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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 (1) : 40-44     DOI: 10.1007/s11703-010-1058-y
RESEARCH ARTICLE |
Construction and transformation for the antisense expression vector of the polyphenol oxidase gene in Yali pear
Guiqin LI1, Jing QI1, Yuxing ZHANG2(), Zhihua GAO1, Dongqian XU1, Huixuan LI1, Chenmin HUO1
1. College of Biology Science and Engineering, Hebei University of Economics and Business, Shijiazhuang 050061, China; 2. College of Horticulture, Agricultural University of Hebei, Baoding 071001, China
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Abstract  

To inhibit the browning process in fruits of Yali pear, in this paper, antisense gene techniques were used to reduce the expression of BoldItalic gene. A cDNA fragment of 450 bp, which is located at the 3′ terminal of the polyphenol oxidase (BoldItalic) gene, was amplified from Yali pear using the RT-PCR method, then the antisense expression vector was constructed by inserting the fragment of the Yali pear BoldItalic gene between the CaMV promoter and NOS terminator of the expression vector pBI121 in a reverse orientation. After that, with the agrobacterium-mediated method, the BoldItalic antisense gene was transformed into Yali pear shoots. Northern blot analysis and enzyme activity assay showed that the PPO activities in the transgenic Yali pear shoots were significantly decreased, compared with the non-transformed Yali pear shoots. This lays a good foundation for breeding new varieties of pears with browning resistance in the future.

Keywords Yali pear      polyphenol oxidase      antisense expression vector      genetic transformation     
Corresponding Authors: ZHANG Yuxing,Email:jonsonzhyx@yahoo.com.cn   
Issue Date: 05 March 2011
URL:  
http://academic.hep.com.cn/fag/EN/10.1007/s11703-010-1058-y     OR     http://academic.hep.com.cn/fag/EN/Y2011/V5/I1/40
Fig.1  Diagram of antisense express vector of pBI121-AsPPO
Fig.2  Generation of adventitious buds from the calli
Fig.3  PCR analysis for gene leaves
Note: Lane M, Marker of DNA ladder; lane 1, non-transformed Yali pear control; lanes 2, plasmid pBI121-AsPPO; lane 3-6, kanamycin-resistant lines No. 1-4.
Fig.4  RNA blot analysis of gene in transgenic shoots and non-transformed shoots
Note: The blot was hybridized with a DIG-labeled PPO probe. Lane A, transgenic shoots; lane B, non-transformed control. The equivalence of RNA was demonstrated by ethidium bromide staining of RNA on the gel.
Fig.5  Analysis of PPO activity in the control and transgenic plants
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