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作物学报 ›› 2022, Vol. 48 ›› Issue (7): 1843-1850.doi: 10.3724/SP.J.1006.2022.13043

• 研究简报 • 上一篇    

基于高通量测序技术的转基因玉米GM11061分子特征研究

杨迎霞1(), 张冠1,2, 王梦梦1,2, 陆国清1, 王倩1, 陈锐1,*()   

  1. 1天津市农业科学院种质资源与生物技术研究所, 天津 300381
    2天津商业大学生物技术与食品科学学院 / 天津市食品生物技术重点实验室, 天津 300134
  • 收稿日期:2021-06-03 接受日期:2021-11-30 出版日期:2022-07-12 网络出版日期:2021-12-27
  • 通讯作者: 陈锐
  • 作者简介:E-mail: yingxiayang@126.com
  • 基金资助:
    农业农村部农业转基因生物安全评价(食用)重点实验室开放课题(SAGM-Y-2020009)

Molecular characterization of transgenic maize GM11061 based on high-throughput sequencing technology

YANG Ying-Xia1(), ZHANG Guan1,2, WANG Meng-Meng1,2, LU Guo-Qing1, WANG Qian1, CHEN Rui1,*()   

  1. 1Institute of Germplasm Resources and Biotechnology, Tianjin Academy of Agricultural Sciences, Tianjin 300381, China
    2Tianjin Key Laboratory of Food Biotechnology / School of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China
  • Received:2021-06-03 Accepted:2021-11-30 Published:2022-07-12 Published online:2021-12-27
  • Contact: CHEN Rui

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摘要：

鉴定外源DNA片段在受体基因组中的插入位点、整合序列与拷贝数信息是转基因植物安全评价体系的关键环节。传统的转基因植物分子特征鉴定技术繁杂费力、耗时低效且具有一定的局限性。本研究利用基因组重测序技术与实验室自建的数据分析流程, 针对转基因玉米GM11061开展了分子特征鉴定研究, 结果表明: 外源DNA片段仅插入受体基因组一个拷贝, 位于5号染色体198,621,57~198,621,620 bp之间, 不含载体骨架序列, 并通过Sanger测序验证了其上下游结合位点。测序数据量梯度分析显示, 最低~5×的重测序原始数据可实现该整合位点的鉴定。本研究证实Illumina高通量测序技术与配套的数据分析方法结合可实现简易、快速、精准的植物分子特征鉴定研究。本研究结果有助于植物功能基因组学基础研究, 同时也为转基因安全评价体系的完善提供技术支撑。

关键词: 高通量测序, 转基因玉米, 分子特征

Abstract:

Insertion site, integrated sequences, and copy number information of exogenous DNA integrations in host genome are key steps in the safety evaluation of genetically modified plants. Traditional identification techniques for molecular characteristics of transgenic plants are complicated, laborious, inefficient and limited. Here, we reported the molecular characterization of one new transgenic maize event GM11061 via a whole genome (paired-end) sequencing approach and laboratory-developed bioinformatics methods. The results showed there was only one copy of the exogenous DNA inserted, which was located within the region of 198,621,571-198,621,620 bp on chromosome 5 in GM11061 genome, without the vector skeleton sequence. Its upstream and downstream binding sites were validated using conventional PCR and sanger sequencing. Gradient analysis of sequencing data revealed that the lowest 5× resequencing raw data could be used to identify the integration site. The study confirmed that the whole genome resequencing approach combined with bioinformatics methods could achieve simple, rapid, and accurate identification of plant molecular characteristics. These results are helpful to the basic research of functional genomics and provide technical support for the safety evaluation of transgenic plants.

Key words: high throughput sequencing, transgenic maize, molecular characterization

图1

基于全基因组测序的转基因玉米GM11061分子特征分析流程及Clean reads过滤读序示意图"

表1

质粒序列与玉米参考基因组blastn比对结果"

查询序列Query 目标序列
Subject		 一致性
百分比
Identity
(%)		 区域长度
Alignment length		 错配数Mismatch Gap数目
Gap
opening		 查询序列
起始位点Query
start site		 查询序列
终止位点Query
end site		 目标序列
起始位点
Subject
start site		 目标序列
终止位点
Subject
end site		 E-value Bit score
GM11061 NC_024463.2 99.950 1993 0 1 6861 8853 84,635,058 84,633,067 0 3674
GM11061 NC_024463.2 89.778 225 12 4 8189 8405 84,633,511 84,633,290 4.30E-71 278
GM11061 NC_024463.2 89.778 225 12 4 8409 8630 84,633,731 84,633,515 4.30E-71 278
GM11061 NC_024466.2 99.876 804 1 0 5135 5938 174,182,636 174,181,833 0 1480

图2

转基因玉米GM11061左侧和右侧接合区Reads覆盖图 A: 左侧侧翼和T-DNA 5'端序列信息, 红色为基因组序列, 黑色为T-DNA序列; B: 右侧侧翼和T-DNA 3'端序列信息, 绿色为基因组序列, 黑色为T-DNA序列。"

