转基因玉米浙大瑞丰8特异性定性PCR检测方法研究

doi:10.13560/j.cnki.biotech.bull.1985.2024-0324

摘要/Abstract

摘要：

【目的】浙大瑞丰8是杭州瑞丰生物科技有限公司开发的抗虫转基因玉米，于2021年12月获得生产应用安全证书。研究浙大瑞丰8的特异性PCR检测方法，为市场监管和产业化推广提供技术支持。【方法】以浙大瑞丰8转化体外源基因插入端侧翼序列为靶标，在5'端（右侧翼）和3'端（左侧翼）分别设计特异性引物，以与玉米内标准基因zSSIIb一致的标准反应体系和反应程序，对左侧翼和右侧翼各20对引物进行了筛选，得到了特异性高、稳定性好的候选引物，通过改变退火温度和引物浓度这两个关键参数，初步建立了浙大瑞丰8转化体PCR检测方法。【结果】 筛选到的浙大瑞丰8 PCR扩增引物RF 8-RB-R4/F1可特异性扩增目标片段265 bp，建立了与玉米内标准基因zSSIIb一致的标准反应体系和反应程序，实现了批量检测的高通量。【结论】建立的特异性定性PCR检测方法稳定性及适应性好，对拷贝数分数为0.1%（约20个拷贝）能稳定检出，可精准、特异地检测出浙大瑞丰8转化体。

关键词: 转基因玉米, 浙大瑞丰8, 定性PCR技术, 特异性检测

Abstract:

【Objective】Zheda Ruifeng 8 is an insect-resistant transgenic maize developed by Hangzhou Ruifeng Biotechnology Co. Ltd., the safety certificate for production and application was obtained in December 2021. The specific PCR detection method for Zheda Ruifeng 8 was investigated to provide technical support for market regulation and industrialization. 【Method】In this study, the flanking sequence of the insertion end of exogenous gene of Zheda Ruifeng 8 event was used as the target sequence, specific primers were designed at the 5'-end（right flanking）and the 3'-end（left flanking）respectively. Twenty pairs of primers for each of the left flanking and the right flanking were screened by the standard reaction system and the reaction procedure that was consistent with that of the standard gene zSSIIb in maize, and the primers with high specificity and fine stability were obtained. A preliminary method for the detection of PCR of the Zheda Ruifeng 8 event was established by changing the two key parameters of the annealing temperature and the concentration of the primer. 【Result】The screened Zheda Ruifeng 8 PCR amplification primer RF 8-RB-R4/F1 may specifically amplify the target fragment of 265 bp, and a standard reaction system and reaction procedure consistent with the standard gene zSSIIb in maize was established, and it achieved high throughput for batch detection. 【Conclusion】The established specific qualitative PCR detection method demonstrated excellent stability and adaptability. It could consistently detect a copy number fraction as low as 0.1%, equivalent to approximately 20 copies. This method proved to be highly accurate and specific, effectively detecting the transformants of Zheda Ruifeng 8.

Key words: genetically modified maize, Zheda Ruifeng 8, qualitative PCR, specific detection

田锦, 张月秋, 张华, 陈子言, 田璐, 王颢潜, 高芳瑞, 梁晋刚, 陈红. 转基因玉米浙大瑞丰8特异性定性PCR检测方法研究[J]. 生物技术通报, 2024, 40(12): 45-52.

TIAN Jin, ZHANG Yue-qiu, ZHANG Hua, CHEN Zi-yan, TIAN Lu, WANG Hao-qian, GAO Fang-rui, LIANG Jin-gang, CHEN Hong. Specific Qualitative PCR Detection Method for Transgenic Maize Zheda Ruifeng 8[J]. Biotechnology Bulletin, 2024, 40(12): 45-52.

