Crops ›› 2022, Vol. 38 ›› Issue (1): 65-69.doi: 10.16035/j.issn.1001-7283.2022.01.009

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Establishing of a Fast and Efficient Testing Method of Transgenic Maize

Zhou Delong1(), Meng Lingcong1, Zheng Shubo1, Wang Nan1, Li Mu1, Wang Xinqi1, Lu Shi1, Wang Min2, Liu Wenguo1, Lu Ming1()   

  1. 1Maize Research Institute, Jilin Academy of Agricultural Sciences/National Engineering Laboratory for Maize (Changchun)/National Engineering Research Center for Maize (Jilin)/Key Laboratory of Biology and Genetic Improvement of Maize in Northeast Region, Ministry of Agriculture and Rural Affairs/Open Laboratory of Jilin Province Crop Breeding of South Breeding Base, Changchun 130033, Jilin, China
    2Jilin Jinong Hi-Tech Development Co., Ltd., Gongzhuling 136100, Jilin, China
  • Received:2021-04-27 Revised:2021-08-23 Online:2022-02-15 Published:2022-02-16
  • Contact: Lu Ming E-mail:1311608097@qq.com;lum7893@163.com

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Abstract:

An efficient target gene detection method is an important technical support to ensure the development and industrialization of biological breeding. In this study, conventional PCR method, TaqMan probe real-time fluorescence PCR method and leaf direct PCR method were used to detect two transgenic elements of CaMV35S promoter and NOS terminator in leaves of transgenic maize plants. The experiment results showed that the leaf direct PCR method saved 62% and 48% time and reduced 25% and 43% cost compared with the conventional PCR method and TaqMan probe real-time fluorescence PCR method, respectively, and the amplified target band was clearly visible, and the fragment size was in accord with the target fragment size. The results were true and reliable, which was suitable for rapid screening and identification of a large number of transgenic maize leaves.

Key words: Maize, Transgenosis, Conventional PCR method, TaqMan probe real-time fluorescence PCR method, Leaf direct PCR method

Table 1

Primer informations for genetically modified detection"

引物名称Primer name 引物序列Primer sequence 目的片段大小Target fragment size (bp)
CaMV35S
F1：5'-GCTCCTACAAATGCCATCATTGC-3'
R1：5'-GATAGTGGGATTGTGCGTCATCCC-3'		 195
NOS
F1：5'-GAATCCTGTTGCCGGTCTTG-3'
R1：5'-TTATCCTAGTTTGCGCGCTA-3'		 182

Fig.1

PCR analysis of CaMV35S promoter M: DL1000 DNA Marker; 1: blank control; 2: negative control; 3: positive quality control; 4-6: transgenic material T561; 7-9: transgenic material T661; 10-12: transgenic material T841, the same below"

Fig.2

PCR analysis of NOS terminator"

Fig.3

Real time expansion reaction curves of CaMV35S"

Fig.4

Real time expansion reaction curves of NOS"

Fig.5

Direct PCR detection of CaMV35S promoter M1: alignment marker (15-600bp); M2: size marker (25-500bp), the same below"

Fig.6

Direct PCR test results of NOS terminator"

Fig.7

Peak results of capillary electrophoresis of CaMV35S promoter and NOS terminator 4, 7 and 10 are transgenic materials T561, T661 and T841, respectively"

Table 2

Time and cost consumed of three methods"

方法Method 步骤Step 时间Time (min) 成本（元）Cost (yuan)
常规PCR Conventional PCR DNA提取（CTAB法） 122 25
PCR扩增 95 15
琼脂糖凝胶电泳及结果分析 90 40
合计Total 307 80
TaqMan探针实时荧光PCR
TaqMan probe real-time fluorescence PCR		 DNA提取（试剂盒） 93 76
实时荧光PCR 130 30
合计Total 223 106
叶片直接PCR Leaf direct PCR 叶片预处理 7 5
PCR扩增 95 15
毛细管电泳及结果分析 15 40
合计Total 117 60
[1] 赵久然, 王帅, 李明, 等. 玉米育种行业创新现状与发展趋势. 植物遗传资源学报, 2018, 19(3):435-446.
[2] 国际农业生物技术应用服务组织. 2018年全球生物技术转基因作物商业化发展态势. 中国生物工程杂志, 2019, 39(8):1-6.
[3] 甄贞, 高学军, 张明辉. 主要农作物种子转基因检测方法及应用. 中国种业, 2018(4):8-10.
[4] 武海斌, 路兴波, 孙红炜, 等. 转基因玉米转化体特异性PCR检测技术研究. 玉米科学, 2009, 17(1):60-70.
[5] 金芜军, 郝旸, 程红梅, 等. 用复合PCR方法检测6种转基因玉米中外源DNA的特异性. 农业生物技术学报, 2005, 13(5):562-567.
[6] 王忻, 聂绪恒, 杨瑾. 多重-降落PCR方法在转基因粮食检测中的应用. 粮食科技与经济, 2017, 42(3):36-38.
[7] 宋欣, 赵淑清. 拟南芥35S:MS606/myb26转基因植物的鉴定. 山西农业科学, 2017, 45(5):680-683.
[8] 牛犇, 邹华文, 吴忠义, 等. 利用微滴式数字PCR技术分析转基因玉米抗除草剂标记基因EPSP拷贝数. 华北农学报, 2017, 32(3):70-76.
[9] 武海斌, 孙红炜, 李宝笃, 等. 转基因玉米多重PCR-基因芯片联用的检测方法. 农业生物技术学报, 2009, 17(6):1075-1082.
[10] 李飞武, 闫伟, 龙丽坤, 等. 应用多重PCR技术筛选检测转Bt基因作物. 现代食品科技, 2014, 30(5):269-273.
[11] 闫伟, 李葱葱, 夏蔚, 等. 应用单管巢式和半巢式PCR检测转基因玉米MON89034. 玉米科学, 2016, 24(4):56-60.
[10] 苏锐, 熊嫣, 庆宏, 等. 转基因作物检测新技术研究进展. 化学通报, 2012(2):27-31.
[12] Fraiture M A, Herman P, Taverniers I, et al. Current and new approaches in GMO detection:challenges and solutions. BioMed Research International, 2015, 2015:392872.
[13] 吴静, 李铁柱, 孙瑶, 等. 应用PCR-DHPLC技术高通量快速检测转基因玉米. 玉米科学, 2012, 20(5):40-44.
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