图3

转基因玉米GM11061接合区IGV分析 粉色框表示玉米基因组与T-DNA边界的接合区。"

图4

转基因玉米GM11061插入位点及侧翼序列实验验证 A: T-DNA插入位点上下游设计的引物对位置; B: PCR扩增结果。1、3泳道为转基因玉米GM11061, 2、4泳道为非转基因受体材料; C: T-DNA插入位点上游PCR扩增产物Sanger测序峰图; D: T-DNA插入位点下游PCR扩增产物Sanger测序峰图。"

表2

megahit组装序列与G11061 T-DNA序列比对结果"

查询序列Query 目标序列Subject 一致性
百分比Identity (%)		 区域长度
Alignment length		 错配数Mismatch Gap数目
Gap
opening		 查询序列
起始位点Query
start site		 查询序列
终止位点Query
end site		 目标序列
起始位点Subject
start site		 目标序列
终止位点
Subject
end site		 E-value Bit score
k141_0 T-DNA 100 408 0 0 1 408 6313 6720 0 754
k141_0 T-DNA 100 266 0 0 1 266 6214 5949 2.61E-142 492
k141_0 T-DNA 99.306 144 1 0 265 408 10,099 10,242 8.01E-73 261
k141_3 T-DNA 100 437 0 0 1 437 10,102 10,538 0 808
k141_3 T-DNA 100 253 0 0 1 253 6580 6832 6.25E-135 468
k141_1 T-DNA 100 141 0 0 1 141 8713 8853 8.31E-73 261
k141_5 T-DNA 99.876 804 1 0 428 1231 5135 5938 0.00E+00 1480
k141_7 T-DNA 100 1993 0 0 1 1993 8853 6861 0 3681
k141_7 T-DNA 89.778 225 12 4 224 445 8405 8189 4.61E-77 278
k141_7 T-DNA 89.778 225 12 4 449 665 8630 8409 4.61E-77 278
k141_6 T-DNA 100 1530 0 0 1 1530 10,242 8713 0 2826
k141_6 T-DNA 99.306 144 1 0 1 144 6720 6577 3.08E-72 261
k141_9 T-DNA 99.761 4602 1 1 141 4732 155 4756 0.00E+00 8429
k141_9 T-DNA 100 498 0 0 4471 4968 4756 5253 0 920

图5

Megahit 拼接contigs与T-DNA序列blastn结果"

