外源基因在转基因玉米中的整合位点分析
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摘要
玉米是我国第一大作物,在保障我国粮食安全中发挥重要作用。通过转基因技术培育具有抗病虫等性状的转基因玉米新品种,可有效减少产量损失。培育的转基因玉米需要鉴定外源基因整合位点,为转基因玉米的安全性评价提供重要依据。以一个抗虫转基因玉米事件IE34为材料,采用热不对称PCR(TAIL-PCR)和遗传定位方法,鉴定外源基因整合位点及旁侧序列。通过TAIL-PCR得到一段长度为776 bp的玉米基因组序列。分别在旁侧序列和外源基因上游序列设计特异性引物,建立了转基因玉米事件特异性的PCR鉴定方法。将旁侧序列在MaizeGDB中进行比对分析,发现此序列是重复序列而且存在于多条染色体上。构建转基因玉米IE34与自交系B73的F2代遗传分离群体,通过BSR-Seq方法确定外源基因整合在玉米第5染色体短臂2.32-2.70Mb区间内。通过精细定位将外源基因整合位点缩小在第5染色体2.35-2.61 Mb约260 kb的区间内。本研究结果表明,对于整合位点旁侧序列复杂的转基因事件,TAIL-PCR结合遗传定位方法能够有效鉴定外源基因的整合位点。
Maize(Zea mays)is the largest crop in China and plays a key role in ensuring food security. Development of transgenic maize varieties with resistance to diseases and insects may effectively reduce the loss of maize yield. In the process of transgenic maize development,it is necessary to analyze the integration sites of exogenous genes,which will provide important basis for the safety evaluation of transgenic maize. In this study,the integration sites and flanking sequences of exogenous genes in a transgenic maize event IE34 were analyzed using thermal asymmetric PCR(TAIL-PCR)and genetic mapping. A segment of maize genomic sequence with 776 bp was obtained using TAIL-PCR,and an event-specific PCR method for the transgenic maize was established with the specific primers in the flanking sequences and upstream sequences of exogenous genes. Sequence BLAST in MaizeGDB showed that the flanking sequence was repeat sequence and hit the locus in several chromosomes. To further confirm the integration chromosome of exogenous gene,a F2 population was generated by crossing IE34 with maize inbred line B73. BSR-Seq analysis results demonstrated that the exogenous gene was located in the interval of 2.32-2.70 Mb on the short arm of chromosome 5. Further,fine mapping results reduced the integration sites of exogenous genes to a 260 kb region of 2.35-2.61 Mb on chromosome 5. The results of this study suggest that,for the transgenic event with complex integration site,combined TAIL-PCR and genetic mapping may effectively identify the integration sites of exogenous genes.
Maize(Zea mays)is the largest crop in China and plays a key role in ensuring food security. Development of transgenic maize varieties with resistance to diseases and insects may effectively reduce the loss of maize yield. In the process of transgenic maize development,it is necessary to analyze the integration sites of exogenous genes,which will provide important basis for the safety evaluation of transgenic maize. In this study,the integration sites and flanking sequences of exogenous genes in a transgenic maize event IE34 were analyzed using thermal asymmetric PCR(TAIL-PCR)and genetic mapping. A segment of maize genomic sequence with 776 bp was obtained using TAIL-PCR,and an event-specific PCR method for the transgenic maize was established with the specific primers in the flanking sequences and upstream sequences of exogenous genes. Sequence BLAST in MaizeGDB showed that the flanking sequence was repeat sequence and hit the locus in several chromosomes. To further confirm the integration chromosome of exogenous gene,a F2 population was generated by crossing IE34 with maize inbred line B73. BSR-Seq analysis results demonstrated that the exogenous gene was located in the interval of 2.32-2.70 Mb on the short arm of chromosome 5. Further,fine mapping results reduced the integration sites of exogenous genes to a 260 kb region of 2.35-2.61 Mb on chromosome 5. The results of this study suggest that,for the transgenic event with complex integration site,combined TAIL-PCR and genetic mapping may effectively identify the integration sites of exogenous genes.
