Quantification of transgenic plant marker gene persistence in the field

被引:73
作者
Widmer, F
Seidler, RJ
Donegan, KK
Reed, GL
机构
[1] DYNAMAC CORP,CORVALLIS,OR 97333
[2] OREGON STATE UNIV,HERMISTON AGR RES & EXTENS CTR,HERMISTON,OR 97838
[3] US EPA,NATL HLTH & ENVIRONM EFFECTS RES LAB,WESTERN ECOL DIV,CORVALLIS,OR 97333
关键词
field experiment; DNA persistence; environmental DNA preparation; quantitative PCR; biotechnology; risk assessment;
D O I
10.1046/j.1365-294X.1997.00145.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Methods were developed to monitor persistence of genomic DNA in decaying plants in the field. As a model, we used recombinant neomycin phosphotransferase II (rNPT-II) marker genes present in genetically engineered plants. Polymerase chain reaction (PCR) primers were designed, complementary to 20-bp sequences of the nopaline synthase promoter in a transgenic tobacco and the cauliflower mosaic virus 35S promoter in a transgenic potato. The PCR reverse primer was complementary to a 20-bp sequence of the N-terminal NPT-II coding region. The PCR protocol allowed for quantification of as few as 10 rNPT-II genes per reaction. We analysed rNPT-II marker gene amounts in samples obtained from two field experiments performed at different locations in Oregon. In transgenic tobacco leaves, buried at 10 cm depth in a field plot in Corvallis, marker DNA amount dropped to 0.36% during the first 14 days and was detectable for 77 days at a final level of 0.06% of the initial amount. Monitoring of residual potato plant litter, from the soil surface of a test field in Hermiston, was performed for 137 days. After 84 days marker gene amounts dropped to 2.74% (leaf and stem) and 0.50% (tuber) of the initially detected amount. At the final sample date 1.98% (leaf and stem) and 0.19% (tuber) were detectable. These results represent the first quantitative analysis of plant DNA stability under field conditions and indicate that a proportion of the plant genomic DNA may persist in the field for several months.
引用
收藏
页码:1 / 7
页数:7
相关论文
共 33 条
[1]   NEW CLONING VEHICLES FOR TRANSFORMATION OF HIGHER-PLANTS [J].
AN, G ;
WATSON, BD ;
STACHEL, S ;
GORDON, MP ;
NESTER, EW .
EMBO JOURNAL, 1985, 4 (02) :277-284
[2]  
[Anonymous], P 3 INT S BIOS RES F
[3]   NUCLEOTIDE-SEQUENCE AND EXACT LOCALIZATION OF THE NEOMYCIN PHOSPHOTRANSFERASE GENE FROM TRANSPOSON TN5 [J].
BECK, E ;
LUDWIG, G ;
AUERSWALD, EA ;
REISS, B ;
SCHALLER, H .
GENE, 1982, 19 (03) :327-336
[4]  
BECKER J, 1994, BIOL SICHERHEIT FORS, P563
[5]   POLYMERASE CHAIN-REACTION USED FOR MONITORING MULTIPLE GENE INTEGRATION IN AGROBACTERIUM-MEDIATED TRANSFORMATION [J].
BLAKE, NK ;
DITTERLINE, RL ;
STOUT, RG .
CROP SCIENCE, 1991, 31 (06) :1686-1688
[6]   APPARENT EUKARYOTIC ORIGIN OF GLUTAMINE SYNTHETASE-II FROM THE BACTERIUM BRADYRHIZOBIUM-JAPONICUM [J].
CARLSON, TA ;
CHELM, BK .
NATURE, 1986, 322 (6079) :568-570
[7]  
DEPICKER A, 1982, Journal of Molecular and Applied Genetics, V1, P561
[8]  
Dietz A., 1993, Transgenic organisms: risk assessment of deliberate release., P209
[9]  
DONEGAN KK, 1995, TRANSGENIC RES, V5, P25
[10]  
DONEGAN KK, 1996, IN PRESS J APPL ECOL