[1] Babekova R, Funk T, Pecoraro S, Engel K H, Busch U. Development of an event-specific real-time PCR detection method for the transgenic Bt rice line KMD1. Eur Food Res Technol, 2009, 228: 707-716.
doi: 10.1007/s00217-008-0981-0
[2] Arai-Kichise Y, Shiwa Y, Nagasaki H, Ebana K, Yoshikawa H, Yano M, Wakasa K. Discovery of genome-wide DNA polymorphisms in a landrace cultivar of Japonica rice by whole-genome sequencing. Plant Cell Physiol, 2011, 52: 274-282.
doi: 10.1093/pcp/pcr003 pmid: 21258067
[3] Bonfini L, Van den Bulcke M H, Mazzara M, Ben E, Patak A. GMOMETHODS: the European Union database of reference methods for GMO analysis. J AOAC Int, 2012, 95: 1713-1719.
[4] Tan G H, Gao Y, Shi M, Zhang X Y, He S P, Chen Z L, An C C. SiteFinding-PCR: a simple and efficient PCR method for chromosome walking. Nucleic Acids Res, 2005, 33: e122.
doi: 10.1093/nar/gni124
[5] Trinh Q, Xu W T, Shi H, Luo Y B, Huang K L. An A-T linker adapter polymerase chain reaction method for chromosome walking without restriction site cloning bias. Anal Biochem, 2012, 425: 62-67.
doi: 10.1016/j.ab.2012.02.029
[6] Trinh Q, Shi H, Xu W T, Hao J R, Luo Y B, Huang K L. Loop-linker PCR: an advanced PCR technique for genome walking. IUBMB Life, 2012, 64: 841-845.
doi: 10.1002/iub.1069
[7] Spalinskas R, Van den Bulcke M, Van den Eede G, Milcamps A. LT-RADE: an efficient user-friendly genome walking method applied to the molecular characterization of the insertion site of genetically modified maize MON810 and rice LLRICE62. Food Anal Methods, 2013, 6: 705-713.
doi: 10.1007/s12161-012-9438-y
[8] Fraiture M A, Herman P, Taverniers I, Taverniers I, Loose M D, Nieuwerburgh F V, Deforce D, Roosens N H. Validation of a sensitive DNA walking strategy to characterize unauthorized GMOs using model food matrices mimicking common rice products. Food Chem, 2015, 173: 1259-1265.
doi: 10.1016/j.foodchem.2014.09.148
[9] Kovalic D, Garnaat C, Guob L, Yan Y P, Groat J, Silvanovich A, Ralston L, Huang M Y, Tian Q, Christian A, Cheikh N, Hjelle J, Padgette S, Bannon G. The use of next generation sequencing and junction sequence analysis bioinformatics to achieve molecular characterization of crops improved through modern biotechnology. Plant Genome, 2012, 5: 149-163.
[10] Wahler D, Schauser L, Bendiek J, Grohmann L. Next-generation sequencing as a tool for detailed molecular characterization of genomic insertions and flanking regions in genetically modified plants: a pilot study using a rice event unauthorized in the EU. Food Anal Methods, 2013, 6: 1718-1727.
doi: 10.1007/s12161-013-9673-x
[11] Yang L T, Wang C M, Holst-Jensen A, Morisset D, Lin Y J, Zhang D B. Characterization of GM events by insert knowledge adapted re-sequencing approaches. Sci Rep, 2013, 3: 2839.
doi: 10.1038/srep02839
[12] Liang C J, van Dijk J P, Scholtens I M J, Staats M, Prins T W, Voorhuijzen M M, da Silva A M, Arisi A C M, den Dunnen J T, Kok E J. Detecting authorized and unauthorized genetically modified organisms containing vip3A by real-time PCR and next-generation sequencing. Anal Bioanal Chem, 2014, 406: 2603-2611.
doi: 10.1007/s00216-014-7667-1
[13] Fraiture M A, Vandamme J, Herman P, Roosens N H C. Development and validation of an integrated DNA walking strategy to detect GMO expressing cry genes. BMC Biotechnol, 2018, 18: 40.
doi: 10.1186/s12896-018-0446-x
[14] Fraiture M A, Saltykova A, Hoffman S, Winand R, Deforce D, Vanneste K, De Keersmaecker S C J, Roosens N H C. Nanopore sequencing technology: a new route for the fast detection of unauthorized GMO. Sci Rep, 2018, 8: 7903.
doi: 10.1038/s41598-018-26259-x
[15] Lepage É, Zampini É, Boyle B, Brisson N. Time- and cost- efficient identification of T-DNA insertion sites through targeted genomic sequencing. PLoS One, 2013, 8: e70912.
doi: 10.1371/journal.pone.0070912
[16] Guo B F, Guo Y, Hong H L, Qiu L J. Identification of genomic insertion and flanking sequence of G2-EPSPS and GAT transgenes in soybean using whole genome sequencing method. Front Plant Sci, 2016, 7: 1009.
[17] Guttikonda S K, Marri P, Mammadov J, Ye L, Soe K, Richey K, Cruse J, Zhang M B, Gao Z F, Evans C, Rounsley S, Kumpatla S P. Molecular characterization of transgenic events using next generation sequencing approach. PLoS One, 2016, 11: e0149515.
doi: 10.1371/journal.pone.0149515
[18] Park D, Park S H, Ban Y W, Kim Y S, Park K C, Kim N S, Kim J K, Choi I Y. A bioinformatics approach for identifying transgene insertion sites using whole genome sequencing data. BMC Biol, 2017, 17: 67.
doi: 10.1186/s12915-019-0676-y
[19] Wang X J, Jiao Y, Ma S, Yang J T, Wang Z X. Whole-Genome Sequencing: an effective strategy for insertion information analysis of foreign genes in transgenic plants. Front Plant Sci, 2020, 11: 573871.
doi: 10.3389/fpls.2020.573871
[20] Zhang Y C, Zhang H W, Qu Z, Zhang X J, Cui J J, Wang C H, Yang L T. Comprehensive analysis of the molecular characterization of GM rice G6H1 using a paired-end sequencing approach. Food Chem, 2020, 309: 125760.
doi: 10.1016/j.foodchem.2019.125760
[21] 马硕, 焦悦, 杨江涛, 王旭静, 王志兴. 基因组测序技术解析耐除草剂转基因水稻G2-7的分子特征. 作物学报, 2020, 46: 1703-1710.
Ma S, Jiao Y, Yang J T, Wang X J, Wang Z X.Molecular characterization identification by genome sequencing of transgenic glyphosate-tolerant rice G2-7. Acta Agron Sin, 2020, 46: 1703-1710.
[22] Cade R M, Burgin K, Schiling K, Lee T J, Ngam P, Devitt N, Fajardo D. Evaluation of whole genome sequencing and an insertion site characterization method for molecular characterization of GM maize. J Regul Sci, 2018, 6: 1-14.
[23] Siddique K, Wei J, Li R, Zhang D B, Shi J X. Identification of T-DNA insertion site and flanking sequence of a genetically modified maize event IE09S034 using next-generation sequencing technology. Mol Biotechnol, 2019, 61: 694-702.
doi: 10.1007/s12033-019-00196-0 pmid: 31256331
[24] Duan L J, Zhang S S, Yang Y X, Wang Q, Lan Q K, Wang Y, Xu W T, Jin W J, Li L, Chen R. A feasible method for detecting unknown GMOs via a combined strategy of PCR-based suppression subtractive hybridization and next-generation sequencing. Food Control, 2020, 119: 107448.
doi: 10.1016/j.foodcont.2020.107448
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