图/表 11

表1 用于PCR检测浙大瑞丰8转化体的候选引物

Table 1 Candidate primers for PCR detecting Zheda Ruifeng 8 event

引物Primer	序列 Sequence（5'-3'）	引物Primer	序列Sequence（5'-3'）
RF 8-LB-F1	GGGTTTCGCTCATGTGTTGAG	RF 8-RB-F1	AGTTGGCGTCTCTCTGTTCG
RF 8-LB-F2	CGTCCGCAATGTGTTATTAAGTTGTCTA	RF 8-RB-F2	GTCGTTACTGAAACCGGTCC
RF 8-LB-F3	AGGAGCTCGAGTTTCTCCAT	RF 8-RB-F3	TCGTTACTGAAACCGGTCCAT
RF 8-LB-F4	AGCGTCAATTTGTTTACACCACA	RF 8-RB-F4	ATCCCAACCATTGCCGTCTT
RF 8-LB-F5	ACTAAAATCCAGATCCCCCGAA	RF 8-RB-F5	CCGTCTTGATGAGTTGGCGT
RF 8-LB-F6	GGTTTCGCTCATGTGTTGAG	RF 8-RB-F6	TGCCGTCTTGATGAGTTGGC
RF 8-LB-R1	GTGTCGACGGCTGAGATGAA	RF 8-RB-R1	AAAAACGTCCGCAATGTGTT
RF 8-LB-R2	AGTGCCAATACATACGCAACTGTTGCAG	RF 8-RB-R2	CTGAGGCCACCGCCTAATA
RF 8-LB-R3	ATACGATCGGATTCGTGGGA	RF 8-RB-R3	CTGAGGCCACCGCCTAAT
RF 8-LB-R4	GAACCAGAGCACTCTCACCA	RF 8-RB-R4	CGTCCGCAATGTGTTATTAAGT
RF 8-LB-R5	GGCCCATGAGTCCAAGAACC	RF 8-RB-R5	CATTAAAAACGTCCGCAATGTGT
RF 8-LB-R6	CAACTGGCGACACAGGGT	RF 8-RB-R6	TTAAAAACGTCCGCAATGTGTT

表2 浙大瑞丰8转化体PCR检测的引物组合

Table 2 Primer pairs for PCR detecting Zheda Ruifeng 8 event

序号 No.	组合 Group	产物大小 Amplication size/bp	序号 No.	组合 Group	产物大小 Amplication size/bp	序号 No.	组合 Group	产物大小 Amplication size/bp
1	RF 8-RB-R1/F1	270	15	RF 8-RB-R5/F5	285	28	RF 8-LB-F2/R6	282
2	RF 8-RB-R1/F4	294	16	RF 8-RB-R5/F6	287	29	RF 8-LB-F3/R3	299
3	RF 8-RB-R1/F5	281	17	RF 8-RB-R6/F1	272	30	RF 8-LB-F4/R4	141
4	RF 8-RB-R1/F6	283	18	RF 8-RB-R6/F4	296	31	RF 8-LB-F5/R1	149
5	RF 8-RB-R2/F2	295	19	RF 8-RB-R6/F5	283	32	RF 8-LB-F5/R2	179
6	RF 8-RB-R2/F3	294	20	RF 8-RB-R6/F6	285	33	RF 8-LB-F5/R3	128
7	RF 8-RB-R3/F2	295	21	RF 8-LB-F1/R1	254	34	RF 8-LB-F5/R4	222
8	RF 8-RB-R3/F3	294	22	RF 8-LB-F1/R2	284	35	RF 8-LB-F5/R5	237
9	RF 8-RB-R4/F1	265	23	RF 8-LB-F1/R3	233	36	RF 8-LB-F5/R6	165
10	RF 8-RB-R4/F4	289	24	RF 8-LB-F1/R6	270	37	RF 8-LB-F6/R1	253
11	RF 8-RB-R4/F5	276	25	RF 8-LB-F2/R1	266	38	RF 8-LB-F6/R2	283
12	RF 8-RB-R4/F6	278	26	RF 8-LB-F2/R2	290	39	RF 8-LB-F6/R3	232
13	RF 8-RB-R5/F1	274	27	RF 8-LB-F2/R3	245	40	RF 8-LB-F6/R6	269
14	RF 8-RB-R5/F4	298

图1 浙大瑞丰8转化体PCR检测引物筛选 M：DL1000 marker；1：1%浙大瑞丰8；2：阴性对照；3：空白对照

Fig. 1 Screening of the primers for PCR detecting Zheda Ruifeng 8 event M：DL1000 marker; 1: 1% Zheda Ruifeng 8 event; 2: negative control; 3: blank control

