一种低成本、高效、操作简便的转基因植株鉴定方法

doi:10.19586/j.2095-2341.2024.0113

生物技术进展 ›› 2024, Vol. 14 ›› Issue (6): 1016-1023.DOI: 10.19586/j.2095-2341.2024.0113

一种低成本、高效、操作简便的转基因植株鉴定方法

彭海霞¹(), 汪世娟², 辛转霞¹, 梅莉¹, 马猛²

^1.西北农林科技大学风景园林艺术学院，陕西杨凌 712100
^2.西北农林科技大学生命科学学院，陕西杨凌 712100

收稿日期:2024-06-11 接受日期:2024-08-01 出版日期:2024-11-25 发布日期:2024-12-27
作者简介:彭海霞 E-mail： penghaixia@nwafu.edu.cn
基金资助:
国家自然科学基金项目(32072059);陕西省自然科学基础研究计划(2024JC-YBQN-0185);西北农林科技大学实验技术研究与创新项目(SY20230222)

A Low-cost, Efficient and Easy-to-operate Method for Transgenic Plant Identification

Haixia PENG¹(), Shijuan WANG², Zhuanxia XIN¹, Li MEI¹, Meng MA²

^1.College of Landscape Architecture and Art，Northwest A&F University，Shaanxi Yangling 712100，China
^2.College of Life Sciences，Northwest A & F University，Shaanxi Yangling 712100，China

Received:2024-06-11 Accepted:2024-08-01 Online:2024-11-25 Published:2024-12-27

摘要/Abstract

摘要：

鉴于特异性PCR、试纸条等常用转基因植株检测方法存在费时费力且需要一定专业技术等局限性，研究希望探索一种低成本、高效、操作简便且适用于小麦全生育期田间大规模筛选的转基因植株鉴定方法。选取具有草铵膦除草剂BASTA抗性的转基因小麦进行最适BASTA溶液浓度筛选，发现在大田环境下200 mg·L^-1的BASTA溶液可分别在苗期和扬花期有效鉴定转基因阳性植株。同时，为了验证BASTA叶片涂抹法的准确性和实用性，选取20个T₀代转基因小麦样本，分别采用Bar试纸条、特异性PCR和BASTA叶片涂抹法3种方法进行转基因阳性鉴定。结果显示，BASTA叶片涂抹法与Bar试纸条鉴定结果一致，并且其检测范围能够覆盖特异性PCR的检测结果。与传统方法相比，BASTA叶片涂抹法成本低、高效、操作便捷，且全生育期可用，尤其适用于田间转基因植株的大规模筛选。

关键词: 转基因植株, 阳性鉴定, BASTA叶片涂抹法, 低成本, 操作简便

Abstract:

Given the time-consuming and technically demanding nature of commonly used methods such as specific PCR and strip tests for detecting transgenic plants， we hoped to explore a simplified and efficient method for identifying transgenic wheat throughout its entire growth cycle in field conditions. We optimized a leaf painting method using BASTA （glufosinate herbicide）， which is low-cost， straightforward， and suitable for large-scale screening of transgenic plants in wheat fields. Selecting transgenic wheat with BASTA resistance， we determined that a 200 mg·L^-1 BASTA solution effectively identified transgenic positive plants during both seedling and flowering stages in field environments. Additionally， we compared this method with Bar strip tests and specific PCR on 20 T₀ generation transgenic wheat plants. Results indicated that the BASTA leaf painting method correlated with the Bar strip test and covered the findings of specific PCR. Compared to traditional methods， the BASTA leaf painting method is cost-effective， efficient， operationally simple， and applicable throughout the entire growth cycle， making it particularly suitable for large-scale field screening of transgenic plants.

Key words: transgenic plants, positive identification, BASTA leaf painting method, low cost, easy-to-operate

中图分类号:

彭海霞, 汪世娟, 辛转霞, 梅莉, 马猛. 一种低成本、高效、操作简便的转基因植株鉴定方法[J]. 生物技术进展, 2024, 14(6): 1016-1023.

Haixia PENG, Shijuan WANG, Zhuanxia XIN, Li MEI, Meng MA. A Low-cost, Efficient and Easy-to-operate Method for Transgenic Plant Identification[J]. Current Biotechnology, 2024, 14(6): 1016-1023.

图/表 7

图1 p35S::Bar表达载体结构示意图注：LB—左边界；35S-T—花椰菜花叶病毒35S (CaMV35S)基因终止子；Bar—抗除草剂基因；p35S—花椰菜花叶病毒35S启动子；Nos T—nos基因终止子；RB—右边界。

Fig. 1 The structure of p35S::Bar vector

图2 苗期和扬花期用不同浓度BASTA溶液涂抹小麦叶片7 d后的表型

Fig. 2 Phenotype of wheat leaves painted with different concentrations of BASTA after 7 days at seedling and flowering stage

图3 T0代转基因株系BASTA筛选液检测WT—野生型；T0-1~T0-20—不同的T0代转基因株系。

Fig. 3 Detection of BASTA screening solution in T0 generation transgenic lines

图4 T0代转基因株系的Bar试纸条检测注：WT—野生型；T0-1~T0-20—不同的T0代转基因株系。

Fig. 4 Bar test strip assay for T0 generation transgenic lines

图5 T0代转基因植株特异片段的PCR检测注：M—D2000 DNA Marker；ddH2O—空白对照；WT—JW1野生型阴性对照；PC—阳性对照；T0-1~T0-20—T0代转基因小麦植株。

Fig. 5 PCR detection of T0 generation transgenic plants

表1 特异性PCR、Bar试纸条和BASTA叶片涂抹鉴定转基因小麦结果比较

Table 1 Comparison of specific PCR, Bar test strips and BASTA leaf coating for identification of transgenic wheat

株系	特异性PCR	Bar试纸条	BASTA叶片涂抹
T_0-1	-	+	+
T_0-2	+	+	+
T_0-3	+	+	+
T_0-4	-	+	+
T_0-5	+	+	+
T_0-6	-	+	+
T_0-7	+	+	+
T_0-8	+	+	+
T_0-9	+	+	+
T_0-10	-	+	+
T_0-11	+	+	+
T_0-12	+	+	+
T_0-13	+	+	+
T_0-14	+	+	+
T_0-15	+	+	+
T_0-16	+	+	+
T_0-17	+	+	+
T_0-18	+	+	+
T_0-19	+	-	-
T_0-20	-	-	-

图6 qRT-PCR检测T1代转基因小麦中靶基因的表达水平注：**代表与WT相比，差异在P<0.01水平具有统计学意义。

Fig. 6 qRT-PCR to detect the expression levels of target genes in T1 generation transgenic wheat

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一种低成本、高效、操作简便的转基因植株鉴定方法

A Low-cost, Efficient and Easy-to-operate Method for Transgenic Plant Identification

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