转基因康乃馨Moonlite品系检测用质粒标准样品研制

doi:10.3969/j.issn.1004-1524.2022.08.13

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (8): 1692-1702.DOI: 10.3969/j.issn.1004-1524.2022.08.13

转基因康乃馨Moonlite品系检测用质粒标准样品研制

杨茹梦¹(), 尹璐², 钱昌元², 左翠花², 于海燕¹^,^*(), 李想²^,^*()

1.上海应用技术大学香料香精技术与工程学院,上海 201418
2.上海海关动植物与食品检验检疫技术中心,上海 200135

收稿日期:2021-10-13 出版日期:2022-08-25 发布日期:2022-08-26
通讯作者: 于海燕,李想
作者简介:李想,E-mail: idealne@163.com
*于海燕,E-mail: hyy@usit.edu.cn;
杨茹梦(1996—),女,河南新乡人,硕士研究生,主要从事转基因检测技术研究。E-mail: 1370560777@qq.com
基金资助:
上海市技术标准项目(21DZ2203000);海关总署科研项目(2020HK164)

Development of plasmid standard material for detection of genetically modified carnation line Moonlite

YANG Rumeng¹(), YIN Lu², QIAN Changyuan², ZUO Cuihua², YU Haiyan¹^,^*(), LI Xiang²^,^*()

1. School of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai 201418, China
2. Technical Center for Animal, Plant and Food Inspection and Quarantine of Shanghai Customs, Shanghai 200135, China

Received:2021-10-13 Online:2022-08-25 Published:2022-08-26
Contact: YU Haiyan,LI Xiang

摘要/Abstract

摘要：

截至2020年底,国际上已有19种转基因康乃馨品系进入商业化种植。为了实现转基因康乃馨的有效检测监管,研制检测标准样品十分必要。目前,国际上还没有该类标准品的相关报道。为此,本文研制了商业化时间最长、种植量较大的转基因康乃馨Moonlite品系质粒标准样品,使其与转基因康乃馨检测技术标准的应用相配套,为口岸转基因产品的检测监管提供必要的技术支撑。首先,将康乃馨内源ANS基因1 279 bp片段和转基因康乃馨Moonlite品系406 bp 5'端特异性序列分别插入到pcDNA3.0载体的酶切位点和距离1 kb以上的多克隆位点上,合成质粒DNA标准样品。后将质粒DNA标准样品进行稀释、分装,对其均匀性、最小取样量和短期、长期稳定性等进行测定,采用数字PCR方法对质粒DNA标准样品进行定值,并进行不确定度评估。质粒DNA标准样品pLite经全基因组测序,与公开发布的序列完全一致。均匀性实验结果表明,分别以ANS基因和Moonlite品系特异性序列为目标进行测定,F值分别为1.61和1.14,均小于95%置信区间的F值,质粒DNA标准样品足够均匀。质粒DNA标准样品的最小取样量为2 μL。稳定性实验结果表明,质粒DNA标准样品pLite在45 ℃高温条件下,可稳定保存14 d;在26 ℃及以下温度条件下,可稳定保存1个月,在4 ℃及以下温度可稳定保存6个月,说明质粒DNA标准样品适宜运输。质粒DNA标准样品在反复冻融25次后,拷贝数浓度无显著变化。定值结果表明,质粒DNA标准样品pLite中康乃馨内源ANS基因拷贝数的参考值为(6.13±0.22)×10⁶ μL^-1,Moonlite品系特异性序列拷贝数的参考值为(6.10±0.24)×10⁶ μL^-1。研制的质粒DNA标准样品pLite具有良好的均匀性和稳定性,获得国家标准样品证书后,可用于转基因康乃馨Moonlite品系的检测。

关键词: 转基因康乃馨, Moonlite品系, 质粒标准样品

Abstract:

By the end of 2020 year, nineteen genetically modified (GM) carnation lines had been commercialized in the world. In order to detect and supervise GM carnations effectively, it is necessary to develop standard materials. Until now, there are no standard materials for GM carnation detection that have been reported around the world. Therefore, this study developed a plasmid standard molecule pLite for GM carnation line Moonlite, which has been commercialized and planted for a long time, so that it could be suitable for the application of the standard for GM carnation detection, and could support the inspection and supervision of GM products imported and exported. To develop a plasmid DNA molecule for Moonlite, two fragments of carnation endogenous ANS gene with 1 279 bp in length and the 5'-end event-specific sequence of GM carnation Moonlite with 406 bp in length were inserted into plasmid pcDNA3.0. The distance between the two inserted sites was more than 1 kb. Then, the plasmid DNA was diluted and packaged. The homogeneity, minimum sampling amount, short-term and long-term stability and reference values of the plasmid were measured, meanwhile, the uncertainty were evaluated. The genome sequencing results showed that the sequence of pLite was consistent with the published sequence. Results of the homogeneity test showed that F values of ANS gene and Moonlite event-specific sequence were 1.61 and 1.14, respectively, which were both less than the F value under 95% confidence interval, indicating that the plasmid DNA molecule was homogeneous. The minimum sampling amount of pLite was 2 μL. Results of stability experiments showed that pLite stayed stable at 45 ℃ for 14 days. At room temperature of 26 ℃ and below, the plasmid DNA molecule could be stably stored for one month, and could be stably stored for 6 months at 4 ℃ and below, indicating that pLite was suitable for transportation. After repeated freeze-thaw 25 times, the copy number of pLite had no significant change, which could meet the requirements of daily detection. The calculation results indicated that the reference values of ANS gene and Moonlite specific sequence were (6.13±0.22)×10⁶ μL^-1 and (6.10±0.24)×10⁶ μL^-1 in pLite, respectively. The developed plasmid DNA molecule pLite had good homogeneity and stability, at the same time it was suitable for the detection of GM carnation event Moonlite.

Key words: genetically modified carnation, Moonlite, plasmid standard material

中图分类号:

S681.9

杨茹梦, 尹璐, 钱昌元, 左翠花, 于海燕, 李想. 转基因康乃馨Moonlite品系检测用质粒标准样品研制[J]. 浙江农业学报, 2022, 34(8): 1692-1702.

YANG Rumeng, YIN Lu, QIAN Changyuan, ZUO Cuihua, YU Haiyan, LI Xiang. Development of plasmid standard material for detection of genetically modified carnation line Moonlite[J]. Acta Agriculturae Zhejiangensis, 2022, 34(8): 1692-1702.

图/表 10

表1 引物和探针序列信息

Table 1 Primers and probes sequence used in this paper

基因/质粒 Gene/plasmid	扩增片段大小 Length of amplified fragment/bp	引物/探针 Primer/Probe	序列 Sequence (5'→3')
ANS	91	ANS-F	cctaaatgtaagaacaacgcaatca
		ANS-R	gcgtagcgctgaacatcgt
		ANS-P	FAM-tataagaccaataaatggttgatggatggag-BHQ1
Moonlite	137	Moonlite-F	cattgcgaaagtgaattgatac
		Moonlite-R	gctgcggacatctacatttttg
		Moonlite-P	FAM-ctctttctttttctccatattgacc-BHQ1

图1 构建的转基因康乃馨Moonlite品系质粒DNA标准样品示意图和序列信息 A,转基因康乃馨Moonlite品系质粒DNA标准样品pLite示意图;B,插入的康乃馨内源ANS基因序列信息;C,插入的转基因康乃馨Moonlite品系特异性序列信息。核酸片段方向均为5'→3'。

Fig.1 Schematic diagram and sequences of the constructed plasmid DNA standard sample pLite for GM carnation line Moonlite A, Schematic diagram of pLite, a plasmid DNA standard sample for GM carnation Moonlite; B, Sequence of the inserted carnation endogenous gene ANS fragment; C, Sequence of the inserted GM carnation Moonlite event-specific sequence. The direction of nucleic acid was 5'to 3'.

图2 质粒DNA标准样品pLite电泳图 1,未线性化质粒DNA标准样品pLite;2,线性化质粒DNA标准样品pLite。

Fig.2 Electrophoretogram of plasmid DNA standard sample pLite 1, Unlinearized plasmid DNA standard sample pLite; 2, Linearized plasmid DNA standard sample pLite.

图3 质粒DNA标准样品pLite的标准曲线 ,ANS基因;B,Moonlite品系特异性序列。

Fig.3 Standard curves of plasmid DNA standard sample pLite A, ANS gene; B, Moonlite specific sequence.

