| In recent years, great progress and even breakthrough in rice resistance to biological and non-biological research have been made,specially, It revealed the composition of the encoded protein, topology and subcellular localization characteristics by cloning the resistance genes and sequence analysis, which provided a clue to open the role of the characteristics of resistance genes. In order to adapt to water stress changes, some water stress-related genes would be Inducible expression.The objective of this research is try to study the function of OsI2 gene (the second Up-regulated expression by the chip results from the cDNA of drought-stressed rice IRAT109), which based on cloning of OsI2gene. To study its expression in plants of OsI2 gene is the focus of the study, which transformed OsI2 gene into the model plant such as tobacco,arabidopsis and rice. The overall results have been summarized as followings:1: The dicotyledonous expression vector pBI121- OsI2 was constructed for transforming Arabidopsis and tobacco through Agrobacterium-mediated genetic transformation method. As a result, 12 genetic transformant tobacco T1 plants with kanamycin-resistance and 15 kanamycin-resistant Arabidopsis T1 plants have been obtained. PCR analysis of OsI2 gene confirmed the fact of successful genetic transformation in these two kinds of model plants. Southern blot analysis in the T1 plants further verified that the OsI2 has been integrated into the genomes of tobacco and Arabidopsis by multi-copy integration of the target gene. Semi-quantitative PCR showed that there were seven tobacco plants expressing the target gene. Salt (NaCl) and anti-mannitol, as well as drought-resistant analysis showed that the salt-tolerant and drought-resistant capacity of transgenic plants was significantly higher than that of wild-type, but lower resistance capacity to mannitol was obaserved in OsI2 gene confirmed plants compared with non-transformed wild type plants. The over-expression of OsI2 gene may lead to the excess sensitivity of mannitol in transgenic plants.2: The over-expression vector pC1300- OsI2 and antisense expression vector pC1300- OsI2 (-) were constructed for transformation of rice which based on the pCAMBIA1300 vector. It obtained 12 over-expression transgenic plants with multiple OsI2 gene insertions. Results showed that 9 transgenic plants were over-expressed by qRT-PCR experiment. 3: The construction of fusion expression vector pBI121- OsI2-GUS was based on the pBI121 vector, and the construction of a GFP reporter vector pC1323- OsI2-GFP was based on the pCAMBIA1323 vector for studying subcellular localization of the target OsI2 gene. It was deduced that the OsI2gene was mainly expressed in roots through GUS-positive staining from transformed Arabidopsis plants.4: The OsI2gene expression analysis of upland rice IRAT109 treated by drought stress by Real-time PCR showed that the expression was markedly increased with the deeper drought stress. In details, we measured the relative expression amount of OsI2 gene after 10d, 12d, 14d drought stress in tillering stage and 5d,7d,9d drought stress in booting stage of IRAT109 and results showed the increased expression after drought stress..5: The results of stomatal investigate by the high-power microscope showed that the stomatal number of transgenic plants was significantly higher than the number of wild-type plants. In the dry process, the transgenic plants with lower critical relative water content have the lower rate to lose water than the wild type.6: The sequence alignment analysis of OsI2 gene in 34 wild rice and cultivated rice materials showed that the genetic evolution was a more conservative. There were nine bases insertioned in the African cultivated rice 10037 and wild rice 10069, S3037, 105888.Which leading to the three amino acids insertion in the frame ORF coding sequences. The cluster analysis of nucleosides showed that IRat109 and 9311, zh11, Nipponbare, 102338, Jinghong, S02001and DX23 had close genetic distance. |
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