Construction and transformation for the antisense expression vector of the polyphenol oxidase gene in Yali pear

doi:10.1007/s11703-010-1058-y

Front Agric Chin

2011, Vol. 5

Issue (1) : 40-44 https://doi.org/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

Download: PDF(162 KB) HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks

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 Author(s): ZHANG Yuxing,Email:jonsonzhyx@yahoo.com.cn

Issue Date: 05 March 2011

Cite this article:

Zhihua GAO,Dongqian XU,Huixuan LI, et al. Construction and transformation for the antisense expression vector of the polyphenol oxidase gene in Yali pear[J]. Front Agric Chin, 2011, 5(1): 40-44.

URL:

https://academic.hep.com.cn/fag/EN/10.1007/s11703-010-1058-y
https://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

1	Bachem C W B, Speckmann G J, van der Linde P C G, Verheggen F T M, Hunt M D, Steffens J C, Zabeau M (1994). Antisense expression of polyphenol oxidase genes inhibits enzymatic browning in potato tubers. Nat Biotechnol , 12(11): 1101–1105 doi: 10.1038/nbt1194-1101
2	Cary J W, Lax A R, Flurkey W H (1992). Cloning and characterization of cDNAs coding for Vicia faba polyphenol oxidase. Plant Mo1 Biol , 20: 245–253
3	Chevalier T, de Rigal D, Mbéguié-A-Mbéguié D, Gauillard F, Richard-Forget F, Fils-Lycaon B R (1999). Molecular cloning and characterization of apricot fruit polyphenol oxidase. Plant Physiol , 119(4): 1261–1270 doi: 10.1104/pp.119.4.1261
4	Dong Z (2007). The study on the ACC oxidase genes clone of pear fruit. Dissertation for the Master Degree . Baoding: Agricultural University of Hebei, 84–85 (in Chinese)
5	Gao M, Matsuta N, Murayama H, Toyomasu T, Mitsuhashi W, Dandekar A, Tao R, Nishimura K (2007). Gene expression and ethylene production in transgenic pear (Pyrus communis cv. ‘La France’) with sense or antisense cDNA encoding ACC oxidase. Plant Sci , 173(1): 32–42 doi: 10.1016/j.plantsci.2007.03.014
6	Gao M, Sakamoto A, Miura K, Murata N, Sugiura A, Tao R (2000). Transformation of Japanese persimmon (Diospyros kaki Thunb.) with a bacterial gene for choline oxidase. Molecular Breeding , 6(5): 501–510
7	Joy R W IV, Sugiyama M, Fukuda H, Komamine A (1995). Cloning and characterization of polyphenol oxidase cDNAs of Phytolacca americana. Plant Physiol , 107(4): 1083–1089 doi: 10.1104/pp.107.4.1083
8	Li G Q, Li H X, Xu D Q, Liu K, Zhang Y X (2008). Cloning and expression of polyphenol oxidase gene of CDS from Yali pear. Journal of Fruit Science , 25(4): 577–580 (in Chinese)
9	Matsuda N, Gao M, Isuzugawa K, Takashina T, Nishimura K (2005). Development of an Agrobacterium-mediated transformation method for pear (Pyrus communis L.) with leaf-section and axillary shoot-meristem explants. Plant Cell Rep , 24(1): 45–51 doi: 10.1007/s00299-005-0924-1
10	Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant , 15(3): 473–497 doi: 10.1111/j.1399-3054.1962.tb08052.x
11	Newman S M, Eannetta N T, Yu H, Prince J P, Vicente M C, Tanksley S D, Steffens J C (1993). Organization of the tomato polyphenol oxidase gene family. Plant Mo1 Biol , 21: 1035–1051
12	Thygesen P W, Dry I B, Robinson S P (1995). Polyphenol oxidase in potato. A multigene family that exhibits differential expression patterns. Plant Physiol , 109(2): 525–531 doi: 10.1104/pp.109.2.525
13	Vámos-Vigyázó L, Haard N (1981). Polyphenol oxidase and peroxidase in fruits and vegetables. Crit Rev Food Sci Nutr , 15(1): 49–127 doi: 10.1080/10408398109527312
14	Van der Krol A R, Lenting P E, Veenstra J, van der Meer I M, Koes R E, Gerats A G M, Mol J N M, Stuitje A R (1988). Antisense chalcone synthrase gene in transgenetic plants inhibits flower pigmentation. Nature , 333(6176): 866–869 doi: 10.1038/333866a0
15	Vaughn K C, Lax A R, Duke S O (1988). Polyphenol oxidase: the chloroplast oxidase with no established function. Physiol Plant , 72(3): 659–665 doi: 10.1111/j.1399-3054.1988.tb09180.x
16	Wang Q, Huang H Y, Ma W F, Wang D (2007). Effect of anti-sense PPO gene on the tuber-browning of Solanum tuberosum L. Acta Agron Sin , 33(11): 1822–1827 (in Chinese)
17	Yamamoto T, Hayasi T (2001). Mapping of peach. In: Sasaki T, Tabata S, Shimamoto K, eds. Protocol for Studying of Plant Genome . Tokyo: Shujunsha Co., Ltd., 222–225

[1]	Yanhui WANG, Yuan LIU, Yuxing ZHANG, Zhanyang XU. Quality evaluation of mixed brewed perries based on PCA and sensory evaluation[J]. Front Agric Chin, 2011, 5(4): 529-533.
[2]	Yongbo WANG, Yuxing ZHANG, Jianghong ZHANG, . Effects of salicylic acid on the behavior of Yali pear infected by Alternaria kikuchiana Tanaka[J]. Front. Agric. China, 2010, 4(1): 79-83.
[3]	Chunli SONG, Junlian MA, Xia TANG, Zide ZHANG, Zhixia HOU, . Strawberry FaEtr2 gene RNAi expression vector construction and genetic transformation[J]. Front. Agric. China, 2009, 3(4): 419-424.
[4]	Haina ZHANG, Fanghua ZHAO, Yuxin ZHAO, Chengjin GUO, Cundong LI, Kai XIAO. Establishment of transgenic cotton lines with high efficiency via pollen-tube pathway[J]. Front. Agric. China, 2009, 3(4): 359-365.
[5]	HAN Shengfang, GU Juntao, XIAO Kai. Improving organic phosphate utilization in transgenic white clover by overexpression of Aspergillus niger PhyA gene[J]. Front. Agric. China, 2007, 1(3): 265-270.

Viewed

Full text

Abstract

Cited

Shared

Discussed