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作物学报 ›› 2011, Vol. 37 ›› Issue (03): 397-404.doi: 10.3724/SP.J.1006.2011.00397

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

转BADH基因苜蓿T-DNA侧翼序列分析及转化事件特异性分析

张艳敏1,张红梅1,相金英2,郭秀林1,刘子会1,李国良1,陈受宜3   

  1. 1 河北省农林科学院遗传生理研究所 / 河北省植物转基因中心, 河北石家庄 050051；2 河北省农林科学院谷子研究所，河北石家庄 050031；3 中国科学院遗传与发育生物学研究所，北京 100101
  • 收稿日期:2010-08-09 修回日期:2010-12-06 出版日期:2011-03-12 网络出版日期:2011-01-17
  • 基金资助:

    本研究由河北省农林科学院科技发展计划项目资助(A09110301)资助。

Analysis of T-DNA Flanking Sequences and Event Specific Detection of Transgenic Alfalfa with Gene BADH

ZHANG Yan-Min1,ZHANG Hong-Mei1,XIANG Jin-Ying2,GUO Xiu-Lin1,LIU Zi-Hui1,LI Guo-Liang1,CHEN Shou-Yi3   

  1. 1 Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences / Plant Genetic Engineering Center of Hebei Province, Shijiazhuang 050051, China; 2 Institute of Millet Research, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050031, China; 3 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
  • Received:2010-08-09 Revised:2010-12-06 Published:2011-03-12 Published online:2011-01-17

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摘要： 为了从分子水平上鉴别不同的转基因株系，以转BADH基因苜蓿的T0代基因组DNA为模版，采用热不对称交错PCR(TAIL-PCR)方法分离其外源基因插入位点的侧翼序列，获得了B127株系的左翼序列和右翼序列，以及B125、B138、B295和B196株系的左翼序列。侧翼序列特征分析表明，有的T-DNA边界序列被删除，有的边界序列被保留，并填充了一段未知来源的核苷酸序列。根据侧翼序列中插入载体序列和紧邻插入序列的基因组序列特征，分别设计PCR扩增的上、下游引物，并对获得的42个转BADH株系分别进行左、右翼序列的扩增，结果表明，转基因植株B106、B125、B138、B157、B158、B289、B295、B305和B127具有相同的扩增条带，B203、B220、B223和B196具有相同的扩增条带，说明这些株系可能仅来源于2个转化事件。本研究建立的事件特异性检测方法可以准确地将不同的转化株系区别开来。

关键词: 转基因苜蓿, BADH, T-DNA, 侧翼序列, 事件特异性检测

Abstract: The gene encoding betaine aldehyde dehydrogenase (BADH), an important osmoregulation gene, has been transformed into many crops, including wheat (Triticum aestivum L.), rice (Oryza sativa L.), maize (Zea maysL.), rape (Brassica campestris L.), and potato (Solanum tuberosumL.). Transgenic crops carring BADH gene enhances tolerance to salinity and drought stresses. Gene badh had been integrated into the most important forage crop alfalfa (Medicago sativa L.) in our previous work, and 42 transgenic plants with improved salt tolerance were obtained. Since they were derived from the same transformant vector, these plants were not able to be distinguished from each other by commonly used methods, such as screening detection, gene specific detection, and vector specific detection. To differentiate these transformants in molecular level, we performed the thermal asymmetric interlaced PCR (TAIL-PCR) to separate the T-DNA flanking sequences for identification of the transgenic plants in event specific detection. A total of six sequences flanking either the left or the right borders of the T-DNA were obtained, which included the flanking sequences at both left and right borders of plant B127, and the left border flanking sequences of plants B125, B138, B295, and B196. The left border sequence of T-DNA was completely deleted from the vector and was thus not integrated into the genome of alfalfa in the transgenic plant B196. Although the left border flanking sequence in the transgenic plant B127 was not changed, it was filled with a DNA sequence of unknown origin. The forward and backward primers for PCR were designed based on the characteristics of the flanking sequences originating from the vector sequence and the alfalfa genomic sequence adjacent to the integrated vector sequence, respectively. The results of amplification in 42 BADH-transgenic alfalfa plants showed that plants B106, B125, B127, B138, B157, B158, B289, B295, and B305 presented the same amplification banding patterns. Plants B196, B203, B220, and B223 produced the same banding pattern which was different from that in other plants. These results indicated that the plants with identical amplification banding pattern may come from the same transformation event. Based on the findings in the present study, we successfully used the flanking sequences separated by TAIL-PCR analysis in developing event specific detection method, which can be used not only to differentiate the origins of various transformants, but also to distinguish the transformants from each other. It is useful in protection of transgenic crops and labeling of transgenic products.

