基于基因组重测序的高含量油酸转基因大豆T-DNA旁侧序列分析及事件特异性PCR检测
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摘要
基于外源T-DNA与植物基因组的连接区域的特异性建立的特异性检测方法,具有非常高的特异性和准确性,是实现对该转基因事件及其衍生品种的有效监督管理、保障转基因产业健康发展的重要技术手段。本研究前期利用反义RNA和RNAi技术,获得2个油酸含量达78%以上的高油酸转基因大豆(Glycine max)事件。由于T-DNA携带大豆内源基因,旁侧序列不易分析,且为进一步推进上述转化事件的安全评价及应用,本研究基于基因组重测序技术并结合PCR,分析上述2个转基因大豆事件外源TDNA整合位点旁侧序列。Southern杂交结果显示,2个高油酸转基因大豆事件E2D9050和EB8072外源T-DNA插入拷贝数分别为1个和2个。采用BWA-Burrows-Wheeler Alignment tool (http://bio-bwa.sourceforge.net/)方法,将基因组重测序分析获得的转基因事件序列信息与参考大豆基因组进行对比。结果表明,转基因大豆事件E2D9050整合位点为Chr04号染色体的51410941位点,整合方式为反向单拷贝插入;EB8072整合位点为大豆基因组Chr19染色体38147218位点,整合方式为单位点双拷贝反向插入,两个拷贝的右边界相接。结合PCR扩增,分别获得E2D9050和EB8072转基因事件整合位点的左、右边界旁侧序列。依据其序列特征,建立了转基因大豆事件两个转化体特异性检测方法,检测灵敏度为0.1%,为上述2个高油酸转基因大豆事件及其衍生产品特异性检测提供依据。
The specific detection method based on the specificity of the connection area between external TDNA and plant genome has very high specificity and accuracy, which is an important technical means to effectively supervise and manage the transgenic event and its derivatives, and ensure the healthy development of the transgenic industry. In the previous study, 2 transgenic soybean(Glycine max) events were obtainedwith >78% oleic acid content. To isolate the flanking sequences of T-DNAs with endogenous genes and promote the safety assessment and application of these 2 transgenic events, genome re-sequencing combined with PCR was carried out to analyze the flanking sequences of T-DNAs of these 2 transgenic events. There were 1 and 2 copies T-DNAs in these 2 transgenic events by Southern blot analysis, respectively. Using genome re-sequencing compared to reference genome information based on BWA-Burrows-Wheeler Alignment tool method(http://bio-bwa. sourceforge. net/), flanking sequences of T-DNAs in the transgenic soybean 'E2 D9050' and 'EB8072' genomes were isolated. The T-DNA of 'E2 D9050' was integrated into the position 51410941 on Chr04 with one copy. The T-DNA of 'EB8072' was integrated into soybean genome with2 copies in position 38147218 on Chr19 and the pattern of integration was inverted insertion with right borders of these copies interfaced. These results were in accord with the results of Southern blot analysis. According to the flanking sequences of these positions, primers were designed and PCR was amplified to verify these flanking sequences of insertion sites. Event-specific primers were designed based on these fragments and the sensitivities of these pairs of primer were both 0.1%. It provides an accurate and fast method for identification of these kinds of transgenic event.
The specific detection method based on the specificity of the connection area between external TDNA and plant genome has very high specificity and accuracy, which is an important technical means to effectively supervise and manage the transgenic event and its derivatives, and ensure the healthy development of the transgenic industry. In the previous study, 2 transgenic soybean(Glycine max) events were obtainedwith >78% oleic acid content. To isolate the flanking sequences of T-DNAs with endogenous genes and promote the safety assessment and application of these 2 transgenic events, genome re-sequencing combined with PCR was carried out to analyze the flanking sequences of T-DNAs of these 2 transgenic events. There were 1 and 2 copies T-DNAs in these 2 transgenic events by Southern blot analysis, respectively. Using genome re-sequencing compared to reference genome information based on BWA-Burrows-Wheeler Alignment tool method(http://bio-bwa. sourceforge. net/), flanking sequences of T-DNAs in the transgenic soybean 'E2 D9050' and 'EB8072' genomes were isolated. The T-DNA of 'E2 D9050' was integrated into the position 51410941 on Chr04 with one copy. The T-DNA of 'EB8072' was integrated into soybean genome with2 copies in position 38147218 on Chr19 and the pattern of integration was inverted insertion with right borders of these copies interfaced. These results were in accord with the results of Southern blot analysis. According to the flanking sequences of these positions, primers were designed and PCR was amplified to verify these flanking sequences of insertion sites. Event-specific primers were designed based on these fragments and the sensitivities of these pairs of primer were both 0.1%. It provides an accurate and fast method for identification of these kinds of transgenic event.
