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Structural Analysis and Identification of Cis-Elements of Rice osRACD Gene

Acta Biochimica et Biophysica Sinica 2004年第3期36卷 191-198页

作者： Yun-Zhe XU, Rui-Lin YOU and Nai-Hu WU Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080, China 2College of Life Sciences, Peking University, Beijing 100871, China Institute of Genetics and Developmental Biology Chinese Academy of Sciences College of Life Sciences Peking University

The osRACD gene correlated with fertility transformation in the photoperiod sensitive genic male sterile rice (PGMR), Nongken 58S, encoded a rice (Oryza sativa L. ssp. japonica) small GTPase belonging to the Rac/Rho family. Inverse PCR was performed to amplify a fragment about 1.4 kb in 5' upstream region of the osRACD promoter. Deletion mutation and gel mobility shift assay characterized two fragments (–799 to –686 nt, and –686 to –431 nt) in the osRACD promoter that could be involved in its transcriptional regulation. When these two deletion fragments were used as probe respectively, a retarded band appeared in the nuclear extracts of fertile 58S rice under short day (58S-SD). Whereas no retarded band was shown in the nuclear extracts of sterile 58S rice under long day (58S-LD). Competition assay indicated that the factors in the retarded bands binding to these two fragments were the same trans-acting factor (termed rice factor, RF). The binding affinity of RF was affected by phosphorylation and was higher in SD-growth rice than that of LD-growth rice.

关键词： photoperiod-sensitive male genic sterile rice osRACD gene photoperiod fertility transformation cis-element

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Biochemical characterization of the protein encoded by rice osRACD gene

引用

Progress in Natural Science:Materials International 2004年第4期 46-51页

作者： XU Yunzhe, MI Zhiyong, QU Hong, YOU Ruilin and WU Naihu (1. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080, China 2. College of Life Sciences, Peking University, Beijing 100871, China) InstituteofGeneticsandDevelopmentalBiology ChineseAcademyofSciencesBeijing100080China CollegeofLifeSciences PekingUniversityBeijing100871China

A recombinant plasmid pE,T-racd was first constructed by cloning osRACD, the development-regulating gene that controls photoperiod fertility transformation in the photoperiod sensitive genie male sterile rice Nongken 58S, into a prokaryote expression vector pET28a( + ). It was then transformed into E. coli BL-21. Cutting with thrombin of the fusion protein extracted from transformants and PAGE separation yielded pure osRACD protein, which was further concentrated using ultrafiltration and renatured using glutathione oxidation/reduction refolding system for later functional study. As demonstrated by in vitro functional assay, the osRACD protein expressed in E . coli B-21 shows remarkable activity in binding GTP specifically and hydrolyzing it.

关键词： rice (Oryza saliva L.) expression of osRACD gene photoperiod fertility transformation small molecular weight GTP-binding protein.

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