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抗嘉磷塞雜草之變異與鑑定
89
A Molecular Characterization of Point-Mutations in
the 5-Enolpyruvylshikimate-3-Phosphate Synthase
(EPSPS) Enzyme of Glyphosate-Resistant Biotypes of
Hedyotis corymbosa
Li-Chang Lin
1
, Jia-Yan Zhang
2
, Chiou-Ing Yuan
1
*
Abstract
Lin, C. L., Zhang, J. Y., and Yuan, C. I. 2016. A molecular characterization of point-mutations in the
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme of glyphosate-resistant biotypes of
Hedyotis corymbosa
. Taiwan Pestic. Sci. 1: 70-90.
Weed resistance to herbicides has become a major concern for crop production
worldwide. We investigated the resistance of
Hedyotis corymbosa
to glyphosate in Taiwan,
focusing on dose-response, shikimic acid level, and the production of target enzyme
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). For this, Hedyotis corymbosa
seedlings at the 12 ~ 15 leaf stage were foliar sprayed with 0.10 to 26.24 kg a.e./ha glyphosate.
Dose-response was established based on the dry weight of seedlings 14 days after glyphosate
treatment. ED
50
was estimated using a log-logistic equation, and was found to be 0.46 or
4.58 kg a.e./ha for susceptible and resistant biotypes, respectively. Susceptible
H. corymbosa
showed obvious accumulation of shikimic acid. Ten days following glyphosate treatment,
shikimic acid content of susceptible
H. corymbosa
was 36.5 times higher than that of resistant
H. corymbosa.
Furthermore, after using real-time polymerase chain reaction (RT-PCR) to
prepare the related target enzyme EPSPS of
H. corymbosa
and treating plants with 1 mM
glyphosate, an EPSPS activity assay showed that the EPSPS content of the resistant biotype
was 3 times higher than that of the susceptible biotype. We also generated EPSPS cDNA
by performing RT-PCR with specific primers designed according to sequence data from
GenBank. In so doing, we determined that EPSPS cDNA was 1,551 bp for both resistant
and susceptible
H. corymbosa.
However, sequence data revealed nucleotides differences in
11 bp between resistant and susceptible biotypes. For example, the resistant biotype had a
Accepted: September 13, 2016.
* Corresponding author, Email:
yci@tactri.gov.tw1
Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Council of Agriculture. Taichung.
2
Graduate Institute of Biochemical Sciences and Technology, Chaoyang University of technology. Taichung.