引文
郭超,何行健,邓力华,等.2017.转基因水稻BarKasalath-01事件特异性检测[J].分子植物育种,15(11):4466-4475.(Guo C,He X J,Deng L H,et al.2017.Event-specific detection of genetically modified rice BarKasalath-01[J].Molecular Plant Breeding,15(11):4466-4475.)
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仇有文,高学军,张明辉,等.2011.抗除草剂转基因大豆插入拷贝数及其旁侧序列分析[J].生物技术,21(6):31-35.(Qiu Y W,Gao X J,Zhang M H,et al.2011.The copy number of transgenic soybean with the EPSPSgene and flanking sequence analysis[J].Biotechnology,21(6):31-35.)
申爱娟,陈松,周晓婴,等.2014.转基因油菜W-4 T-DNA旁侧序列分析与事件特异性检测[J].江苏农业学报,30(1):10-20.(Shen A J,Chen S,Zhou X Y,et al.2014.Analysis of the flanking sequence and event-specific detection of transgenic line W-4 of Brassica napus[J].Jiangsu Journal of Agricultural Science,30(1):10-20.)
汪巧,谢家建,苏长青,等.2011.转cry1Ac基因抗虫棉鄂杂棉1号的旁侧序列和品系特异性检测[J].农业生物技术学报,3(19):427-433.(Wang Q,Xie J J,Su C Q,et al.2011.The genome flanking sequence and event-specific qualitative detection of the transgenic cry1Ac cotton Ezamian 1[J].Journal of Agricultural Biotechnology,3(19):427-433.)
魏岁军,邓力华,肖国樱.2014.转基因水稻EB7001S事件特异性检测方法的建立[J].农业生物技术学报,2014,22(2):621-631.(Wei S J,Deng L H,Xiao G Y.2011.Establishment of an event-specific method to detect transgenic rice(Oryza sativa)EB7001S[J].Journal of Agricultural Biotechnology,22(2):621-631.)
杨静,邢国杰,牛陆,等.2017.反义RNA介导GmFAD2-1B基因沉默增强大豆种子中油酸的高效累积[J].作物学报,43(11):1588-1595.(Yang J,Xing G J,Niu L,et al.2017.Antisense RNA-mediated GmFAD2-1B gene silencing enhances accumulation of oleic acid in transgenic soybean seeds[J].Acta Agronomica Sinica,2017,43(11):1588-1595.)
杨瑞芳,白建江,朴钟泽,等.2014.转cryAc1基因抗虫水稻的培育[J].分子植物育种,12(6):1103-1111.(Yang RF,Bai J J,Piao Z Z,et al.2014.Development of insectresistant transgenic rice with cyr1Ac1 gene[J].Molecular Plant Breeding,12(6):1103-1111.)
袁磊,孙红炜,杨崇良,等.2010.转基因玉米MON88017旁侧序列分析及定性PCR检测[J].作物学报,36(2):361-364.(Yuan L,Sun H W,Yang C L,et al.Analysis of junction sequence in the transgenic maize MON88017and the methods of qualitative PCR detection[J].Acta Agronomica Sinica,2010,36(2):361-364.)
Alolabi A S,Worland B,Snape J W,et al.2004.A large-scale study of rice plants transformed with different T-DNAs provides new insights into locus composition and T-DNA linkage configurations[J].Theoretical and Applied Genetics,4(19):815-826.
Chen S,Jin W,Wang M,et al.2003.Distribution and characterization of over 1000 T-DNA tags in rice genome[J]Plant Journal,36(1):105-113.
Chen S,Shen A J,Zhou X Y,et al.2014.Analysis of the flanking sequence and event-specific detection of transgenic line W-4 of Brassica napus[J].Agricultural Science&Technology,15(7):1089-1094.
Demorest L Z,Coffman A,Baltes J N,et al.2016.Direc stacking of sequence-specific nuclease-induced mutations to produce high oleic and low linolenic soybean oi[J].BMC Plant Biology,16(1):225.
Goodwin S,McPherson J D,McCombie W R.2016.Coming of age:Ten years of next-generation sequencing technologies[J].Nature Reviews and Genetics,17(6):333-351.
