SCIENTIA SILVAE SINICAE. BoBZF RT-PCR RACE BoBZF RT-PCR

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1 SCIENTIA SILVAE SINICAE Vol. 48 No. 10 Oct BoBZF RT-PCR RACE 1 BoBZF GenBank EU BoBZF cdna bp B-Box B-Box BoBZF BoBZF CaMV 35S pbi121 RT-PCR BoBZF BoBZF S A Cloning and Primary Functional Analysis of a Zinc Finger Protein Gene of BoBZF from Bambusa oldhamii Gao Zhimin Liu Qing Mu Shaohua Li Xueping Hu Tao Key Laboratory on the Science and Technology of Bamboo and Rattan International Center for Bamboo and Rattan Beijing Abstract Zinc finger proteins play important roles in regulating gene expression in both prokaryote and eukaryotice. A zinc finger protein gene was isolated from leaves of Bambusa oldhamii with RT-PCR and RACE methodsand designed as BoBZF GenBank No. EU The full length cdna of BoBZF is bp containing an open reading frame which encodes 256 amino acids. The protein encoded by BoBZF has two B-Box domainswhich indicates BoBZF belongs to B-Box zinc finger protein. Tissue specific expression showed that BoBZF expressed in leafsheathstem and rootwith the highest level in leaf. BoBZF gene was subcloned into the multiple cloning sites of pbi121 vector driven by 35S promoterand transferred into Arabidopsis thaliana. RT-PCR analysis showed that BoBZF was expressed in the Arabidopsis. The transgenic plant was more drought tolerance than controlindicating that BoBZF was related to the drought tolerance ability of the bamboo. Key words Bambusa oldhamii zinc finger protein gene tissue specific expression ectopic expression drought tolerance 1 / Pleioblastus kongosanensis f. aureostriatus > Sasa zinc finger protein fortunei > S. auricoma > S. argenteostriatus 2010 SOD 2005 Huai et al.2009 MDA 3 5 Bambusa multiplex var. riviereorum Bambusa

2 50 48 multiplex var. multiplex 10 μmol L μl 10 μmol Sasa glabra f. alba-striata L μl dntp 2. 5 mmol 3 μl 2 Buffer 10 Bambusa multiplex min 61 1 min 72 1 min PCR Promega pgem-t easy DNA Bambusa oldhamii DH5α RACE 5'-RACE BoZ5-1 5'-ACGGCGGAAAGAACGGCGACGACGGC AC-3' BoZ5-2 5'-TGGTGCTTGCTGGCGAGCTTGT TGGCGG-3' 3'-RACE BoZ3-1 5'-TTCGTGT N P TCTGCGTGGAGGACAGGGCG-3' BoZ3-2 5'-TGGG K Na TTCACGGAGCTGGAGTGGTTCGC-3' 3'cDNA ' cdna BoZ5-1 BoZ3-1 UPM 1 PCR s 72 3 min s s 72 3 min s s 72 3 min 25 Arabidopsis 1 PCR BoZ5-2 thaliana BoZ3-2 NUP PCR NCBI 15 cm BoZ-F BoZ-R RT-PCR 25 / = 16 h /8 h 1. 2 RNA cdna Invitrogen Trizol reagent 94 5 min 94 1 min 58 1 min 72 RNA Gao et al.2006 Promega cdna 1. 5 Clontech SMART TM 3'cDNA 5'cDNA 1. 3 RACE BoBZF pbi121 5' Phyllostachys edulis GGGGAGAGGGAGAGTTG-3' 5' FP cdna Sac Ⅰ BoZ-R1 5'-gagctcCAATAGTTGAA BoZ-F 5'-ATGAG TCTGTTCTTGC-3' GATCCAGTGCGACG-3' BoZ-R 5'-TCATCCAAG DNA ATCAGGAACGATGAAG-3' PCR 94 5 min 94 PCR cdna μg L μl μl Taq 5 U μl μl 20 Esherichia coli s s s 25 PCR DNASTAR5. 01 SMART cdna BoBZF Actin 2007 PCR 1 min min Xba Ⅰ BoZ-F1 5'-tctagaAT 1 min 62 1 min 72 1 min 35 72

