Construction and application of a novel multiple target plasmid used for identification of 18 genetically modified soybean transformants

doi:10.3785/j.issn.1008-9209.2019.08.122

Journal of Zhejiang University (Agriculture and Life Sciences)

2020, Vol. 46

Issue (3): 280-290 DOI: 10.3785/j.issn.1008-9209.2019.08.122

Biological sciences & biotechnology

Construction and application of a novel multiple target plasmid used for identification of 18 genetically modified soybean transformants

Zongyong SHI¹,Ziyan CHEN²,Chen QI¹,Cheng WANG¹,Mengxiao ZHAO¹,Xiaying LI²,Wenbin WANG¹,Jianqin YUAN¹,Dongmei XU¹,Yonggang QIAO¹,Jiandong LIU¹,Xiujie ZHANG²(

),Jianhua GAO¹(

)

^1.College of Life Sciences, Shanxi Agricultural University, Jinzhong 030801, Shanxi, China
^2.Development Center for Science and Technology, Ministry of Agriculture and Rural Affairs, Beijing 100025, China

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Abstract

A novel multiple target plasmid (MTP) molecule that satisfies the identification of specific transgenic soybean transformants in China was constructed. Fourteen transformants approved for import into China and four ones unauthorized but have important application prospects, were chosen as the targets. The transformants’ specific sequences described in corresponding national detection standard and the detection sequence of the soybean endogenous reference gene Lectin were properly arranged and spliced, and then was inserted to linearized pUC18 plasmid to produce a multi-target plasmid pDDID-1905. Among the insertion sequences, plenty of restriction endonuclease recognition sites were dispersed uniquely, which could be used for later revision and/or update of the plasmid. Polymerase chain reaction (PCR) detection targeted on the 19 inserted sequences was performed to verify the utility of the plasmid. The results showed that all target sequences were successfully amplified and the products met the expected sizes, which indicated that the multi-target plasmid pDDID-1905 suitable for the identification of 18 transgenic transformants was constructed. Due to the widest target coverage of pDDID-1905, it will greatly simplify the cumbersome preparation of positive materials for identifying these transgenic soybean transformants and their derivates.

Key words： genetically modified soybean plasmid positive molecule transformant specific detection transformant specific sequence

Received: 12 August 2019 Published: 17 July 2020

CLC:

S 565.1

Corresponding Authors: Xiujie ZHANG,Jianhua GAO E-mail: zhxj7410@sina.com;gaojh_edu@163.com

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	Articles by authors
	Zongyong SHI
	Ziyan CHEN
	Chen QI
	Cheng WANG
	Mengxiao ZHAO
	Xiaying LI
	Wenbin WANG
	Jianqin YUAN
	Dongmei XU
	Yonggang QIAO
	Jiandong LIU
	Xiujie ZHANG
	Jianhua GAO

Cite this article:

Zongyong SHI,Ziyan CHEN,Chen QI,Cheng WANG,Mengxiao ZHAO,Xiaying LI,Wenbin WANG,Jianqin YUAN,Dongmei XU,Yonggang QIAO,Jiandong LIU,Xiujie ZHANG,Jianhua GAO. Construction and application of a novel multiple target plasmid used for identification of 18 genetically modified soybean transformants. Journal of Zhejiang University (Agriculture and Life Sciences), 2020, 46(3): 280-290.

URL:

http://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2019.08.122 OR http://www.zjujournals.com/agr/Y2020/V46/I3/280

一种用于鉴定18种转基因大豆转化体的多靶标质粒的构建与应用

本文拟构建一种可满足我国转基因大豆转化体鉴定需求的多靶标质粒（multiple target plasmid, MTP）分子。根据相应的国家农业检测标准，选定我国已经批准进口的14种独立转化体和尚未批准但具有重要应用前景的4种重要转化体，将这18种转化体的特征序列以及大豆内标准基因Lectin的检测序列合理排列后，接入pUC18质粒，构建一种多靶标质粒pDDID-1905。在插入序列之间，分布着多种位点单一的限制性内切酶识别序列，可用于后期对该质粒的修订和更新。对pDDID-1905质粒中包含的19种靶标序列进行聚合酶链式反应检测，以验证该质粒的实用性。结果显示，所有靶标序列均被成功扩增，其大小与预期一致。总之，本文构建的适用于18种转基因大豆转化体鉴定的阳性质粒分子pDDID-1905，是目前包含大豆转化体特征序列最多的多靶标质粒，其构建和应用将极大地简化烦琐的阳性物质准备工作。

