature embryos were suitable explants. The MS II mediumbased on the MS medium, was appropriate for inducing callus. Agrobacterium tumefaciens strain EHA105 was much more efficient than that of AGLO for indica rice cultivar Zhongxian 3037 transformation, and its transformation efficiency was 18.0 percent while AGLO's transformation efficiency was 5.5 percent when using immature embryos as explants. While the optimal concentration of Agrobacterium were OD₆₀₀=0-6 and the incursion time were 14min. The efficiency of callus regeneration was much higher when 20g/L sorbitol was added to the regeneration medium MSR than that of the control. Using this transformation system, the HPT gene was introduced into Zhongxian 3037 and its mutant A and B, and many transgenic plants were obtained. Some of these transgenic rice plants were confirmed by PCR and Southern blotting analysis which indicating the T-DNA had been integrated into the genome of transgenic rice plants, Genetic analysis showed that the transgenes were segregated in a Mendelian fashion in the T₁ generation.2. For well investigation of rice centromere function, GFP-OsCENH3 chimeric gene was constructed and transformed to an indica rice Zhongxian3037, mediated with Agrobacturium. The integration of exogenous genes in the transgenic rice plants was further confirmed by both PCR and Southern blotting analysis. The overlapping of GFP signals and anti-CENH3 foci revealed both in mitotic and meiotic cells from T₀ and T₁ generation plants, indicating that GFP had successfully fused with CENH3, and the location of GFP signals could represent that of CENH3 region. To explore the application of the transgenic plants, fluorescence in situ hybridization (FISH) using rice centromeric tandem repetitive sequence CentO as a probe was conducted on pollen mother cells (PMCs) at zygotene stage. It was showed that GFP signals were overlapped with CentO FISH signals, proving that CentO is one of the key factors composed of rice functional centromeres. Immuno-fluorescent staining results by anti-α-tublin antibody and anti-PAIR2 antibody during mitosis and meiosis of these transgenic plants further approved that GFP-OsCENH3 transgenic rice plants could be widely applied in rice molecular biological study, especially for tagging of functional centromeres in both living cells and tissues.3. According to the biological informatics of the rice telomerase, the telomerase reverse transcriptase was inactivated in the transgenic rice by RNA interference technique. The total 165 transgenic plants were obtained from 78 transformants by Agrobacterium-mediated transformation, when the plant expressed vector pCam23A-l-2 introduced into the japonica rice cultivar Nipponbare. Some of transgenic rice plants were confirmed by PCR and Southern blotting analysis, indicating that the T-DNA had been integrated into the genome of transgenic rice plants. Terminal restriction fragment (TRF) and fluorescence in situ hybridization (FISH) showed that the length of telomere DNA are getting short in the offspring of transgenic rice plants in the successive generations. However, the main agronomical characters of the transgenic rice have not changed in the T₀ and T₁ generation and without severe developmental defects.4. With Agrobacterium-mediated transformation, the plant expression vector pBTER with 602 bp rice telomeric DNA was introduced into the japonica rice cultivar, Wuxiangjing 9, about 1782 transgenic plants were obtained from 652 the resistant callus. Some of these transgenic rice plants were confirmed by PCR and Southern blotting and the phenotype of these plants were investigated, showing that the T-DNA had been integrated into the genome of transgenic rice plants, many mutants were obtained in the progenies of the transgenic rice plants, such as: chromosome number variation, chromosome translocation, sterile, early maturing, delayed heading and dwarf and so on.5. A dwarf mutant was identified from a T-DNA insertion population derived from a japonica rice variety, Wuxiangjing 9. Molecular characterization and genetic analysis was conducted for the mutant and its related genetic populations. Genetic analysis of the mutant showed that two kinds of phenotype, such as dwarf and normal height plants in the segregating population derived from the T-DNA insertion heterozygote plant. They were fit for the ratio of 3(dwarf type):1 (normal type), indicating the dwarf gene is dominant mutation. The dwarf gene was further mapped on rice chromosome 4 using a segregation population derived from the mutant crossed with an indica rice variety, Longtepu. Test for HPT resistance showed the dwarf plants were all resistant while the normal height plants were susceptible, and the ratio of resistance and susceptible plants was 3:1, indicating that the dwarf mutation was co-segregated with HPT resistance. These results were consistent with only one copy exogenous gene was detected in the transgenic rice genome revealed by Southern analysis. So the dwarf mutant phenotype was controlled by a dominant gene mutation, which was caused by T-DNA insertion.