Genetic Transformation and Detection of the Cymbidium hybridum Modified by Coat Protein Gene of ORSV

PENG Bo; WEI Li; YANG Kai; LI Lu-bin

[Objective]To optimize the transformation system and identification methods for obtaining the germplasm resources of anti-virus Cymbidium hybridum by plant transgenic technology. [Method]The coat protein gene (CP) of ORSV was cloned from the cDNA of infected C. hybridum. After sequencing, the gene was constructed into pBI121 expression vector and transformed to the protocorm-like bodies (PLBs) of C. hybridum by Agrobacterium tumefaciens-mediated method. [Result]The genetic transformation system was optimized and the identification method of the transgenic plants was established by nest polymerase-chain-reaction (Nest-PCR). There were 32 transgenic plants of C. hybridum detected by Nest-PCR. [Conclusion]The Agrobacterium tumefaciens-mediated transformation method of C.hybridum was optimized using PLBs as explants. The antibiotics(kanamycin)screening concentration was determined as 5% to 10% PLBs survival rate. Using the Nest-PCR to detected the transgenic plants was more sensitive and accurate than conventional PCR.

1. State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China

2. Environmental Monitoring Center of Handan, Handan 056002, Hebei, China

3. Beijing Key laboratory for Agricultural Application and New Technique, Beijing University of Agriculture, Beijing 102206, China

Received Date: 2015-02-02

Abstract: [Objective]To optimize the transformation system and identification methods for obtaining the germplasm resources of anti-virus Cymbidium hybridum by plant transgenic technology. [Method]The coat protein gene (CP) of ORSV was cloned from the cDNA of infected C. hybridum. After sequencing, the gene was constructed into pBI121 expression vector and transformed to the protocorm-like bodies (PLBs) of C. hybridum by Agrobacterium tumefaciens-mediated method. [Result]The genetic transformation system was optimized and the identification method of the transgenic plants was established by nest polymerase-chain-reaction (Nest-PCR). There were 32 transgenic plants of C. hybridum detected by Nest-PCR. [Conclusion]The Agrobacterium tumefaciens-mediated transformation method of C.hybridum was optimized using PLBs as explants. The antibiotics(kanamycin)screening concentration was determined as 5% to 10% PLBs survival rate. Using the Nest-PCR to detected the transgenic plants was more sensitive and accurate than conventional PCR.

HTML

Reference (15)

[1]	卢思聪.中国兰与洋兰[M].北京:金盾出版社,1994:10-16.
[2]	吴应祥.中国兰花[M].北京:中国林业出版社,1991:141.
[3]	Hu J S,Ferreira S,Wang M,et al. Detection of cymbidium mosaic virus, odontoglossum ringspot virus, tomato spotted wilt virus, and potyviruses infecting orchids in Hawaii [J].Plant Disease, 1993, 77(5):464-468.
[4]	Zettler F W,Ko N J,Wisler G C,et al. Viruses of orchids and their control [J].Plant Disease, 1990, 74(9):621-626.
[5]	肖火根,郑冠标,张曙光,等.广东兰花病毒病的鉴定和检测研究[J].华南农业大学学报,1996,17(1):21-24.
[6]	Powell A P,Nelson R S,De B,et al. Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene [J].Science, 1986, 232:738-743.
[7]	Rubino L,Capriotti G. Resistance to Cymbidium ringspot tombusvirus infection in transgenic Nicotiana benthamiana plants expressing the virus coat protein gene[J].Plant Molecular Biology, 1993, 21(4):665-672.
[8]	Ferreira S A,Pitz K Y,Manshardt R,et al. Virus coat protein transgenic Papaya provides practical control of Papaya ringspot virus in Hawaii [J].Plant Disease, 2002, 86(2):101-105.
[9]	王涛.兰花转CiDREB₁、PeDREB₂、CyMV-CP基因研究[D].保定:河北农业大学,2010.
[10]	Murray M G,Thompson W F. Rapid isolation of high molecular weight plant DNA [J].Nucleic acids research, 1980, 8(19):4321-4326.
[11]	Liao L J,Pan I Ch,Chan Y L,et al. Transgene silencing in Phalaenopsis expressing the coat protein of Cymbidium mosaic virus is a manifestation of RNA-mediated resistance [J].Molecular Breeding, 2004, 13(3):229-242.
[12]	Chan Y L,Lin K H,Jay A S,et al. Gene stacking in Phalaenopsis orchid enhances dual tolerance to pathogen attack [J].Transgenic Research, 2005, 14:279-288.
[13]	程保平,彭埃天,宋晓兵,等.三种PCR方法检测柑橘黄龙病菌的效果比较[J].植物保护,2014,40(5):106-110.
[14]	吴琼,陈枝楠,范怀忠,等.16S nested-PCR技术检测玉米细菌性枯萎病菌[J].植物病理学报,2005,35(5):420-427.
[15]	沈万宽,杨湛端,刘睿,等.基于PCR和巢式PCR技术的甘蔗黑穗病早期检测[J].热带作物学报,2013,34(9):1756-1760.

Genetic Transformation and Detection of the Cymbidium hybridum Modified by Coat Protein Gene of ORSV

Abstract

References

Proportional views

通讯作者: 陈斌, bchen63@163.com

Proportional views