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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) : 333-337     DOI: 10.1007/s11703-011-1094-2
RESEARCH ARTICLE |
Construction of RNAi vector for flower-related gene and verifications of the mutant in Arabidopsis thaliana
Jing ZHANG, Jihong XING(), Jingao DONG()
Molecular Plant Pathology Laboratory, Agricultural University of Hebei, Baoding 071001, China
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Abstract  

The timing of floral transition is tightly controlled by a combination of endogenous and environmental signals. One early flowering mutant plant was screened from Arabidopsis library of T-DNA insertion to accelerate flowering under short-day condition, and a related-gene EFS1 (AT4G36680.1) was isolated and identified as a novel flowering-time gene of Arabidopsis in our preliminary studies. To investigate the function and the specific mechanism of EFS1 in the flower process control, the RNAi expression vector containing EFS1 gene-specific sequences in the sense and antisense orientations was constructed and transferred into Arabidopsis by using the floral-dip method, with 11 transgenic plants obtained through hygromycin B screening and PCR assays. The results showed that the expression level of EFS1 in transgenic lines was significantly lower than that in wild type and efs1 mutant. The flowering time of the efs1 mutant and RNAi transgenic plants was much earlier than that of wild-type plants. This result further verified that the EFS1 gene played an important role in flowering, and its specific mechanisms need further study. These work provided a foundation to further regulatory mechanisms of EFS1 in the control of floral transition.

Keywords Arabidopsis thaliana      RNAi      EFS1 gene      early flowering     
Corresponding Authors: XING Jihong,Email:xingjihong2000@126.com; DONG Jingao,Email:shmdjg@hebau.edu.cn   
Issue Date: 05 September 2011
URL:  
http://academic.hep.com.cn/fag/EN/10.1007/s11703-011-1094-2     OR     http://academic.hep.com.cn/fag/EN/Y2011/V5/I3/333
CategoryPrimersPrimer sequence (5?–3?)
RNAi specific primersAT4L+5?-CGCTCTAGAATAATGAGTGCTCAAAAGGAA-3?
AT4R+5?-TAAGGATCCCCAACAACAAACCGTCAA-3?
AT4L-5?-TGTGAGCTCATAATGAGTGCTCAAAAGGAA-3?
AT4R-5?-TAGGGATCCAGCCCGAGTTCCTCCTC-3?
M13 primers pMD-19 vector for PCRM13F5?-CGCCAGGGTTTTCCCAGTCACGAC-3?
M13R5?-AGCGGATAACAATTTCACACAGGA-3?
Specific primers of EFS gene for PCREFSF5′-TACGACCACCGCTCCCT-3?
EFSR5′-CTCAAAATGGTGGTGAAAGCA-3′
Hyg primersHYGF5′-AAGCCTGAACTCACCGCGAC-3′
HYGR5′-CTATTTCTTTGCCCTCGGAC-3′
RT-PCREFS-15?-ATAATGAGTGCTCAAAAGGAA-3?
EFS-25?-CCAACAACAAACCGTCAA-3?
18S-15?-GTTGCAGTTAAAAAGCTCGT-3?
18S-25?-TTGATTTCTCATAAGGTGCC-3?
Tab.1  Primers used for specific PCR and RT-PCR
Fig.1  Illustration for construction of RNAi plant expression vector of gene.
Fig.2  Construction of RNAi expression vector of .
Note: A is PCR identification sense and antisense fragments of genes. B is restriction enzyme digestion analysis of T19-F and T19-R. C and D are restriction enzyme digestion analysis of RNAi expression vector of .
Fig.3  Phenotype of transgenic plants on Petri dish.
Note: Arrows represent transgenic plants.
Fig.4  PCR identification of transgenic line.
Note: M is DL2000 DNA Marker. 1 and 2 represent RNAi transformant and Col-0.
Fig.5  The expression of was examined by RT-PCR analysis.
Note: 1-3 represent Col-0, RNAi plants and mutant, respectively.
Fig.6  Flowering times of Col-0, mutants and RNAi plant under SD.
Note: 1-3 represent Col-0, mutant and RNAi plants, respectively.
Fig.7  Flowering time of the wild-type (Col-0) and the mutant () (RNAi)
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