Cloning Of Rice OsI2 Gene And Its Prelimary Function Study

Drought stress is a limiting factor that affects crop growth and development. It is theoretically and practically important to study drought resistance of rice for the purpose of developing water-saving agriculture. In this study, drought stressed seedlings and booting plants of rice cv. IRAT109 have been used as experiment materials and the Affymetrix 57K cDNA array has been applied to study the expression profile of rice under drought stess. As a result, the second most up-regulated gene in the chip results has been cloned and named as OsI2. The preliminary function of OSI2 gene has been explored through genetic transformation into tobacco and expression analysis in rice plant. To improve the efficiency of tissue culture in genetic transformation, plant growth regulator Picloram has been used to replace 2,4-D to explore its effect on callus growth. The overall results have been summarized as followings:1. Full length cDNA of the second most up-regulated gene (named OSI2) was cloned by using RT-PCR and RACE methods from total RNA of drought-stressed rice IRAT109, and the gene sequence homology and the ORF was analyzed. The sequence of gene OSI2 showed 99% homology with a cDNA CT836140.1 conserved in the full-length cDNA library. Its putative translation product corresponds to an unknown function protein. Southern blotting showed that OSI2 gene exists in rice genome as a single copy gene.2. Overexpression vector in plant was constructed by fusing OSI2 gene sequence with promoter CaMV35 in plant expression vector pBI121. And through freeze-thaw method, the recombinant plasmid was successfully transformed into the Agrobacterium tumefaciens strain LBA4404; Agrobacterium tumefaciens-mediated transform tobacco, Gained transgenic plants; PCR and Southern blotting experiments proved that OSI2 gene has been integrated into tobacco genome.3. Rice seedlings had been sampled after drought stress traeatments for 0 min, 30 min, 3 h, 8 h, 20 h and total RNA were extracted from the leaves and roots respectively. Quantitative PCR analysis showed that the expression levels of OSI2 gene under stresses were all significantly higher than that with no stress in both leaves and roots. Expression in leaves was much higher than in roots. Both the cDNA microarray result (up-regulated fold change of OSI2 gene was 8.9) and quantitative PCR analysis indicated that OSI2 gene is related to drought stress. This study provided preliminary experiment basis for further study of OSI2 gene function. 4. By subsitituting Picloram for 2, 4-D in callus induction and callus maintenance culture of five rice cultivars namely Zhonghua 11, Nipponbare, Zhenshan 97B, Mianhui 725, Zhong413, it was found that Picloram was better than 2, 4-D in callus induction from mature seeds. The number of induced embryogenic callus was significantly increased, and the shoot differentiation rate and survival rates were increased also. The result showed that the application of picloram in callus induction and maintenance culture provided an alternative method to improve regenereation rate of rice especially for Oryza sativa indica subspecies.