Imoto Y,Yamada T,KitamuraK,et al.2008.Spatial and temporal control of transcription of the soybeanβ-conglycininαsubunit gene is conferred by its proximal promoter region and accounts for the unequal distribution of the protein during embryogenesis[J].Genes&Genetic Systems,83(6):469-476
Kinney A J,Cahoon E B,Hitz W D.2002.Manipulating desaturase activities in transgenic crop plants[J].Biochemical Society Transactions,30(6):1099-1103.
Liu B,Su Q,Tang M Q,et al.2006.Progress of the PCR amplification techniques for chromosome walking[J].Hereditas,28(5):587-595.
Liu Y G and Chen Y L.2007.High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J].BioTechniques,43(5):649-656.
Liu Y G,Mitsukawa N,Oosumi T,et al.1995.Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR[J].The Plant Journal,8(3):457-463.
Liu Y G and Whittier R F.1995.Thermal asymmetric interlaced PCR:Automatable amplification and sequencing of insert and fragments from P1 and YAC clones for chromosome walking[J].Genomics,25(3):674-681.
Pham Anh-Tung,Lee J D,Shannon J G,et al.2010.Mutant alleles of FAD2-1A and FAD2-1B combine to produce soybeans with the high oleic acid seed oil trait[J].BMCPlant Biology,10:195.
Tel-zur N,Abbo S,Myslabodski D,et al.1999.Modified CTAB procedure for DNA isolation from epiphytic cacti of the genera Hylocereus and Selenicereus(Cactaceae)[J].Plant Molecular Biology Reporter,17:249-254.
Warner K,Orr P,Parrot L,et al.1994.Effects of frying oil composition on potato chip stability[J].Journal of the American Oil Chemists'Society,71(10):1117-1121.
Yang J,Xing G J,Niu L,et al.2018.Improved oil quality in transgenic soybean seeds by RNAi-mediated knockdown of GmFAD2-1B[J].Transgenic Research,27(2):155-166.
金永梅,马瑞,于志晶,等.2016.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,41(1):14-19.(Jin Y M,Ma R,Yu ZJ,et al.Identification of the T-DNA flanking sequences and event-specific PCR detection of transgenic rice'Jishengjing 2'[J].Journal of Northeast Agricultural Sciences,41(1):14-19.)
李付鹏,伍宝朵,马朝芝,等.2010.基于PCR的染色体步移技术研究进展[J].中国生物工程杂志,30(12):87-94.(Li F P,Wu B D,Ma C Z,et al.2010.Progress of chromosome walking by PCR amplification techniques[J].China Biotechnology,30(12):87-94.)
梁成真,张锐,郭三堆.2009.染色体步移技术研究进展[J].生物技术通报,(10):75-82.(Liang C Z,Zhang R,Guo S D.2009.Progress of chromosome walking[J].Biotechnology Bulletin,(10):75-82.)
刘营,张明辉,霍楠,等.2012.转基因大豆OSDREB3品系特异性定性PCR检测方法的建立[J].中国农业大学学报,2012,17(4):34-39.(Liu Y,Zhang M H,Huo N,et al.2012.Establishment of event-specific qualitative PCR for detecting transgenic soybean OsDREB3[J].Journal of China Agricultural University,17(4):34-39.)
仇有文,高学军,张明辉,等.2011.抗除草剂转基因大豆插入拷贝数及其旁侧序列分析[J].生物技术,21(6):31-35.(Qiu Y W,Gao X J,Zhang M H,et al.2011.The copy number of transgenic soybean with the EPSPSgene and flanking sequence analysis[J].Biotechnology,21(6):31-35.)
申爱娟,陈松,周晓婴,等.2014.转基因油菜W-4 T-DNA旁侧序列分析与事件特异性检测[J].江苏农业学报,30(1):10-20.(Shen A J,Chen S,Zhou X Y,et al.2014.Analysis of the flanking sequence and event-specific detection of transgenic line W-4 of Brassica napus[J].Jiangsu Journal of Agricultural Science,30(1):10-20.)
汪巧,谢家建,苏长青,等.2011.转cry1Ac基因抗虫棉鄂杂棉1号的旁侧序列和品系特异性检测[J].农业生物技术学报,3(19):427-433.(Wang Q,Xie J J,Su C Q,et al.2011.The genome flanking sequence and event-specific qualitative detection of the transgenic cry1Ac cotton Ezamian 1[J].Journal of Agricultural Biotechnology,3(19):427-433.)
