Molecular studies of transcriptional regulation of the rolC gene in higher plants

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書誌事項

タイトル
Molecular studies of transcriptional regulation of the rolC gene in higher plants
著者
松木, 吏弓
著者別名
  • Matsuki, Rikyu
学位授与大学
北海道大学
取得学位
博士(理学)
学位授与番号
甲第3307号
学位授与年月日
1994-03-25

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説明

Transgenic rice plants were obtained from protoplasts treated with two plasmids by electroporation． Primary transformants were selected on the basis of resistance to hygromycin， which was conferred by one of the co-transferred plasmids． Two plants out of 26 hygromycin resistant plants showed the reporter gene activity due to another plasmid possessing a chimeric gene consisting of a promoter region （about 900 bp upstream non-coding strand） of ORF12 gene （rolC） of Ri plasmid and the coding region for β-glucuronidase （GUS）． GUS activity was strong in leaves and roots, but weak in callus． Colorimetric reaction using the GUS enzyme always showed the localization of the gene expression to vascular tissues only． Furthermore， transgenic rice plants possessing the rolC gene were obtained． Their progeny plants showed the reduction of plant height． Gel shift analysis revealed that sequence specific binding factors interacting with the 5’-upstream region of the rolC gene exist in nuclear extracts from both leaves and calli． The binding site visualized by DNase Ⅰ footprinting experiments possesses AT-1 boxlike－sequence ”ATATTTTTAT”， located from -76 bp to -67 bp． ln a further upstream region， an AT-rich region from -203 bp to -164 bp that was protected by DNase I digestion was seen using leaf nuclear proteins， but not with callus nuclear proteins． A novel DNA binding protein RCS2 （rolC single stranded DNA binding protein 2） was identified in the nuclear extract of tobacco seedlings that interacted with the region from -136 to -111 nt of the rolC gene promoter． DNA－protein gel shift and competition assays demonstrated that RCS2 bound to single－stranded DNA in a sequence－specific manner． A five－base direct repeat （GCATC） wasshown to be important for the DNA binding of RCS2． South－Western blot analysis suggested that the size of RCS2 is approximately 43 kDa. In the rolC promoter， three G－box like elements are found at -136， -219， and -364 positions． Parsley CPRF2 and CPRF3， which belong to G－box binding factor （GBF） group， interacted with these Gbox like elements． Rice cDNA clone （RGBFI： rice GBFI） was isolated from root cDNA library by DNA hybridization using CPRF3 cDNA as a probe． RGBFI contains a proline－rich region at its N－terminus and a bZIP （basic region－leucine zipper） motif at its C－terminus． A high degree of conservation was detected between the rice cDNA and GBF group proteins in both regions of the bZIP domain and N-terminal acidic domain．

目次

Contents

Summary

General Introduction

Part I.Analysis of the rolC gene expression in transgenic rice plants

Introduction

Materials and Methods

Results and Discussion

Tables

Figures

Part II.Interaction of rice nuclear proteins with 5'-upstream region of the rolC gene

Results

Discussion

Part III.Detection of the sequence specific single-stranded DNA binding protein

Part IV.Isolation of rice G-box binding factor

General Conclusion

Figure

Acknowledgments

References

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