| Polyploidy is recognized as a crucial factor in plant evolution and breeding. Rapid progress has been made in crops, such as wheat, cotton, and rape, the yields will be doubled when their genomes are duplicated. Rice is considered as an ancient aneuploid, and polyploid resulted in tre-mendous evolutionary dominance both as far as yield and stress resistance. However, most polyploid rice whose genomes were duplicated can have disorganized meiosis behavior,low seed set rate and pollen fertility. Therefore, if polyploid breeding wants to obtain a breakthrough, increasing the seed set rate is the key. The problem of low seed set rate has became a bottleneck for polyploid rice breeding,which is looking forward to excavating new rice lines.By employing the so-called strategy "breed super rice varieties via two superiority of distant hybridization and polyploidization", our lab has bred two lines of polyploid rice, which have high seed set rate and meiosis stablity, called PMeS lines (Polyploid Meiosis Stablity).The PMeS HN2026-4X, whose meiotic behavior, pollen fertility and seed set rate are very similar with the diploid, is considered as a Ph-like(pairing homoeologous) gene materials.While crossed with other polyploid rice lines, their seed set rate generally increased, breaking through the bottleneck of polyploid rice breeding,which has been confirmed the characteristic of the high seed set rate of PMeS can be stably inherited. Till up now, the reason for unstability of the molecular mechanism of polyploid rice meiosis is still unknown. Therefore, it's urgent to study the molecular mechanism of PMeS lines.OsMND1, involved in rice meiosis, is the homologous gene of yeast (S.cerevisiae) MND1 (meiotic nuclear divisions 1).It has been reported a high homology gene MND1 in Arabidopsis thaliana, has played a very important role in the process of meiotic homologous chromosome pairing, synapsis and recombination, but it has never been reported in the rice.In this study, clone and expression of OsMND1, constructing OsMND1 gene over-express vector and RNAi vector aim at OsMND1 gene and expressing OsMND1 gene in E.coli and S.pombe successfully have been finished before.This study has completed the following experiment list:1.We use two methods about PCR and restriction enzyme digestion to test and verify vectors, and the result is correct. 2.In order to explore the function of OsMNDl, we carry on the transgene research in gen over-express and gene silencing, proves the gene's function from forward and reverse, we use th OsMNDl gene over-express vector to induce callus tissue of line BLL-2X. After optimizing ric transgene system, we transform the over-express vector into ployploid rice mediated b Agrobactium tumefaciens and get green plantlets. We do PCR examination to select positiv plants,and then the result of real-time quantitative PCR examination for positive plants has bee proved that target gene expression has certain upward.We carry on analyses on Transgeni plants's agronomic characters of To& T1, in order to investigate the impact on rice after it over-expression, showing that pollen fertility and seed rates have no significant changes. It may b due to the selection of the BLL-2X which is a high setting of the material itself, and whos meiosis is normal, so over-expression of the gene will not bring a significant change in pollei fertility. We need to do a subsequent work to make BLL-2X doubled into tetraploid and thei analysis its agronomic traits. It is also found that increases in the number of tillers of transforme plants are significantly abnormal.The specific reasons needs to be done in the further research.3.we transform the RNAi vector into the PMeS HN2026-4X mediated by Agrobactiun tumefaciens and get green plantlets.We do PCR examination to select positive plants,and thei carry on analyses on Transgenic plants's agronomic characters of TO, which shows that pollei fertility and seed rates have reduced significantly.Compared with the control plants,the average seed set rate is down by 92.53%,the pollen fertility is down by 81.91%,which show tha interfering with gene expression of OsMND1 significantly reduces pollen fertility, suggesting tha the gene may be involved in the regulation of meiosis of the polyploid rice, but the specific regulatory mechanism needs a further experiment to verify.4.In this study, by optimizing the conditions of OsMNDl gene expression in E.coli and choosing the best protein purification condition, we success in preparation of high purity targe protein, which has laid a good foundation for later experiments.We can investigate the gene in the temporal and spatial expression during pollen development through the immune experiment. Bu this part is not fully completed because of the time. |
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