抗除草剂大豆GE-J12实时荧光定量PCR检测方法的建立

doi:10.13560/j.cnki.biotech.bull.1985.2021-1330

摘要/Abstract

摘要：

GE-J12是中国农业科学院研发的转G2-EPSPS基因和GAT基因的抗除草剂大豆新品系。根据抗除草剂大豆GE-J12品系外源基因插入位点5'端转化体特异性序列设计引物和探针,经引物探针筛选、特异性测试、反应体系和程序优化、标准曲线构建、检测极限和定量极限测试等试验,建立了基于TaqMan水解探针的实时荧光定量PCR检测方法。该方法特异性强、准确度高,在RSD小于25%的情况下检测极限为0.032%,定量极限为0.16%,线性度大于0.992,经5%、3%和1%定值样品测试,与真实值平均偏差为2.87%-16.67%。可用于转基因耐除草剂大豆GE-J12转化体特异性序列含量的精准定量,为GE-J12大豆新品系的检测提供了新方法。

关键词: 实时荧光定量PCR, GE-J12转化体, 特异性序列

Abstract:

GE-J12 is a new herbicide-resistant soybean line with G2-EPSPS gene and GAT gene developed by the Chinese Academy of Agricultural Sciences. In this study,primers and probes were designed based on the specific sequence of the transformant at the 5' end of the insertion site of the exogenous gene in the herbicide-resistant soybean GE-J12 strain. A realtime fluorescent quantitative PCR detection method based on TaqMan hydrolysis probe was established,after primer probe screening,specificity testing,reaction system and program optimization,and standard curve construction,detection limit and quantitative limit test and other tests. This method presents strong specificity and high accuracy. When the RSD was < 25%,the detection limit was 0.032%,the quantification limit was 0.16%,and the linearity was > 0.992. After testing with 5%,3%,and 1% fixed-value sample,the average deviation of the true value was 2.87%-16.67%. This method can be used to accurately quantify the specific sequence content of genetically modified herbicide-tolerant soybean GE-J12 transformants,which provides a new method for the detection of new soybean lines of GE-J12.

Key words: real-time fluorescent quantitative PCR, GE-J12 transformant, specific sequence

曹英芳, 赵新, 刘双, 李瑞环, 刘娜, 徐石勇, 高芳瑞, 马卉, 兰青阔, 檀建新, 王永. 抗除草剂大豆GE-J12实时荧光定量PCR检测方法的建立[J]. 生物技术通报, 2022, 38(7): 146-152.

CAO Ying-fang, ZHAO Xin, LIU Shuang, LI Rui-huan, LIU Na, XU Shi-yong, GAO Fang-rui, MA Hui, LAN Qing-kuo, TAN Jian-xin, WANG Yong. Establishment of Real-time Fluorescent Quantitative PCR Detection Method for Genetically Modified Herbicide-tolerant Soybean GE-J12[J]. Biotechnology Bulletin, 2022, 38(7): 146-152.

图/表 7

表1 引物和探针

Table 1 Primers and probes used in this study

基因名称 Gene name	引物和探针序列 Primer and probe sequence	扩增片段大小 Amplified fragment size/bp	来源 Source
GE-J12转化体 GE-J12 transformant	5'-GGCTTTACTAAAATATAAATCCTAA-3'	91	自行设计
	5'-GGCGTTAATTCAGTACATTA-3'
	5'-FAM-TGACGCTTAGACAACTTAATAACACAT-BHQ1-3'
Lectin	5'-GCCCTCTACTCCACCCCCA-3'	118	农业部2031号公告-8-2013
	5'-GCCCATCTGCAAGCCTTTTT-3'
	5'-FAM-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1-3'

图1 实时荧光定量PCR特异性检测结果 1：转基因大豆GE-J12;2：大豆受体Jack,其他转基因大豆,转基因玉米,转基因油菜,转基因水稻,转基因棉花,其他非转基因大豆混样