Key words: Transgenic alfalfa, BADH, T-DNA, Flanking sequence, Event specific detection

[1]Liu F-H(刘凤华), Guo Y(郭岩), Gu D-M(谷冬梅), Xiao G(肖岗), Chen Z-H(陈正华), Chen S-Y(陈受宜). Salt tolerance of transgenic plants with BADH cDNA. Acta Genet Sin (遗传学报), 1997, 24(1): 54–58 (in Chinese with English abstract)
[2]Guo B-H(郭北海), Zhang Y-M(张艳敏), Li H-J(李洪杰), Du L-Q(杜立群), Li Y-X(李银心), Zhang J-S(张劲松), Chen S-Y(陈受宜), Zhu Z-Q(朱至清). Transformation of wheat with a gene encoding for the betaine aldehyde dehydrogenase (BADH). Acta Bot Sin (植物学报), 2000, 42(3): 279–283 (in Chinese with English abstract)
[3]Han D-J(韩德俊), Chen Y-F(陈耀锋), Li C-L(李春莲), Guo D-W(郭东伟), Li Z-Q(李振岐). Agrobacterium-mediated transformation with a gene encoding for betaine-aldehyde dehydrogenase (BADH) in Brassica napus. Agric Res Arid Areas (干旱地区农业研究), 2007, 25(4): 6–11 (in Chinese with English abstract)
[4]Zhang N(张宁), Si H-J(司怀军), Li L(栗亮), Yang T(杨涛), Zhang C-F(张春凤), Wang D(王蒂). Drought and salinity tolerance in transgenic potato expressing the betaine aldehyde dehydrogenase gene. Acta Agron Sin (作物学报), 2009, 35(6): 1146–1150 (in Chinese with English abstract)
[5]Berdal K G, Holst-Jensen A. Roundup ready soybean event specific real-time quantitative PCR assay and estimation of the practical detection and quantification limits in GMO analyses. Eur Food Res Technol, 2001, 213: 432–438
[6]Terry C F, Harris N. Event specific detection of Roundup Ready soybean using two different real time PCR detection chemistries. Eur Food Res Technol, 2001, 213: 425–431
[7]Taverniers I, Windels P, van Bockstaele E, de Loose M. Use of cloned DNA fragments for event-specific quantification of genetically modified organisms in pure and mixed food products. Eur Food Res Technol, 2001, 213: 417–424
[8]Holck A, Vaitilingom M, Didierjean L, Rudi K. 5'-nuclease for quantitative event specific detection of the genetically modified Mon810 Maisgard maize. Eur Food Res Technol, 2002, 214: 449–454
[9]Zimmermann A, Lüthy J, Pauli U. Event specific transgene detection in BT11 corn by quantitative PCR at the integration site. Lebensmittel-wissenschaft und-technologie-food Sci Technol, 2000, 33: 210–216
[10]Yang L T, Xu S C, Pan A H, Yin C S, Zhang K W, Wang Z Y, Zhou Z G, Zhang D B. Event specific qualitative and quantitative polymerase chain reaction detection of genetically modified MON863 maize based on the 5'-transgene integration sequence. J Agric Food Chem, 2005, 53: 9312–9318
[11]Yang R, Xu W T, Luo Y B, Guo F, Lu Y, Huang K L. Event-specific qualitative and quantitative polymerase chain reaction detection of roundup ready event GT73 based on the 3’-transgene integration sequence. Plant Cell Rep, 2007, 26: 1821–1831
[12]Liu B(刘博), Su Q(苏乔), Tang M-Q(汤敏谦), Yuan X-D(袁晓东), An L-J(安利佳). Progress of the PCR amplification techniques for chromosome walking. Hereditas (遗传), 2006, 28(5): 587–595(in Chinese with English abstract)
[13]Liu Y G, Mitsukawa N, Oosumi T, Whittier R F. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J, 1995, 8: 457–463
[14]Liu Y G, Whittier R F. Thermal asymmetric interlaced PCR: Automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics, 1995, 25: 674–681
[15]Liu Y G, Huang N. Efficient amplification of insert end sequences from bacterial artificial chromosome clones by thermal asymmetric interlaced PCR. Plant Mol Biol Rep, 1998, 16: 175–181
[16]Terauchi R, Kahl G. Rapid isolation of promoter sequences by TAIL-PCR: The 5-flanking regions of Pal and Pgi genes from Yams (Dioscorea). Mol Gen Genet, 2000, 263: 554–560
[17]Fang J(方进), Zhai W-X(翟文学), Wang W-M(王文明), Li S-W(李素文), Zhu L-H(朱立煌). Amplification and analysis of T-DNA flanking sequences in transgenic rice. Acta Genet Sin (遗传学报), 2001, 28(4): 345–351 (in Chinese with English abstract)
[18]Ying G(应革), Wu W(武威), He C-Z(何朝族). Cloning of Xanthomonas campestris pv. campestris pathogenicity related gene sequences by TAIL-PCR. Chin J Biotechnol (生物工程学报), 2002, 18(2): 182–186 (in Chinese with English abstract)
[19]Rai M, Datta K, Parkhi V, Tan J, Oliva N, Chawla H S, Datta S K. Variable T-DNA linkage configuration affects inheritance of carotenogenic transgenes and carotenoid accumulation in transgenic indica rice. Plant Cell Rep, 2007, 26: 1221–1231
[20]Kononov M E, Bassuner B, Gelvin S B. Integration of T-DNA binary vector “backbone” sequences into the tobacco genome: evidence for multiple complex patterns of integration. Plant J, 1997, 11: 945–957
[21]Kononov M E, Bassuner B, Gelvin S B. Integration of T-DNA binary vector ‘backbone’ sequences into the tobacco genome: evidence for multiple complex patterns of integration. Plant J, 1997, 11: 945–957
[22]Forsbach A, Schubert D, Lechtenberg B, Gils M, Schmidt R. A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome. Plant Mol Biol, 2003, 52: 161–176
[23]Windels P, De Buck S, Van Bockstaele E, De Loose M, Depicker A. T-DNA integration in Arabidopsis chromosomes. Presence and origin of ?ller DNA sequences. Plant Physiol, 2003, 133: 2061–2068
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