48 10 2 0 1 2 10 SCIENTIA SILVAE SINICAE Vol. 48 No. 10 Oct. 2 0 1 2 BoBZF 100102 RT-PCR RACE 1 BoBZF GenBank EU606025 BoBZF cdna 1 076 bp 256 2 B-Box B-Box BoBZF BoBZF CaMV 35S pbi121 RT-PCR BoBZF BoBZF S718. 46 A 1001-7488 2012 10-0049 - 06 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 100102 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. EU606025. The full length cdna of BoBZF is 1 076 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 /2 2008 4 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 2011-08 - 05 2012-08 - 15 201004005
50 48 multiplex var. multiplex 10 μmol L - 1 1 μl 10 μmol Sasa glabra f. alba-striata L - 1 1 μl dntp 2. 5 mmol 3 μl 2 Buffer 10 Bambusa multiplex 2010 94 1 min 61 1 min 72 1 min35 2011 52 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 2003 5' cdna BoZ5-1 BoZ3-1 UPM 1 PCR 94 30 s 72 3 min 5 94 30 s 70 30 s 72 3 min 5 94 30 s 68 30 s 72 3 min 25 Arabidopsis 1 PCR BoZ5-2 thaliana BoZ3-2 NUP PCR 1 1. 1 2 NCBI 15 cm 1. 4 7 3 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' FP100973 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 0. 04 μg L - 1 1 μl μl Taq 5 U μl - 1 0. 2 μl 20 Esherichia coli 94 30 s 68 30 s 72 50 s 25 PCR DNASTAR5. 01 SMART cdna BoBZF Actin 2007 PCR 1 min28 72 10 min Xba Ⅰ BoZ-F1 5'-tctagaAT 1 min 62 1 min 72 1 min 35 72
10 BoBZF 51 10 min A pgem-t easy 1 B-Box DH5α Slack et al.1998 BoBZF pbi121 1. 6 BoBZF EHA105 2005 50 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-6000 2006 2. 3 7 BoBZF 2 3 2. 1 AZF2 STZ BoZ-F BoZ-R PCR Sakamoto et al.2004 1% EB 2. 4 BoBZF 750 bp 1 BoBZF BoZ-F1 BoZ-R1 PCR 61. 8 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 1 1 076 bp cdna 2. 2 GenBank EU606025 NCBI blastx BoBZF PeBZ1 ACC85612 89. 1% BoBZF Zea Cucumis sativus Glycine max Xba Ⅰ tctaga Sac Ⅰ gagctc 12 4 2. 5 BoBZF DNASTAR cdna T0 5' 156 bp 3' 15 T1 149 bp 771 bp 256 T3 3 4. 875 27 099. 71 RT-PCR BoBZF 3 u G + C 69. 13% Gramineae 5 Campbell et al.1990 60 2 B-Box 6 CK 3 4 47 53 100 6A B C CHC3H2 2 5 15 ~ 20
52 48 1 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 1 000 Numbers on major branches indicated bootstrap estimates for 1 000 replicates.
10 BoBZF 53 3 BoBZF RT-PCR Fig. 3 RT-PCR analysis of BoBZF gene 1 Leaf 2 Sheath 3 Stem 4 Root. PEG 2006 30% PEG-6000 MS 10 7 7A 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.1995 1996 1998 AZF2 STZ ABA AZF2 STZ Sakamoto et al.2004 B-Box BoBZF AZF2 STZ BoBZF BoBZF Song et al.1998
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