The Expression Regulation Of OsRhoGDI2 Gene In Rice

Identification of fertility genes in rice has always been one of significant topics to which investigators working on genetics and breeding pay attention. Experiments have shown the Rho gene Os Rac D is associated with reproductive development in rice. The previous systemic study in our lab on the proteins that interacted with Os Rac D demonstrated the factor Os Rho GDI2 that inhibits Rho GDP separation interacted and had synergic expression with Os Rac D. Those indicated there was essential functional relationship between Os Rho GDI2 and Os Rac D in the process of rice growth and development.This study aims to explore the pattern of Os Rho GDI2 expression, identify the hormones and abiotic stress factors impacting the gene expreesion, and screen T1 generation rice that contains RNAi vector interfering the gene via identification of the function of it’s promotor, which will lay a solid foundation for further study on its role for growth, development and fertility control in rice.The effects six kinds of hormones IAA, 6-BA, GA, Me J-A, ABA and SA, and two kinds of abiotic stresses drought and high salinity had on Os Rho GDI2 expression within aboveground part of rice during seedling stage were analyzed by q RT-PCR. The reusults showed Os Rho GDI2 expression increased after treatment with IAA, ABA, Me J-A, 6-BA and SA by 5-fold, 9-fold, 7-fold, 5-fold and 9-fold with the peaks achored at 24 h, 8h, 4h, 8h, and 4h, respectively. GA inhibted Os Rho GDI2 expression demonstrated by decreasing to 30% after 8h-treatment with GA. Os Rho GDI2 expression also increased after treatment with drought and high salinity by 7-fold and 18-fold with the peaks achored at 8h and 12 h, respectively. Together, Os Rho GDI2 expression was induced by IAA, ABA, Me J-A, 6-BA, SA, drought and high salinity.The promoter comprised of 2266 bp was sucessfully constructed by PCR, and cis-elements within the promoter were predicted and analyzed by bioinformatics. We found that, besides primary elements, several kinds of reponse elements to hormones and adversity including ABA, GA, IAA, Me J-A, light and injury were contained within the promotor, which corresponded to the mentioned results by q RT-PCR from the foregoing.The expression vector p BI121-p Os Rho GDI2-GUSwas constructed by DNA recombination of Os Rho GDI2 gene promoter with GUS,and the transgenic plants were screened after transfromation by the vector. The expression pattern of GUS reporter gene was detected by q RT-PCR after treatments of transgenic rice with the hormones and abiotic stresses. The results showed that the expression of GUS gene increased after treatments with Me J-A, IAA and ABA with the maximum values found at the 8h, 4h, 24 h, and the expression levels up to 6 times, 8 times and 16 times respectively. The expression of the gene was not changed significantly after treatments with 6-BA or SA, and inhibited by GA with the greatest inhibition degree after 8h when the expression of the quantity was less than 20% of the control. These results indicated that the promoter of the Os Rho GDI2 gene contained the response elements to ABA, GA, Me J-A,IAA, drought and salt stress, but might not contain the key response elements to SA and 6-BA.The transgenic rice vegetative organs roots, stems, leaves, and reproductive organs pollen, glume and seedswere analyzed by GUS histochemical staining after the heading stage of rice. The results showed that a strong color reaction was found in the sheath, petiole and pollen, but undetectable in the leaf, glume and neck section. Further analysis of the distribution of GUS activity in leaf and petiole by freehand sections showed Gus blue are mainly located in the xylem of the epidermal and vascular bundle, not in the phloem and mesophyll cells. This suggests that the Os Rho GDI2 gene might be expressed in those tissues and regulated the growth and development of rice.The T1 generations of Os Rho GDI2 RNAi rice were screened by PCR, and the expression of the gene in 7 transgenic rice lines were determined by real time PCR. The results showed that the expression of the gene was down-regulated by 70%-20%.