外源抗草膦EPSPs基因在大豆基因组中的整合与定位

doi:10.3724/SP.J.1006.2010.00365

Abstract

Abstract:

The detection of genetically modified crops (GMCs) is becoming both food labels and legal necessity. The aim of this study was to identify integration and insertion locus of foreign EPSPs gene in genetically modified soybean for the safety assessment of genetically modified crop. The EPSPs gene fragment was detected using gene specific primes and immunochromatographic strip was used to detect the EPSPs protein. The result showed that the EPSPs gene was integrated into soybean genome and EPSPs protein could be expressed normally. Genome walking was used to analyze the flanking sequences of both 35S promoter and NOS terminator, and soybean genome database (Phytozome) was used to analyze the insertion site and study the effect of the insertion on soybean genome. The Dra I and EcoR V restriction enzyme were found that they could be used to digest the soybean genome completely, and the library with adaptor was established and nest PCR was used to amplify the flanking sequences of 35S and NOS genes in genetically modified soybean genome. The result showed that the start site of flanking region of either 35S promoter or NOS terminator was Gm02:7912740 or Gm02:7777705, respectively, which means the foreign gene may not insert into a fixed position, and one 135 kb DNA fragment may be translocated. Two unknown sequences and two soybean DNA fragments with physical distance of 24 kb in opposite directions were found at the flanking region of NOS terminator. We also found that there were high AT content (about 70%) and low gene density in 90kb flanking regions of the insertion locus, and one gene coding HEC1 and HEAT repeat domain (Glyma02g09790) was found to be rearranged. RT-PCR and qRT-PCR showed that the gene was down-regulated during PEG and ABA treatment. In this study, we found the genetically engineered soybean genome was rearranged because of the insertion of foreign genes and one gene coding HEC1 and HEAT repeat domain (Glyma02g09790) which may response to drought stress through ABA signal pathway was identified for the first time.

Key words: Genetically modified soybean, Insertion locus, Genome, EPSPs, Glyphosate-Resistance, Location, Flanking sequence

WANG Xiao-Bo,JIANG Ling-Xue,WEI Li,LIU Lin,LU Wei,Li Wen-Xin,WANG Jun,CHANG Ru-Z. Integration and Insertion Site of EPSPs Gene on the Soybean Genome in Genetically Modified Glyphosate-Resistant Soybean[J].Acta Agron Sin, 2010, 36(3): 365-375.

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Integration and Insertion Site of EPSPs Gene on the Soybean Genome in Genetically Modified Glyphosate-Resistant Soybean

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