Fig. 1 Specific detection results via real-time fluorescence quantitative PCR 1：Genetically modified soybean GE-J12. 2：Soybean recipient Jack,other genetically modified soybeans,genetically modified corn,genetically modified rapeseed,genetically modified rice,genetically modified cotton,and other non-transgenic soybean mixtures

图2 实时荧光定量PCR体系优化结果 0：空白对照;1：引物0.2 µmol/L、探针0.1 µmol/L;2：引物0.4 µmol/L、探针0.2 µmol/L;3：引物0.5 µmol/L、探针0.25 µmol/L;4：引物0.6 µmol/L、探针0.3 µmol/L;5：引物0.8 µmol/L、探针0.4 µmol/L：6：引物1.0 µmol/L、探针0.5 µmol/L

Fig. 2 Optimization results of real-time fluorescent quanti-tative PCR system 0：Blank control;1：primer 0.2 µmol/L,and probe 0.1 µmol/L;2：primer 0.4 µmol/L,and probe 0.2 µmol/L;3：primer 0.5 µmol/L,and probe 0.25 µmol/L;4：primer 0.6 µmol/L,and probe 0.3 µmol/L;5：primer 0.8 µmol/L,and probe 0.4 µmol/L;6：primer 1.0 µmol/L,and probe 0.5 µmol/L

图3 标准曲线建立1：100%;2：20%;3：4%;4：0.8%;5：0.16%;6：0.032%;7：0.006 4%

Fig. 3 Standard curve establishment

表2 样品定值结果

Table 2 Results of sample setting

测试样品Testing sample	重复1 Repeat 1			重复2 Repeat 2			重复3 Repeat 3			平均偏差 Mean deviation
	拷贝数 Number of copies		含量 Content/%	拷贝数 Number of copies		含量 Content/%	拷贝数 Number of copies		含量 Content/%
	GE-J12	Lectin	含量 Content/%	GE-J12	Lectin	含量 Content/%	GE-J12	Lectin	含量 Content/%
GE-J12（5%）	336.20	6 322.67	5.32	318.27	6 496.33	4.90	333.47	6 660.00	5.01	2.87
GE-J12（3%）	125.97	4 719.33	2.67	256.67	8 025.00	3.20	238.33	8 155.67	2.92	6.78
GE-J12（1%）	117.60	9 451.00	1.24	53.11	4 722.33	1.12	51.87	4 538.67	1.14	16.67

图4 转基因大豆GE-J12实时荧光定量PCR检出限测试

Fig. 4 Detection limit via real-time fluorescence quantita-tive PCR for genetically modified soybean GE-J12

表3 实时荧光定量PCR灵敏度及可重复性测试

Table 3 Sensitivity and repeatability test of real-time fluorescence quantitative PCR

DNA模板量 DNA template amount	重复1 Repeat 1			重复2 Repeat 2			重复3 Repeat 3
DNA模板量 DNA template amount	Ct mean	SD	RSD/%	Ct mean	SD	RSD/%	Ct mean	SD	RSD/%
100%	24.13	0.05	0.23	24.09	0.07	0.31	23.92	0.08	0.35
20%	26.29	0.07	0.26	26.24	0.03	0.12	26.19	0.07	0.26
4%	28.51	0.04	0.15	28.47	0.06	0.23	28.57	0.15	0.51
0.8%	30.73	0.06	0.20	30.69	0.06	0.20	30.75	0.18	0.59
0.16%	32.89	0.14	0.43	32.96	0.20	0.59	33.11	0.16	0.48
0.032%	35.21	0.42	1.20	35.22	0.37	1.04	35.44	0.67	1.89
0.006 4%	37.32	1.04	2.79	36.94	0.86	2.33	37.67	0.99	2.62
Formula	Y=-3.158X+36.784			Y=-3.132X+36.683			Y=-3.286X+37.113
R²	0.992			0.995			0.992
Amplification efficiency	107.3%			108.6%			101.5%