表2 质粒DNA标准样品pLite均匀性方差分析

Table 2 Variance analysis of homogeneity of the plasmid DNA standard sample pLite

基因/质粒 Gene/plasmid		离差平方和 Sum of squares of deviations	自由度 Degree of freedom	方差 Variance	F	F_0.05(14,30)
ANS	组间Intergroup	1.13	14	0.08	1.61	2.04
	组内Intragroup	1.50	30	0.05
Moonlite	组间Intergroup	2.79	14	0.20	1.14	2.04
	组内Intragroup	5.23	30	0.17

表3 质粒DNA标准样品pLite最小取样量测定

Table 3 Determination of the minimum sampling volume of plasmid DNA standard sample pLite

取样量Sampling volume/μL	基因/质粒Gene/plasmid	$X ¯$ /10⁶ μL^-1	N	F	F_0.05(2,24)
0.5	ANS	1.36	27	10.39	3.40
	Moonlite	1.29	27	9.09	3.40
1.0	ANS	3.12	27	4.63	3.40
	Moonlite	3.01	27	4.59	3.40
2.0	ANS	6.30	27	0.13	3.40
	Moonlite	6.30	27	0.26	3.40

表3 质粒DNA标准样品pLite最小取样量测定

Table 3 Determination of the minimum sampling volume of plasmid DNA standard sample pLite

取样量Sampling volume/μL	基因/质粒Gene/plasmid	$X ¯$ /10⁶ μL^-1	N	F	F_0.05(2,24)
0.5	ANS	1.36	27	10.39	3.40
	Moonlite	1.29	27	9.09	3.40
1.0	ANS	3.12	27	4.63	3.40
	Moonlite	3.01	27	4.59	3.40
2.0	ANS	6.30	27	0.13	3.40
	Moonlite	6.30	27	0.26	3.40

图4 质粒DNA标准样品pLite在不同温度条件下的稳定性变化曲线 A,pLite在-20、4、26 ℃放置4个星期的稳定性变化曲线;B,pLite在45 ℃放置14 d的稳定性变化曲线。

Fig.4 Stability curves of plasmid DNA standard sample pLite under different temperatures A, Stability curves of pLite under -20, 4, 26 ℃ within 4 weeks; B, Stability curves of pLite under 45 ℃ within 14 days.

表4 质粒DNA标准样品pLite长期稳定性

Table 4 Long-term stability determination of plasmid DNA standard sample pLite 106 μL-1

温度/℃	基因/质粒 Gene/plasmid	储存不同时间(月)的拷贝数浓度 Copy number at different storage time (month)/10⁶ μL^-1						β₁	s(β₁)	t_0.95,_n_-2	t_0.95,_n_-2*s (β₁)
温度/℃	基因/质粒 Gene/plasmid	0	1	2	3	4	6	β₁	s(β₁)	t_0.95,_n_-2	t_0.95,_n_-2*s (β₁)
-20	ANS	6.45	6.24	6.43	6.34	6.39	6.38	0.001	0.02	2.78	0.048
	Moonlite	6.46	6.34	6.43	6.36	6.38	6.33	-0.015	0.01	2.78	0.025
4	ANS	6.52	6.37	6.41	6.28	6.23	6.38	-0.026	0.02	2.78	0.054
	Moonlite	6.36	6.39	6.39	6.33	6.30	6.38	-0.004	0.01	2.78	0.024

图5 质粒DNA标准样品pLite数字PCR一维散点图 A,康乃馨内源ANS基因;B,Moonlite品系特异性序列。

Fig.5 1-D scatter plot of plasmid DNA standard sample pLite by digital PCR A, Carnation endogenous gene ANS; B, Moonlite specific sequence.

表5 质粒DNA标准样品pLite浓度的数字PCR结果

Table 5 Concentration of plasmid DNA molecule pLite detected by digital PCR 106 μL-1

样品编号 Sample No.	ANS的4次重复测定值 Test values of four repeats of ANS				Moonlite的4次重复测定值 Test values of four repeats of Moonlite
样品编号 Sample No.	1	2	3	4	1	2	3	4
1	5.89	6.04	6.12	6.20	5.98	5.80	5.89	5.71
2	5.91	6.04	5.95	6.01	6.04	6.30	6.22	5.75
3	6.15	6.12	6.19	6.13	6.27	5.81	6.25	5.99
4	6.29	6.19	6.08	6.30	6.35	6.19	6.29	6.08
5	6.32	6.31	6.22	6.09	6.23	6.20	6.34	6.24

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转基因康乃馨Moonlite品系检测用质粒标准样品研制

Development of plasmid standard material for detection of genetically modified carnation line Moonlite

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