魏岁军,邓力华,肖国樱.2014.转基因水稻EB7001S事件特异性检测方法的建立[J].农业生物技术学报,2014,22(2):621-631.(Wei S J,Deng L H,Xiao G Y.2011.Establishment of an event-specific method to detect transgenic rice(Oryza sativa)EB7001S[J].Journal of Agricultural Biotechnology,22(2):621-631.)
杨静,邢国杰,牛陆,等.2017.反义RNA介导GmFAD2-1B基因沉默增强大豆种子中油酸的高效累积[J].作物学报,43(11):1588-1595.(Yang J,Xing G J,Niu L,et al.2017.Antisense RNA-mediated GmFAD2-1B gene silencing enhances accumulation of oleic acid in transgenic soybean seeds[J].Acta Agronomica Sinica,2017,43(11):1588-1595.)
杨瑞芳,白建江,朴钟泽,等.2014.转cryAc1基因抗虫水稻的培育[J].分子植物育种,12(6):1103-1111.(Yang RF,Bai J J,Piao Z Z,et al.2014.Development of insectresistant transgenic rice with cyr1Ac1 gene[J].Molecular Plant Breeding,12(6):1103-1111.)
袁磊,孙红炜,杨崇良,等.2010.转基因玉米MON88017旁侧序列分析及定性PCR检测[J].作物学报,36(2):361-364.(Yuan L,Sun H W,Yang C L,et al.Analysis of junction sequence in the transgenic maize MON88017and the methods of qualitative PCR detection[J].Acta Agronomica Sinica,2010,36(2):361-364.)
Alolabi A S,Worland B,Snape J W,et al.2004.A large-scale study of rice plants transformed with different T-DNAs provides new insights into locus composition and T-DNA linkage configurations[J].Theoretical and Applied Genetics,4(19):815-826.
Chen S,Jin W,Wang M,et al.2003.Distribution and characterization of over 1000 T-DNA tags in rice genome[J]Plant Journal,36(1):105-113.
Chen S,Shen A J,Zhou X Y,et al.2014.Analysis of the flanking sequence and event-specific detection of transgenic line W-4 of Brassica napus[J].Agricultural Science&Technology,15(7):1089-1094.
Demorest L Z,Coffman A,Baltes J N,et al.2016.Direc stacking of sequence-specific nuclease-induced mutations to produce high oleic and low linolenic soybean oi[J].BMC Plant Biology,16(1):225.
Goodwin S,McPherson J D,McCombie W R.2016.Coming of age:Ten years of next-generation sequencing technologies[J].Nature Reviews and Genetics,17(6):333-351.
Imoto Y,Yamada T,KitamuraK,et al.2008.Spatial and temporal control of transcription of the soybeanβ-conglycininαsubunit gene is conferred by its proximal promoter region and accounts for the unequal distribution of the protein during embryogenesis[J].Genes&Genetic Systems,83(6):469-476
Kinney A J,Cahoon E B,Hitz W D.2002.Manipulating desaturase activities in transgenic crop plants[J].Biochemical Society Transactions,30(6):1099-1103.
Liu B,Su Q,Tang M Q,et al.2006.Progress of the PCR amplification techniques for chromosome walking[J].Hereditas,28(5):587-595.
Liu Y G and Chen Y L.2007.High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J].BioTechniques,43(5):649-656.
Liu Y G,Mitsukawa N,Oosumi T,et al.1995.Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR[J].The Plant Journal,8(3):457-463.
Liu Y G and Whittier R F.1995.Thermal asymmetric interlaced PCR:Automatable amplification and sequencing of insert and fragments from P1 and YAC clones for chromosome walking[J].Genomics,25(3):674-681.
Pham Anh-Tung,Lee J D,Shannon J G,et al.2010.Mutant alleles of FAD2-1A and FAD2-1B combine to produce soybeans with the high oleic acid seed oil trait[J].BMCPlant Biology,10:195.
Tel-zur N,Abbo S,Myslabodski D,et al.1999.Modified CTAB procedure for DNA isolation from epiphytic cacti of the genera Hylocereus and Selenicereus(Cactaceae)[J].Plant Molecular Biology Reporter,17:249-254.
Warner K,Orr P,Parrot L,et al.1994.Effects of frying oil composition on potato chip stability[J].Journal of the American Oil Chemists'Society,71(10):1117-1121.
Yang J,Xing G J,Niu L,et al.2018.Improved oil quality in transgenic soybean seeds by RNAi-mediated knockdown of GmFAD2-1B[J].Transgenic Research,27(2):155-166.