参考文献 24

[1]	伍圣文, 贾成刚, 崔春. 脱壳处理对火麻蛋白提取、功能特性和消化性的影响[J]. 食品科学技术学报, 2021, 39(4):87-94.
	Wu SW, Jia CG, Cui C. Effects of hulling treatment on extraction, functional properties and digestibility of hemp protein[J]. J Food Sci Technol, 2021, 39(4):87-94.
[2]	袁明, 韩冬伟, 王淑荣, 等. 有机物料替代化学肥料对大豆产量及品质的影响[J]. 大豆科技, 2020(6):15-19.
	Yuan M, Han DW, Wang SR, et al. Effect of organic material replacing chemical fertilizer on yield and quality of soybean[J]. Soybean Sci Technol, 2020(6):15-19.
[3]	Sato H, Miura M, Fujieda T, et al. Growth performance responses to increased tryptophan supplementation in growing barrows fed three different very low crude protein corn and soybean meal-based diets fortified with essential amino acids[J]. Anim Sci J, 2021, 92(1):e13605.
[4]	何艳, 黄晓伟, 程中一, 等. 新形势下大豆产地土壤环境保护与功能提升的研发建议[J]. 土壤学报, 2021, 58(2):269-280.
	He Y, Huang XW, Cheng ZY, et al. Proposals for research on protection and functional improvement of soil environment in soybean producing area in face of the new situation[J]. Acta Pedol Sin, 2021, 58(2):269-280.
[5]	杨京. 2020年中国大豆市场分析[N]. 粮油市场报, 2021-02-20( B04).
	Yang J. Analysis of China’s soybean market in 2020[N]. Agricultural Science & Technology, 2021-02-20( B04).
[6]	Russo C, Simeone M, Perito MA. Educated millennials and credence attributes of food products with genetically modified organisms:knowledge, trust and social media[J]. Sustainability, 2020, 12 (20):8534.
[7]	梁晋刚, 贺晓云, 武玉花, 等. 中国农业转基因生物安全标准体系现状与展望[J]. 农业生物技术学报, 2020, 28(5):911-917.
	Liang JG, He XY, Wu YH, et al. Current status and prospects of safety standard system for agricultural genetically modified organisms in China[J]. J Agric Biotechnol, 2020, 28(5):911-917.
[8]	雷杰, 包慧敏, 方彩云, 等. 在分析化学课程中引入病毒及其检测方法的思考[J]. 大学化学, 2020, 35(12):13-20.
	Lei J, Bao HM, Fang CY, et al. Introduction of viruses and its detection methods in the course of analytical chemistry[J]. Univ Chem, 2020, 35(12):13-20. doi: 10.3866/PKU.DXHX201907020 URL
[9]	王子骞, 陈彦宇, 齐俊生. 转基因食品安全性分析[J]. 农业与技术, 2020, 40(12):167-169.
	Wang ZQ, Chen YY, Qi JS. Safety analysis of genetically modified food[J]. Agric Technol, 2020, 40(12):167-169.
[10]	Hasell A, Stroud NJ. The differential effects of knowledge on perceptions of genetically modified food safety[J]. Int J Public Opin Res, 2020, 32(1):111-131. doi: 10.1093/ijpor/edz020 URL
[11]	兰青阔, 赵新, 沈晓玲, 等. 基于代谢组学的转基因水稻生物安全评价方法研究[J]. 生物技术通报, 2020, 36(11):222-229. doi: 10.13560/j.cnki.biotech.bull.1985.2020-0198
	Lan QK, Zhao X, Shen XL, et al. Biosafety assessment technology research for genetically modified rice based on metabolomics[J]. Biotechnol Bull, 2020, 36(11):222-229.
[12]	Karalis DT, Karalis T, Karalis S, et al. Genetically modified products, perspectives and challenges[J]. Cureus, 2020, 12(3):e7306.