关键词： 转基因大豆, 质粒, 阳性物质, 转化体特异性检测, 转化体特征序列

Table 1 Eighteen genetically modified soybean events and their modified traits

序号 No.	引物名称 Primer name	引物序列（5′→3′） Primer sequence (5′→3′)	产物长度 Product length/bp	检测方法出处 Source of detection method
1	Lec-1672F	GGGTGAGGATAGGGTTCTCTG	210	农业部公告第2031号-8-2013
1	Lec-1881R	GCGATCGAGTAGTGAGAGTCG	210	农业部公告第2031号-8-2013
2	A2704-F	TGAGGGGGTCAAAGACCAAG	239	农业部公告第1485号-7-2010
2	A2704-R	CCAGTCTTTACGGCGAGT	239	农业部公告第1485号-7-2010
3	A5547-F	CGCCATTATCGCCATTCC	317	农业部公告第1485号-8-2010
3	A5547-R	GCGGTATTATCCCGTATTGA	317	农业部公告第1485号-8-2010
4	CV127-F	CCTTCGCCGTTTAGTGTATAGG	238	农业部公告第1782号-5-2012
4	CV127-R	AGCAGGTTCGTTTAAGGATGAA	238	农业部公告第1782号-5-2012
5	44406-F	GGGGCCTGACATAGTAGCT	259	农业农村部公告第111号-11-2018
5	44406-R	TAATATTGTACGGCTAAGAGCGAA	259	农业农村部公告第111号-11-2018
6	305423-F	CGTCAGGAATAAAGGAAGTACAGTA	235	农业部公告第1782号-4-2012
6	305423-R	GCCCTAAAGGATGCGTATAGAGT	235	农业部公告第1782号-4-2012
7	356043-F	CTTTTGCCCGAGGTCGTTAG	145	农业部公告第1782号-1-2012
7	356043-R	GCCCTTTGGTCTTCTGAGACTG	145	农业部公告第1782号-1-2012
8	FG72-F	TCGGGCTGCAGGAATTAATGT	150	农业部公告第2259号-8-2015
8	FG72-R	TTTGGAGCAATAAACATGTGATAGC	150	农业部公告第2259号-8-2015
9	GTS40-3-2-F	TTCAAACCCTTCAATTTAACCGAT	370	农业部公告第1861号-2-2012
9	GTS40-3-2-R	AAGGATAGTGGGATTGTGCGTC	370	农业部公告第1861号-2-2012
10	MON87701-MF	GCACGCTTAGTGTGTGTGTCAAAC	150	农业部公告第2259号-7-2015
10	MON87701-MR	GGATCCGTCGACCTGCAGTTAAC	150	农业部公告第2259号-7-2015
11	MON87705-F	CGCCAAATCGTGAAGTTTCTCATCT	318	农业部公告第2122号-4-2014
11	MON87705-R	CAGTGATAACAACACCCTGAGTCT	318	农业部公告第2122号-4-2014
12	MON87708-F	CCATCATACTCATTGCTGATCCA	233	农业部公告第2259号-6-2015
12	MON87708-R	AGCCAATCAATCTCAGAACTGTC	233	农业部公告第2259号-6-2015
13	MON87769-F	CCGGACATGAAGCCATTTAC	298	农业部公告第2122号-5-2014
13	MON87769-R	TCCTTGGAGGTCGTCTCATT	298	农业部公告第2122号-5-2014
14	MON89788-F	CTGCTCCACTCTTCCTTT	223	农业部公告第1485号-6-2010
14	MON89788-R	AGACTCTGTACCCTGACCT	223	农业部公告第1485号-6-2010
15	SYHT0H2-F	GAGGCACCAACATTCTT	234	农业农村部公告第111号-10-2018
15	SYHT0H2-R	TATCCGCAATGTGTTATTAA	234	农业农村部公告第111号-10-2018
16	DAS-68416-4-F	CCGCTACTTGCTCTTGTCGT	221	农业部公告第2630号-5-2017
16	DAS-68416-4-R	CGGTTAGGATCCGGTGAGTA	221	农业部公告第2630号-5-2017
17	DAS-81419-2-F	CCCATGTGAAGAAAATCCAACCAT	252	农业农村部公告第111号-9-2018
17	DAS-81419-2-R	CCGAGAAGGTCAAACATGCTAA	252	农业农村部公告第111号-9-2018
18	SHZD32-1F	GAGCAGCTTGAGCTTGGA	234	农业部公告第2630号-15-2017
18	SHZD32-1R	CGAATTTCACCAAAACACTAA	234	农业部公告第2630号-15-2017
19	MON87751-F	TAGAGGAATAGTTAGCGAATGTGAC	268	待发布
19	MON87751-R	GACAGACCTCAATTGCGAGC	268	待发布

Table 2 PCR detection of 19 target sequences

Fig. 1 Insertion sequence in pDDID-1905 plasmidThe main restriction sites were indicated in bold font with underlines.

Fig. 2 Diagram of pDDID-1905 plasmid construction

Fig. 3 Identification of pDDID-1905 plasmidA. Restriction map of pDDID-1905 plasmid (M: 1 kb plus DNA ladder; Lane 1: Restriction map by EcoRⅠ and HindⅢ; Lane 2: Restriction map by EcoRⅠ, HindⅢ and XhoⅠ); B. Result of the assembled sequencing contigs of pDDID-1905 plasmid.

Fig. 4 PCR products of specific sequences of pDDID-1905 plasmidM: DL2000 DNA marker (2 000, 1 000, 750, 500, 250 and 100 bp from top to bottom); －: Blank control (ddH₂O was used as the template for PCR); pUC18: Unmodified pUC18 plasmid; Lane 1-2: pDDID-1905 plasmid.

Fig. 5 PCR products of the transgenic soybean mixturesM: DL2000 DNA marker (2 000, 1 000, 750, 500, 250 and 100 bp from top to bottom); －: Blank control (ddH₂O was used as the template for PCR); N: Non-transgenic soybean; Lane 1-2: Transgenic soybean mixtures; +: pDDID-1905 plasmid.


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