图2 浙大瑞丰8转化体PCR检测引物组合二次筛选 M：DL1000 marker；1：1%浙大瑞丰8；2：0.1%浙大瑞丰8；3：阴性对照（受体）；4：空白对照

Fig. 2 Secondary screening of the primers for PCR detection of Zheda Ruifeng 8 event M: DL1000 marker; 1: 1% Zheda Ruifeng 8 event; 2: 0.1%; 3: negative control; 4: blank control

图3 浙大瑞丰8转化体引物反应浓度优化 M：DL1000 marker；1：引物浓度0.2 μmol/L；2：引物浓度0.4 μmol/L；3：引物浓度0.6 μmol/L；4：引物浓度0.8 μmol/L；5：引物浓度1.0 μmol/L；6：空白对照

Fig. 3 Optimization of primer concentration for Zheda Ruifeng 8 event M: DL1000 marker; 1: primer concentration 0.2 μmol/L; 2: primer concentration 0.4 μmol/L; 3: primer concentration 0.6 μmol/L; 4: primer concentration 0.8 μmol/L; 5: primer concentration 1.0 μmol/L; 6: blank control

图4 浙大瑞丰8转化体PCR特异性测试 A：各被检样品内标准基因扩增结果。M：DL1000 marker；1-8分别对应B图1-8样品的内标准基因（其中玉米内标准基因zSSIIb扩增片段151 bp；大豆内标准基因Lectin扩增片段210 bp；水稻内标准基因SPS扩增片段287 bp；油菜内标准基因HMG I/Y扩增片段206 bp；棉花内标准基因ACP扩增片段210 bp）；B：浙大瑞丰8特异性引物扩增结果。M：DL1000 marker；1：1% 浙大瑞丰8；2：受体浙大瑞丰1；3：空白对照；4：其他转基因玉米混样；5：转基因大豆混样；6：转基因水稻混样；7：转基因油菜混样；8：转基因棉花混样

Fig. 4 Specificity PCR test for Zheda Ruifeng 8 event A：Results of standard gene amplification in each sample. M：DL1000 marker；1-8 corresponds to the internal standard genes in figure B, respectively. B: Results of specific primer amplification by Zheda Ruifeng 8. M：DL1000 marker；1：1% Zheda Ruifeng 8；2：receptor maize of Ruifeng 1；3：blank control；4：other genetically modified（GM）corn mixed samples；5：GM soybean mixed samples；6：GM rice mixed samples；7：GM rape mixed samples；8：GM cotton mixed samples

图5 浙大瑞丰8转化体PCR检测方法灵敏度测试 A：玉米内标准基因电泳图；B：浙大瑞丰8转化体电泳图。M：DL1000 marker；1：1%；2：0.5%；3：0.1%；4：0.05%；5：0.01%；6：阴性对照；7：空白对照

Fig. 5 Sensitivity test for PCR detection method of Zheda Ruifeng 8 event A: Electrophoresis diagram of standard gene in maize. B: Electrophoresis diagram of Zheda Ruifeng 8 event. M: DL1000 marker; 1: 1%; 2: 0.5%; 3: 0.1%; 4: 0.05%; 5: 0.01%; 6: negative control; 7: blank control

图6 浙大瑞丰8转化体检出限测试 A：玉米内标准基因电泳图；B：浙大瑞丰8转化体电泳图。M：DL1000 marker；1：阴性对照；2：空白对照；3-62：0.1%浙大瑞丰8

Fig. 6 Quantitative limit test for Zheda Ruifeng 8 event A: Electrophoresis diagram of standard gene in maize; B: electrophoresis diagram of Zheda Ruifeng 8 event. M: DL1000 marker; 1: negative control; 2: blank control; 3-62: 0.1% Zheda Ruifeng 8

图7 浙大瑞丰8转化体再现性测试

Fig. 7 Reproducibility test for Zheda Ruifeng 8 event M：DL1000 marker；1：5%；2：1%；3：0.1%；4：0.05%；5：0%；6：negative control

