Functional Analysis Of A Rice B-Box Zinc Finger Protein OsBBX25and The Molecular Identification Of Double T-DNA Vector Transformed Rice Plants

Zinc finger is one of the most common transcription factor, and expresses widely in eukaryotes. It plays an important role in regulating cell development and cell apoptosis. According to the researches, some zinc finger proteins can regulate the stress tolerance of plant. Numbers of zinc finger proteins are devided into several subfamilies by their different functions. B-box zinc finger contains one or more B-box domain which consists of cysteines and histidines. B-box enhances its specific tertiary structure stability by combining with zinc iron, which suggests that B-box may take part in the interaction between proteins. There are32B-BOX zinc finger family members in Arabidopsis thaliana, and most of them regulate photomorphism. But some are responsed to salt tolerance. However, up to now there are few reports in rice. So in this report we cloned a B-box gene from rice and named as OsBBX25.OsBBX25is a B-box zinc finger domain rice gene from low temperature chip, which has a B-box domain at N terminal. Arabidopsis thaliana Salt Tolerance Homologous1(STH1) is the nearest homolog of OsBBX25, so we suggest that maybe OsBBX25is response to aboitic stress. The results showed that OsBBX25was induced by dought, salt, ABA and cold by real-time PCR analysis. To further analyze the function of OsBBX25, we transformed the gene into Arabidopsis thaliana. When treated with drought and salt, the overexpression lines survived more than the wide type. OsBBX25can be proved to reduce the content of Reactive Oxygen Species (ROS) in overexpression lines by testing the Proline, MDA and H2O2contents. Under salt and drought stresses, OsBBX25can regulate the expression of stress related marker genes, such as KIN1, RD29A, RD22and COR15. So we infer that OsBBX25may be a cotranscript regulator to improve the stresses tolerance by interacting with other transcription factors to regulate the expression of related genes.In the previous work of our lab, we have cloned six cold stress genes. They are OsMYB, OsRAN2, OsUGE, OsMYB, OsGPCR, OsCION and OsbHLH2. We combined these cold stress genes to the pCDMAR vector respectively, such as CDMAR-OsRAN2, CDMAR-OsGPCR (with MYB and GPCR promoter), CDMAR-OsMYB, CDMAR-OsCION, CDMAR-OsUGE, CDMAR-OsbHLH2. We transformed these constructs into rice (Dongdao) and screened the transgenic seedlings with hygromycin. Then we identified the seedlings whether it has the cold stress gene or not by RT-PCR. The purpose is to find the seedlings with the cold stress gene while without the hygromycin.