3 10 BoBZF min A pgem-t easy 1 B-Box DH5α Slack et al.1998 BoBZF pbi BoBZF EHA mg L - 1 Oryza sativa BoBZF 81. 0% 49. 0% MEGA4 RNA cdna BoZ-F BoZ-R RT-PCR DNA mays cdna 5 cm 1 Solanum sogarandinum Brassica rapa 2 BoBZF MS 10 30% PEG BoBZF AZF2 STZ BoZ-F BoZ-R PCR Sakamoto et al % EB 2. 4 BoBZF 750 bp 1 BoBZF BoZ-F1 BoZ-R1 PCR pgem-t easy 780 bp BoBZF 768 bp 771 bp 1 PCR 2 PCR bp GUS pbi121 XbaⅠ SacⅠ 5' RACE3' RACE 284 bp BoBZF 367 bp BoBZF bp cdna 2. 2 GenBank EU NCBI blastx BoBZF PeBZ1 ACC % BoBZF Zea Cucumis sativus Glycine max Xba Ⅰ tctaga Sac Ⅰ gagctc BoBZF DNASTAR cdna T0 5' 156 bp 3' 15 T1 149 bp 771 bp 256 T RT-PCR BoBZF 3 u G + C % Gramineae 5 Campbell et al B-Box 6 CK A B C CHC3H ~ 20

4 Fig. 1 BoBZF Nucleotide sequence and deduced amino acid sequence of BoBZF B-box1 Single underline B-box2 Double underline. Fig. 2 2 B-box Phylogenetic tree based on the zinc finger proteins coded by B-box genes Numbers on major branches indicated bootstrap estimates for replicates.

5 10 BoBZF 53 3 BoBZF RT-PCR Fig. 3 RT-PCR analysis of BoBZF gene 1 Leaf 2 Sheath 3 Stem 4 Root. PEG % PEG-6000 MS A 7B BoBZF 4 BoBZF Fig. 4 Plant expression vector of BoBZF gene 5 BoBZF T3 RT-PCR Fig. 5 BoBZF transgenics checked by RT-PCR 1-3 Transgenics 4 Wild type control 5 Plasmid control 6 Water control. 6 BoBZF Fig. 6 Phenotype of BoBZF transgenics by water control A B C Transgenics CK Wild type control. 7 PEG BoBZF Fig. 7 Phenotype of BoBZF transgenics treated with PEG A Wild type control B Transgenics. 3 B-Box RNA B-Box RNaseⅠ5S RNA Borden et al AZF2 STZ ABA AZF2 STZ Sakamoto et al.2004 B-Box BoBZF AZF2 STZ BoBZF BoBZF Song et al.1998

6 BoBZF PEG BoBZF BoBZF 2009 Sci Gao ZhiminLi XuepingLi Lubinet al An effective method for total RNA isolation from bamboo. Chinese Forestry Science and Technology O - Huai JZheng JWang G Overexpression of a new Cys2 / His2 COMT zinc finger protein ZmZF1 from maize confers salt and drought tolerance in transgenic Arabidopsis. Plant CellTissue and Organ Culture Sakamoto HMaruyama KSakuma Yet al Arabidopsis Cys2 / His2-type zinc-finger proteins function as transcription repressors under droughtcoldand high-salinity stress conditions. Plant Physiology Slack F JRuvkun G A novel repeat domain that is often associated with RING finger and B-box motifs. Trends in Biochemical Sciences Song JYamanoto KShomura Aet al Isolation and mapping of Floral a family of putative zinc-finger protein cdnas from rice. DNA Dip Research β Na + / H Borden K LLally J MMartin S Ret al Novel topology of a zinc-binding domain from a protein involved in regulating early Xenopus development. EMBO Journal Borden K LLally J MMartin S Ret al In vivo and in vitro characterization of the B1 and B2 zinc-binding domains from the acute promyelocytic leukemia protooncoprotein PML. Proc Natl Acad Borden K L RING fingers and B-boxes zinc-binding proteinprotein interaction domains. Biochem Cell Biol76 2 / Campbell W HGowri G Codon usage in higher plantsgreen algae and cyanobacteria. Plant Physiology