[13]	Yu YT. The study of the impact of genetically modified soybean imports on China’s food safety management[J]. Int J Metrol Qual Eng, 2021, 12:18. doi: 10.1051/ijmqe/2021016 URL
[14]	张先文, 黄冲平, 王东芳, 等. 新形势下高校转基因作物田间试验生物安全管理机制的思考[J]. 农业科技管理, 2021, 40(3):72-77.
	Zhang XW, Huang CP, Wang DF, et al. Considerations on the biosafety management mechanism of field experiment of genetically modified crops in university under the new situation[J]. Manag Agric Sci Technol, 2021, 40(3):72-77.
[15]	Gao Y, Tie YQ, Zhao LQ, et al. Rapid internal control reference recombinase-aided amplification assays for EBV and CMV detection[J]. Biomed Environ Sci, 2021, 34(8):650-655.
[16]	Wang JY, Mi TZ, Yu ZG, et al. Species-specific detection and quantification of scyphomedusae in Jiaozhou Bay, China, using a quantitative real-time PCR assay[J]. J Oceanol Limnol, 2021, 39(4):1360-1372. doi: 10.1007/s00343-020-0160-0 URL
[17]	于园, 刘国强, 苏圣淋, 等. 玉米转基因成分的检测[J]. 江苏农业学报, 2020, 36(4):836-841.
	Yu Y, Liu GQ, Su SL, et al. Detection of transgenic ingredients in maize[J]. Jiangsu J Agric Sci, 2020, 36(4):836-841.
[18]	陈兆贵, 叶新友, 邢澍祺, 等. 马铃薯卷叶病毒实时荧光定量PCR检测技术研究[J]. 湖南农业科学, 2018(9):9-12.
	Chen ZG, Ye XY, Xing SQ, et al. Study on real time fluorescence quantitative PCR detection technology of potato leaf roll virus[J]. Hunan Agric Sci, 2018(9):9-12.
[19]	Fan HY, Hu JY, Luo ZW, et al. Establishment of a real-time fluorescent quantitative RTPCR method for Pineapple mealybug wilt associated virus-2[J]. Plant Dis Pests, 2016, 7(1):22-26.
[20]	李瑞环, 刘双, 兰青阔, 等. 转基因耐除草剂大豆ZH10-6多重PCR检测方法的建立[J]. 农业生物技术学报, 2021, 29(8):1640-1648.
	Li RH, Liu S, Lan QK, et al. Establishment of multiplex PCR detection method for genetically modified herbicide tolerant soybean(Glycine max)ZH10-6[J]. J Agric Biotechnol, 2021, 29(8):1640-1648.
[21]	冀志庚, 高学军, 敖金霞, 等. SYBR Green实时定量PCR检测转基因大豆中外源基因拷贝数[J]. 东北农业大学学报, 2011, 42(10):11-15.
	Ji ZG, Gao XJ, Ao JX, et al. Establishment of SYBR Green-base quantitative real-time PCR assay for determining transgene copy number in transgenic soybean[J]. J Northeast Agric Univ, 2011, 42(10):11-15.
[22]	邢珍娟, 董立明, 龙丽坤, 等. 应用多重荧光PCR快速筛查作物中转基因成分研究[J]. 玉米科学, 2021, 29(4):35-42.
	Xing ZJ, Dong LM, Long LK, et al. Rapid screening of genetically modified ingredient in crops by multiplex real-time PCR[J]. J Maize Sci, 2021, 29(4):35-42.
[23]	汪小福, 陈笑芸, 缪青梅, 等. 食品分析水平测试计划(FA-PAS)转基因检测能力验证中的方法分析与质量控制[J]. 中国食品学报, 2016, 16(7):224-230.
	Wang XF, Chen XY, Miao QM, et al. Analysis and quality control for the detection of transgenic in FAPAS proficiency tests[J]. J Chin Inst Food Sci Technol, 2016, 16(7):224-230.
[24]	Bogožalec Košir A, Demšar T, Štebih D, et al. Digital PCR as an effective tool for GMO quantification in complex matrices[J]. Food Chem, 2019, 294:73-78. doi: 10.1016/j.foodchem.2019.05.029 URL