引文
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[12]Trinh Q, Zhu P, Shi H, et al. A-T linker adapter polymerase chain reaction for determining flanking sequences by rescuing inverse PCR or thermal asymmetric interlaced PCR products[J].Analytical Biochemistry, 2014, 466:24-26.
[13]Zhang L, Huang X, Zhu S. An event-specific real-time PCR detection system for the transgenic rice line 114-7-2 of producing functional human serum albumin[J]. European Food Research&Technology, 2014, 239:403-408.
[14]Guo B, Guo Y, Hong H, et al. Identification of genomic insertion and flanking sequence of G2-EPSPS and GAT transgenes in soybean using whole genome sequencing method[J]. Frontiers in Plant Science, 2016, 7:1009.
[15]郭超,何行健,邓力华,等.转基因水稻BarKasalath-01事件特异性检测[J].分子植物育种, 2017, 15:4466-4475.
[16]杨阳,王叶,范金杰,等.转基因棉花MON757转化体特异性PCR检测方法及应用[J].农业生物技术学报, 2016, 24:908-918.
[2]Liu YG, Mitsukawa N, Oosumi T, et al. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR[J]. Plant J, 1995, 8:457-463.
[3]Fujimoto S, Matsunaga S, Murata M. Mapping of T-DNA and Ac/Ds by TAIL-PCR to analyze chromosomal rearrangements[J].Methods Mol Biol, 2016, 1469:207-216.
[4]Ochman H, Gerber AS, Hartl DL. Genetic applications of an inverse polymerase chain reaction[J]. Genetics, 1988, 120:621-623.
[5]Liu YG, Chen Y. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J].Biotechniques, 2007, 43:649-656.
[6]Yuanxin Y, Chengcai A, Li L, et al. T-linker-specific ligation PCR(T-linker PCR):an advanced PCR technique for chromosome walking or for isolation of tagged DNA ends[J]. Nucleic Acids Res, 2003, 31:e68.
[7]Xu J, Wang X, Chen X, et al. Qualitative detection and quantification of a Cry1A(b)transgene present in rice cv. Zhejing 22[J].European Food Research&Technology, 2011, 233:259-266.
[8]Yang C, Zhang D, Yang L. Development of event-specific PCR detection methods for genetically modified tomato Huafan No. 1[J].Journal of the Science of Food&Agriculture, 2013, 93:652-660.
[9]Liu YJ, Zhang YW, Liu Y, et al. Metabolic effects of glyphosate on transgenic maize expressing a G2-EPSPS gene from Pseudomonas fluorescens[J]. Journal of Plant Biochemistry and Biotechnology.2015, 24:233-241.
[10]Zhang YW, Liu YJ, Ren Y, et al. Overexpression of a novel Cry1Ie gene confers resistance to Cry1Ac-resistant cotton bollworm in transgenic lines of maize[J]. Plant Cell Tissue Organ Culture,2013, 115:151-158.
[11]Murray MG, Thompson WF. Rapid isolation of high molecular weight plant DNA[J]. Nucleic Acids Res, 1980, 8:4321-4325.
[12]Trinh Q, Zhu P, Shi H, et al. A-T linker adapter polymerase chain reaction for determining flanking sequences by rescuing inverse PCR or thermal asymmetric interlaced PCR products[J].Analytical Biochemistry, 2014, 466:24-26.
[13]Zhang L, Huang X, Zhu S. An event-specific real-time PCR detection system for the transgenic rice line 114-7-2 of producing functional human serum albumin[J]. European Food Research&Technology, 2014, 239:403-408.
[14]Guo B, Guo Y, Hong H, et al. Identification of genomic insertion and flanking sequence of G2-EPSPS and GAT transgenes in soybean using whole genome sequencing method[J]. Frontiers in Plant Science, 2016, 7:1009.
[15]郭超,何行健,邓力华,等.转基因水稻BarKasalath-01事件特异性检测[J].分子植物育种, 2017, 15:4466-4475.
[16]杨阳,王叶,范金杰,等.转基因棉花MON757转化体特异性PCR检测方法及应用[J].农业生物技术学报, 2016, 24:908-918.