表3 浙大瑞丰8转化体特异性验证

Table 3 Event-specific verification for Zheda Ruifeng 8 event

Lab	样品名称/编号 Sample name/No.	1	2	3	4	5	6
Lab1 - Lab8	阳性对照 Positive control	+	+	+	+	+	+
	阴性对照 Negative control	-	-	-	-	-	-
	1%浙大瑞丰8 1% Zheda Ruifeng 8	+	+	+	+	+	+
	其他转基因玉米混样 Transgenic corn mixture samples	-	-	-	-	-	-
	转基因大豆混样 Transgenic soybean mixture samples	-	-	-	-	-	-
	转基因水稻混样 Transgenic rice mixture samples	-	-	-	-	-	-
	转基因棉花混样 Transgenic cotton mixture samples	-	-	-	-	-	-
	转基因油菜混样 Transgenic rape mixture samples	-	-	-	-	-	-
	非转基因玉米混样 Non-transgenic maize mixture samples	-	-	-	-	-	-
	0.1%浙大瑞丰8 0.1% Zheda Ruifeng 8	+	+	+	+	+	+

表4 浙大瑞丰8转化体检出限循环测试结果

Table 4 Cyclic verification for quantitative limit test for Zheda Ruifeng 8 event

Lab	0.1%浙大瑞丰8 0.1% transgenic maize Zheda Ruifeng 8
Lab	1	2	3	4	5	6
Lab1	+	+	+	+	+	+
Lab2	+	+	+	+	+	+
Lab3	+	+	+	+	+	+
Lab4	+	+	+	+	+	+
Lab5	+	+	+	+	+	+
Lab6	+	+	+	+	+	+
Lab7	+	+	+	+	+	+
Lab8	+	+	+	+	+	+

