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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front Agric Chin    2009, Vol. 3 Issue (3) : 259-265     DOI: 10.1007/s11703-009-0021-2
RESEARCH ARTICLE |
Improvement of organic phosphate acquisition in transgenic tobacco plants by overexpression of a soybean phytase gene Sphy1
Li GUO1, Yuxin ZHAO1, Shuhua ZHANG2, Haina ZHANG1, Kai XIAO1()
1. College of Agronomy, Agricultural University of Hebei, Baoding 071001, China; 2. College of Life Science, Agricultural University of Hebei, Baoding 071001, China
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Abstract  

Due to the huge amount in the soil, phytate is an important potential source for providing the plants with available phosphorus (Pi) by the involved catalytic reaction of phytase. In this study, a construct fusing the open reading frame (ORF) of Sphy1 into corresponding positions in the fragment of binary expression vector pBI121 was created and used to transform tobacco. Molecular identification by PCR and RT-PCR indicated the target gene Sphy1 in the transgenic tobacco plants was transcribed under the regulation of an upstream promoter. Compared with the control plants, the phytase activities in all the transgenic plants were increased, with the increased range consistent with the expression levels in the transgenic plants. Under the growth conditions with phytate as the sole phosphorus source, the transgenic line 1 plants displayed a high expression level of Sphy1 and shows notable improved growth performance, such as higher fresh weight and dry weight, as well as higher total P content and more accumulative P amount per plant than CK. This clearly indicated that overexpression of Sphy1 could improve the phosphorus acquisition by the extruded Sphy1 phytase in the rhizosphere, where this enzyme could catalyze the degradation of the phytate and release the available Pi for plants. The Sphy1 gene seemed to have a potential value in the creation of new crop cultivars with high phosphorus use efficiency.

Keywords Sphy1 gene      overexpression      phosphorus utilization      transgenic tobacco     
Corresponding Authors: XIAO Kai,Email:xiaokai@hebau.edu.cn   
Issue Date: 05 September 2009
URL:  
http://academic.hep.com.cn/fag/EN/10.1007/s11703-009-0021-2     OR     http://academic.hep.com.cn/fag/EN/Y2009/V3/I3/259
Fig.1  The diagram of binary expression vector fusing the open reading frame (ORF) of
Fig.2  PCR amplification of open reading frame (ORF) of (a), double restricted digestion of recombinants (b) and pBI121 vector plasmid (c), isolation of the target fragments (d), double restricted digestion of pBI121-Sphy1 (e), and PCR identification of the construct (f)
Note: The arrows triangles represent the ORF fragment of and target pBI121 large segment, respectively.
Fig.3  The regeneration process of transgenic tobacco plants which were fused
Note: In (a), the calli around the explant were appeared one week after infection. In (b), the buds were differentiated from the calli one month after the infection. In (c), the young seedlings were generated after 15 d growth in rooting growth medium.
Fig.4  The extracted genome DNA (a), PCR amplification of ORF (b), isolated total RNA (c) and RT-PCR results (d) in tobacco plants transformed by and empty binary vector PBI121
Fig.5  The phytase activities in tobacco plants transformed by and empty binary vector pBI121
Fig.6  The plant fresh weight (a), dry weight (b), total P content and accumulated P amount in tobacco plants transformed by and empty binary vector PBI121
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