花粉管通道法转<i>EPSPS</i>基因创制耐草甘膦玉米种质

李向龙; 郑登俞; 张春; 张中保; 吴忠义

doi:10.19303/j.issn.1008-0384.2023.05.002

花粉管通道法转EPSPS基因创制耐草甘膦玉米种质

doi: 10.19303/j.issn.1008-0384.2023.05.002

北京市农林科学院生物技术研究所/农业基因资源与生物技术北京市重点实验室，北京　100097

基金项目: 北京市农林科学院科技创新能力建设专项（KJCX20230603）

详细信息

作者简介:
李向龙（1981−），男，硕士，助理研究员，主要从事玉米分子育种研究（E-mail：lixianglong2004@sina.com）

通讯作者:
吴忠义(1969−)，男，博士，研究员，主要从事玉米分子育种研究（E-mail：zwu@126.com）

中图分类号: S513
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出版历程
- 收稿日期: 2023-03-30
- 修回日期: 2023-04-15
- 网络出版日期: 2023-05-24
- 刊出日期: 2023-05-28

Glyphosate-tolerant Maize Plants Generated by Pollen Tube Pathway Method

Institute of Biotechnology, Beijing Academy of Agriculture and Forestry Sciences/Beijing Key Laboratory of Agricultural Genetic Resources and Biotechnology, Beijing　100097, China

摘要

摘要: 目的对T₀-T₃代转基因植株进行抗草甘膦EPSPS基因的筛选检测，以期得到遗传稳定的转基因株系。方法利用花粉管通道法将EPSPS基因转入优良自交系京92中，通过田间草甘膦筛选和分子检测鉴定外源基因在世代间的遗传表达。结果在田间用200 mg·L^-1的草甘膦除草剂筛选，T₀代15个抗性植株经PCR鉴定，共获得10个阳性植株，转化率为1.03%；对各株系从T₁代到T₃代逐代筛选淘汰，株系内阳性株数逐渐增多，在T₃代5个株系中分离出K3和K8两个整合了EPSPS外源基因的稳定遗传株系；进一步对K3和K8两个株系的T₃代采用试纸条进行功能表达分析，发现目的基因均能正常表达。结论 K3和K8两个转基因玉米株系后期可作为培育耐草甘膦的基础材料。
- 玉米 /
- 花粉管通道法 /
- EPSPS基因 /
- 草甘膦耐性
Abstract: Objective Stable transgenic glyphosate-tolerant strains of maize were obtained by screening T₀-T₃ generations of the genetically modified plants for the presence of EPSPS after an artificial exposure to glyphosate in the field. Method The gene, EPSPS, responsible for a maize plant to resist the herbicide was transferred into the excellent inbred line Jing 92 by means of the pollen tube pathway method. Expression of the introduced gene in maize was verified by molecular detection after exposing to glyphosate in the field. Result Fifteen resistant plants from T₀ generation of the transgenic maize plants were identified after a treatment of 200 mg·L⁻¹ glyphosate, and 10 by PCR. The genetic transformation rate was 1.03%. The identified plants were further scrutinized to eliminate non-resistant-gene-carriers. The proportion of confirmed carriers among the plants increased from the T₁ to the T₃ generation. Out of 5 strains in T₃ generation, K3 and K8 were confirmed to have positively integrated EPSPS and be hereditarily stable. A test strip functional analysis on the two strains further verified the presence of the target gene. Conclusion The K3 and K8 transgenic lines identified by this study could plausibly be used to breed glyphosate-tolerant maize plants for cultivation.
- Maize /
- pollen tube pathway method /
- EPSPS /
- glyphosate-tolerance

HTML全文

图 1 T₀代植株草甘膦筛选

Figure 1. Screening T₀ plants for glyphosate-tolerance

下载: 全尺寸图片幻灯片

图 2 T₀代草甘膦抗性植株PCR检测

M：DL2000 Marker，-：阴性对照，W：无菌水，+：质粒对照，1-15：除草剂抗性植株。

Figure 2. PCR detection of T₀ glyphosate-resistant plants

M: DL2000 marker; -: negative control; W: sterile water; +: positive control; 1-15: herbicide-resistant plants.

下载: 全尺寸图片幻灯片

图 3 不同世代转基因植株草甘膦筛选

a：T₁代，b：T₂代，c：T₃代。

Figure 3. Screening transgenic plants in generations for glyphosate-tolerant plants

a: T₁ generation; b: T₂ generation; c: T₃ generation.

下载: 全尺寸图片幻灯片

图 4 不同世代草甘膦抗性植株PCR检测

a：T₁代，b：T₂代，c：T₃代。M：DL2000 Marker，-：阴性对照，W：无菌水，+：质粒对照，1-21：除草剂抗性植株。

Figure 4. PCR detection of glyphosate resistant plants in different generations

a: T₁ generation; b: T₂ generation; c: T₃ generation. M: DL2000 marker; -: negative control; W: sterile water; +: positive control; 1-21: glyphosate-resistant plants.

下载: 全尺寸图片幻灯片

图 5 EPSPS蛋白活性试纸条检测

CK：受体对照，1-16：T₃代转基因阳性植株。

Figure 5. Activity of EPSPS detected by strip test

CK: Negative control; 1-16: transgenic plants in T₃ generation.

下载: 全尺寸图片幻灯片

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