参考文献 16

[1]	黎裕, 徐辰武, 秦峰, 等. 玉米生物育种:现状与展望[J]. 中国基础科学, 2022, 24(4): 18-28.
	Li Y, Xu CW, Qin F, et al. Maize breeding: advances and prospects[J]. China Basic Sci, 2022, 24(4): 18-28.
[2]	徐晓丽, 姜媛媛, 王鹏飞, 等. 表达Cry1Ab和Cry2Ab蛋白的转基因玉米GAB-3对四种主要鳞翅目害虫的抗性评价[J]. 中国农业科技导报, 2020, 22(12): 97-104. doi: 10.13304/j.nykjdb.2020.0388
	Xu XL, Jiang YY, Wang PF, et al. Resistance evaluation of genetically modified maize GAB-3 expressing Cry1Ab and Cry2Ab against four major lepidopteran pests[J]. J Agric Sci Technol, 2020, 22(12): 97-104.
[3]	雷展, 王建成, 张晨, 等. 转基因油菜NS-B50027-4定性定量检测方法的建立[J]. 农业生物技术学报, 2021, 29(9): 1825-1835.
	Lei Z, Wang JC, Zhang C, et al. Establishment of qualitative and quantitative detection method for transgenic Brassica napus NS-B50027-4[J]. J Agric Biotechnol, 2021, 29(9): 1825-1835.
[4]	斯能武, 李俊, 武玉花, 等. 数字PCR在转基因定量检测中的研究进展[J]. 中国油料作物学报, 2021, 43(1): 40-50.
	Si NW, Li J, Wu YH, et al. Research progress of digital PCR in quantitative detection of genetically modified organism[J]. Chin J Oil Crop Sci, 2021, 43(1): 40-50. doi: 10.19802/j.issn.1007-9084.2020265
[5]	刘欣, 祝长青, 王毅谦, 等. 大豆转基因检测中DNA提取方法的比较研究[J]. 食品科学, 2013, 34(4): 199-203.
	Liu X, Zhu CQ, Wang YQ, et al. Methodology comparison of DNA extraction from soybean for detection of genetically modified organism[J]. Food Sci, 2013, 34(4): 199-203.
[6]	Gryson N. Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review[J]. Anal Bioanal Chem, 2010, 396(6): 2003-2022. doi: 10.1007/s00216-009-3343-2 pmid: 20012944
[7]	中华人民共和国农业部.转基因植物及其产品成分检测定性PCR方法制定指南: 农业部2259号公告-4—2015[S]. 北京: 中国农业出版社, 2015.
	Ministry of Agriculture of the People's Republic of China.Detection of Genetically Modified Plants and Derived Products Guidelines for Establishing qualitative PCR Method: Department of Agriculture Notice 2259-4-2015[S]. Beijing: China Agriculture Press, 2015.
[8]	中华人民共和国农业部.转基因植物及其产品成分检测玉米内标准基因定性PCR方法: 农业部1861号公告-3—2012[S]. 北京: 中国农业出版社, 2013.
	Ministry of Agriculture of the People's Republic of China.Detection of genetically modified plants and derived products. Target-taxon-specifis qualitative PCR method for maize: Department of Agriculture Notice 1861-3-2012[S]. Beijing: China Agriculture Press, 2013.
[9]	李凌燕, 张旭冬, 陈子言, 等. 转基因抗虫耐除草剂玉米MON87411精准定量检测方法的建立[J]. 生物安全学报, 2023, 32(1): 38-45.
	Li LY, Zhang XD, Chen ZY, et al. Establishment of accurate quantitative detection method for insect-resistant and herbicide-tolerant maize MON87411[J]. J Biosaf, 2023, 32(1): 38-45.
[10]	Gatto F, Savini C, Baillie C, et al. Definition of minimum performance requirements for analytical methods of GMO testing[J]. Luxembourg: Publications Office, 2023.
[11]	阳丽, 操志林. 转基因玉米种子的PCR检测[J]. 江西农业学报, 2014, 26(4): 98-99, 106.
	Yang L, Cao ZL. PCR detection of transgenic maize seeds[J]. Acta Agric Jiangxi, 2014, 26(4): 98-99, 106.
[12]	尹全, 李忆, 宋君, 等. 多重PCR筛查检测进口转基因玉米[J]. 核农学报, 2016, 30(6): 1045-1053. doi: 10.11869/j.issn.100-8551.2016.06.1045
	Yin Q, Li Y, Song J, et al. Multiplex PCR detection of exogenous gene in the imported genetically modified maize[J]. J Nucl Agric Sci, 2016, 30(6): 1045-1053. doi: 10.11869/j.issn.100-8551.2016.06.1045
[13]	李葱葱, 王青山, 李飞武, 等. 用实时荧光PCR方法定量检测Bt176转基因玉米[J]. 吉林农业科学, 2007, 32(5): 24-27.
	Li CC, Wang QS, Li FW, et al. Quantitative detection of the genetically modified Bt176 maize using real-time fluorescent PCR method[J]. J Jilin Agric Sci, 2007, 32(5): 24-27.
[14]	金永梅, 马瑞, 于志晶, 等. 转基因水稻吉生粳2号的外源基因旁侧序列分离及事件特异性PCR检测方法[J]. 东北农业科学, 2016, 41(1): 14-19.
	Jin YM, Ma R, Yu ZJ, et al. Identification of the T-DNA flanking sequences and event-specific PCR detection of transgenic rice‘jishengjing 2’[J]. J Northeast Agric Sci, 2016, 41(1): 14-19.
[15]	郑子繁, 王颢潜, 高佳奇, 等. 耐除草剂转基因大豆SHZD32-1数字PCR绝对定量方法的建立[J]. 计量学报, 2023, 44(3): 415-421.
	Zheng ZF, Wang HQ, Gao JQ, et al. Development of digital PCR absolute quantification method for herbicide-tolerant genetically modified soybean SHZD32-1[J]. Acta Metrol Sin, 2023, 44(3): 415-421.
[16]	林萍萍, 史云鹏, 安鹏天, 等. 影响实时荧光PCR检测转基因作物的因素分析[J]. 植物检疫, 2022, 36(5): 50-53.
	Lin PP, Shi YP, An PT, et al. Analysis of influence factors on detection of transgenic crops by real-time PCR[J]. Plant Quar, 2022, 36(5): 50-53.

编辑推荐 0

Metrics

阅读次数

全文

HTML			PDF

最新录用	在线预览	正式出版	最新录用	在线预览	正式出版
0	0	8	0	0	55

来源	本网站	其他网站

次数	56	7
比例	89%	11%

摘要

最新录用	在线预览	正式出版

0	0	92

	来源	本网站

	次数	92
	比例	100%