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Preliminary Identification of the Function of OsRHOGDI2 Gene as a Rice Rho GDP Dissociation Inhibitor

Author: PengWeiFeng
Tutor: LiangWeiHong
School: Henan Normal
Course: Cell Biology
Keywords: rice OsRhoGDI2gene subcellular localization transgene rice fertility
CLC: S511
Type: Master's thesis
Year: 2011
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Abstract


OsRacD belonging to the rice Rho family of small GTP-binding protein, previous results showed one of its function involved in rice photoperiod fertility conversion of photoperiod sensitive genic male sterile rice Nongken 58S, which influences rice fertility via controlling the pollen tube growth. To further investigate OsRacD-related signaling pathway and partners, using OsRacD as bait, the rice panicle cDNA library were screened by yeast two-hybrid system, and some cDNA sequences encoding putative targets were obtained. In this study, the functions of the gene OsRhoGDI2, a putative partner of OsRacD, which encoding a GDP dissociation inhibitor in rice fertility control had been identified by transgenic methods.Using bioinformatics analysis, the physicochemical characteristics, modification site and subcellular localization of OsRhoGDI2 and OsRacD protein had been predicted and analyzed. The GFP-fused binary plant expression vector pCAMBIA1302-OsRhoGDI2-GFP which controlled by the CaMV35S promoter was constructed. By Agrobacterium-mediated method, the recombinant vector pCAMBIA1302-OsRhoGDI2-GFP was transformed into onion epidermal cells, the subcellular localization of the fusion protein were observed under the fluorescence microscopy. The results showed that OsRhoGDI2 and OsRacD shared some similarities in physical and chemical properties, post-translational modification sites, and OsRhoGDI2 mainly distributed in the cytoplasm, nucleus and cell membrane in onion epidermal cells, suggest that OsRhoGDI2 may be an important partner of OsRacD which regulated its subcellular localization and activities.In order to identify the function of OsRhoGDI2 gene, the binary plant expression vector pCAMBIA1302-OsRhoGDI2-GFP was transformed into rice callus by Agrobacterium-mediated method, after infection, screening and re-differentiation, twelve transgenic rice were obtained, and seven of them were further verified by PCR amplification. RT-PCR detection showed the expression level of the gene OsRhoGDI2 were higher than that of control, suggest that the gene over-expressed in transgenic rice. The phenotype analysis showed that the tiller numbers and total rice grain numbers were similar between transgenic rice and control, but the pollination grain number and maturing rate of transgenic rice were decreased significantly compared with that of control. The statistic analysis data showed the pollination grain number and maturing rate of the transformation rice was less than 10%, while the control was up to 96%, suggested that function of OsRhoGDI2 may related with rice fertility. In order the study the relationships between OsRhoGDI2 and OsRacD in rice fertility control, the RNA interference binary vector pART27-OsRhoGDI2 was constructed, help the further study on the effects of gene silencing of OsRhoGDI2 and OsRhoGDI2-OsRacD interaction in the growth and development of rice panicle, which participated in the process of rice fertility regulation.

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