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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front Agric Chin    2011, Vol. 5 Issue (3) : 322-327     DOI: 10.1007/s11703-011-1084-4
RESEARCH ARTICLE |
Identification of a gene responsible for the 60-day delay in flowering time of Arabidopsis
Jihong XING1, Ye ZHANG1, Jing ZHANG1, Qiaoyun WENG2, Jiao JIA1, Jingao DONG1()
1. Molecular Plant Pathology Lab, Agricultural University of Hebei, Baoding 071001, China; 2. Department of Agricultural and Forest Technology, Hebei North University, Zhangjiakou 075131, China
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Abstract  

Identification of genes related to flowering-time in Arabidopsis is very important and meaningful contribution to the flowering process control. One late flowering mutant plant, which exhibits 60-day delay in flowering, was screened from Arabidopsis library of T-DNA insertion. Southern blotting was used to confirm the single copy of exogenetic T-DNA in the genome of the mutant. The flanking sequence of T-DNA insert was obtained by TAIL-PCR and then analyzed by BLAST to confirm that the insertion site locates at the sixth exon of AT2G19520.1 (FVE gene). FVE is considered as a classical flowering time gene in Arabidopsis. It is a component of the autonomous pathway that encodes AtMSI4, which is a putative retinoblastoma-associated protein. The late-flowering mutant is named as fve-4, which is similar to fve-3 of Columbia and allelic with fve-1 and fve-2 of Landsberg erecta. The fve-4 mutant’s delay of flowering was longer than that of fve-3 mutant, whose T-DNA insertion is located at the first exon of FVE gene, suggesting that the sixth exon of FVE gene may play a more important role in the control of floral transition.

Keywords delay of flowering      FVE gene      Arabidopsis      gene identification     
Corresponding Authors: DONG Jingao,Email:shmdjg@hebau.edu.cn   
Issue Date: 05 September 2011
URL:  
http://academic.hep.com.cn/fag/EN/10.1007/s11703-011-1084-4     OR     http://academic.hep.com.cn/fag/EN/Y2011/V5/I3/322
PurposePrimersPrimer sequence
Probe preparationN15?-TCGGCTATGACTGGGCACAACAGA-3?
N25?-AAGAAGGCGATAGAAGGCGATGCG-3?
TAIL-PCRLexA25?-CTAATCGCATTATCATCCCCTCG-3?
LexA45?-CTGGTTTTATATACAGCAGTCGACG-3?
LexA55?-AGTCGAGGTAAGATTAGATATGG-3?
AD15?-(AGCT)TCGA(G/C)T(A/T)T(G/C)G(A/T)GTT-3?
PCR identificationM13F5?-CGCCAGGGTTTTCCCAGTCACGAC-3?
M13R5?-AGCGGATAACAATTTCACACAGGA-3?
Cloning of FVEFVEL5?-GAAGAGAGAGAGATATAG-3?
FVER5?-GCACAGAGAAGGAATCATTAGG-3?
RT-PCRFVE-15?-GCTCGGTTTGGTAACAAG-3?
FVE-25?-GTTTCCTCGCTGTAAATC-3?
18S-15?-GTTGCAGTTAAAAAGCTCGT-3?
18S-25?-TTGATTTCTCATAAGGTGCC-3?
Tab.1  Primers used for probe of Southern blotting, TAIL-PCR, PCR, and RT-PCR
PlantNo. of rosette leafDays of flowering
Mutant 221-150±0.3 A86±0.3 A
Col-0 (wild type)12±0.2 B27±0.2 B
Tab.2  The numbers of rosette leaf and flowering days under long-day condition
Fig.1  Phenotype of the wild type (Col-0) and the mutant. A is the mutants screened flower later than Col-0 plants in long days; B is the plant of Col-0 (30 days after sowing); C is the plant of the mutant (30 days after sowing); D is the plant of the mutant (90 days after sowing).
Fig.2  Clone of late flowering-related gene. M: DL2000; 1: the second gene amplification; 2, 3: the third gene amplifications.
Fig.3  Structure of mutant and mutations of the alleles. Boxes denote exons, and lines denote introns.
Fig.4  Conserved domain analysis of FVE.
Fig.5  DNA blot hybridization analysis of the mutant.
Fig.6  The result of PCR of gene. 1: the mutant; 2: wide type; M: Wide Range DNA Marker (100-6000 bp).
Fig.7  The expression of was examined by RT-PCR analysis.
Fig.8  The genetic pathways of flowering in . Positive (arrows) and negative (T-lines) interactions are described. Dotted lines show undescribed interaction (Komeda, 2004).
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