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基于 PCR 加限制酶技术检测抗草甘膦的黑麦草中 Pro-106 位置靶酶突变。

A PCR plus restriction enzyme-based technique for detecting target-enzyme mutations at position Pro-106 in glyphosate-resistant Lolium perenne.

机构信息

School of Agriculture and Environment, Massey University, Palmerston North, New Zealand.

AgResearch Grasslands Research Centre, Palmerston North, New Zealand.

出版信息

PLoS One. 2021 Feb 2;16(2):e0246028. doi: 10.1371/journal.pone.0246028. eCollection 2021.

DOI:10.1371/journal.pone.0246028
PMID:33529261
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7853469/
Abstract

The first step in managing herbicide-resistant weeds is to confirm their resistance status. It is, therefore, crucial to have a rapid, reliable and cost-effective technique to assess samples for herbicide resistance. We designed and evaluated three derived cleaved amplified polymorphic sequence (dCAPS) markers for detecting glyphosate resistance in Lolium perenne. conferred by non-synonymous mutations at codon-106 in the enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. The dCAPS markers involve amplification of the target region, digestion of the amplified products with restriction enzymes and gel-based visualisation of the digested products. The results showed that all three dCAPS markers could successfully detect mutations at codon-106 in the target enzyme. The dCAPS markers can also inform us of the zygosity state of the resistance allele and was confirmed by sequencing the target region of the EPSPS gene. The markers described here are effective quick tests for the monitoring and evaluation of the target-enzyme mechanism of glyphosate resistance in Lolium perenne.

摘要

管理抗除草剂杂草的第一步是确认其抗性状态。因此，拥有一种快速、可靠且具有成本效益的技术来评估样本中的除草剂抗性至关重要。我们设计并评估了三个衍生的切割扩增多态性序列 (dCAPS) 标记物，用于检测 Lolium perenne 中由烯醇丙酮酰莽草酸-3-磷酸合酶 (EPSPS) 基因 106 密码子的非同义突变引起的草甘膦抗性。dCAPS 标记物涉及目标区域的扩增、用限制酶消化扩增产物以及基于凝胶的消化产物可视化。结果表明，这三个 dCAPS 标记物都可以成功检测到目标酶中 106 密码子的突变。dCAPS 标记物还可以告知我们抗性等位基因的杂合状态，并通过对 EPSPS 基因的目标区域进行测序来确认。这里描述的标记物是监测和评估 Lolium perenne 中草甘膦靶酶抗性机制的有效快速测试方法。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3429/7853469/968c643ca31c/pone.0246028.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3429/7853469/b85904eb7a30/pone.0246028.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3429/7853469/ad6305610a1a/pone.0246028.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3429/7853469/a2be293b3f26/pone.0246028.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3429/7853469/968c643ca31c/pone.0246028.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3429/7853469/b85904eb7a30/pone.0246028.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3429/7853469/ad6305610a1a/pone.0246028.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3429/7853469/a2be293b3f26/pone.0246028.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3429/7853469/968c643ca31c/pone.0246028.g004.jpg

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本文引用的文献

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Sci Rep. 2020 Mar 6;10(1):4231. doi: 10.1038/s41598-020-61104-0.
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Target-Site Mutations Conferring Herbicide Resistance.赋予除草剂抗性的靶位点突变
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Evolution of a double amino acid substitution in the 5-enolpyruvylshikimate-3-phosphate synthase in Eleusine indica conferring high-level glyphosate resistance.稗草中5-烯醇丙酮酰莽草酸-3-磷酸合酶双氨基酸取代的进化赋予其高水平草甘膦抗性。
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Pest Manag Sci. 2015 Dec;71(12):1617-22. doi: 10.1002/ps.3968. Epub 2015 Jan 19.
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