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

doi:10.3969/j.issn.1004-1524.2022.08.13

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (8): 1692-1702.DOI: 10.3969/j.issn.1004-1524.2022.08.13

• Horticultural Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S681.9

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.

Figures/Tables 10

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

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'.

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

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

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

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	$\bar{X}$ /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

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	$\bar{X}$ /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

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.

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

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

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

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