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| PCR Detection and T-DNA Flanking Sequence Analysis of GmWRKY70 in Transgenic Soybean (Glycine max) |
| YANG Qing-Hua1,*, DONG De-Kun1, CAO Jing2, MEI Na2, CHEN Fen3, HU Xing-Wang3, ZHU Dan-Hua1 |
1 Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China;
2 School of Horticulture and Gardening, Yangtze University, Jinzhou 434025, China;
3 College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China |
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Abstract The PCR detection of transgenic lines and the in-depth analysis of T-DNA flanking genome sequence can help to understand the specific molecular characteristics of transgenic lines. Three GmWRKY70 transgenic soybean (Glycine max) lines were developed using Agrobacterium-mediated soybean cotyledonary nod transformation method. Transformation events were confirmed by PCR detections of selective marker bialaphos resistance (Bar) gene, target gene GmWRKY70, as well as vector-construction-specific fragments and the integration site of T-DNA flanking sequence was analyzed by high-efficient thermal asymmetric interlaced PCR (Hi-TAIL PCR). The results showed that all 3 GmWRKY70 transgenic soybean lines were positive. T-DNA was reversely integrated into soybean chromosome Chr07 between 43 119 699 and 43 119 712 bp. The T-DNA integration led to a 12 bp deletion at the insertion site of soybean chromosome Chr07. For the introduced T-DNA, it was found that the 80 bp sequence at the end of the left border (LB) and the 22 bp sequence at the end of the right border (RB) were truncated. Micro-homologies of 2 base pairs were found between the introduced LB end and soybean genomic DNA at the integration site, while no homology was found for RB. New integration-site-specific primers were designed to validate this transformation events and could further facilitate molecular assisted selections in breeding program.
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Received: 16 December 2019
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Corresponding Authors:
* tsingyang2009@163.com
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[1] 吴国栋, 修宇, 王华芳. 2018. 优化子叶节转化法培育大豆MtDREB2A转基因植株[J]. 植物学报, 53(1): 59-71.
(Wu G D, Xiu Y, Wang H F.2018. Breeding of MtDREB2A transgenic soybean by an optimized cotyledonary-node method[J]. Chinese Bulletin of Botany, 53(1): 59-71.)
[2] Antal Z, Rascle C, Fevre Met al.2004. Single oligonucleotide nested PCR: A rapid method for the isolation of genes and their flanking regions from expressed sequence tags[J]. Current Genetics, 46(4): 240-246.
[3] Cai Y, Chen X, Xie K, et al.2014. Dlf1, a WRKY transcription factor, is involved in the control of flowering time and plant height in rice[J]. PLOS ONE, 9(7): e102529.
[4] Chen L, Song Y, Li S, et al.2012. The role of WRKY transcription factors in plant abiotic stresses[J]. Biochimica et Biophysica Acta, 1819(2): 120-128.
[5] de Buck S, Podevin N, Nolf J, et al.2009. The T-DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell[J]. The Plant Journal, 60(1): 134-145.
[6] Foster J M, Christodoulou Z, Cowan G M, et al.1999. Thermal asymmetric interlaced PCR amplification of YAC insert end fragments for chromosome walking in Plasmodium falciparum and other A/T-rich genomes[J]. Biotechniques, 27(2): 240, 244, 246.
[7] Gheysen G, Villarroel R, Van Montagu M.1991. Illegitimate recombination in plants: A model for T-DNA integration[J]. Genes ＆ Development, 5(2): 287-297.
[8] Huang H, Wang G, Zhao Y, et al.2010. Direct and efficient cloning of full-length genes from environmental DNA by RT-qPCR and modified TAIL-PCR[J]. Applied Microbiology and Biotechnology, 87: 1141-1149.
[9] Ishiguro S, Nakamura K.1994. Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 5' upstream regions of genes coding for sporamin and beta-amylase from sweet potato[J]. Molecular and General Genetics, 244: 563-571.
[10] Jones D H, Winistorfer S C.1992. Sequence specific generation of a DNA panhandle permits PCR amplification of unknown flanking DNA[J]. Nucleic Acids Research, 20(3): 595-600.
[11] Kayum M A, Jung H J, Park J I, et al.2015. Identification and expression analysis of WRKY family genes under biotic and abiotic stresses in Brassica rapa[J]. Molecular and General Genetics, 290(2): 79-95.
[12] Liu Y G, Chen Y.2007. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J]. Biotechniques, 43(5): 649-656.
[13] Liu Y G, Whittier R F.1995. Thermal asymmetric interlaced PCR: Automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking[J]. Genomics, 25(3): 674-681.
[14] Mayerhofer R, Koncz-Kalman Z, Nawrath C, et al.1991. T-DNA integration: A mode of illegitimate recombination in plants[J]. The EMBO Journal, 10(3): 697-704.
[15] Nthangeni M B, Ramagoma F, Tlou M G, et al.2005. Development of a versatile cassette for directional genome walking using cassette ligation-mediated PCR and its application in the cloning of complete lipolytic genes from Bacillus species[J]. Journal of Microbiological Methods, 61(2): 225-234.
[16] Ochman H, Gerber A S, Hartl D L.1988. Genetic applications of an inverse polymerase chain reaction[J]. Genetics, 120(3): 621-633.
[17] Perucho M, Hanahan D, Lipsich L, et al.1980. Isolation of the chicken thymidine kinase gene by plasmid rescue[J]. Nature, 285(5762): 207-210.
[18] Pillai M M, Venkataraman G M, Kosak S, et al.2008. Integration site analysis in transgenic mice by thermal asymmetric interlaced (TAIL)-PCR: Segregating multiple-integrant founder lines and determining zygosity[J]. Transgenic Research, 17(4): 749-754.
[19] Rushton D L, Tripathi P, Rabara R C, et al.2012. WRKY transcription factors: Key components in abscisic acid signalling[J]. Plant Biotechnology Journal, 10(1): 2-11.
[20] Schluttenhofer C, Yuan L.2015. Regulation of specialized metabolism by WRKY transcription factors[J]. Plant Physiology, 167(2): 295-306.
[21] Triglia T, Peterson M G, Kemp D J.1988. A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences[J]. Nucleic Acids Research, 16(16): 8186.
[22] Tzfira T, Li J X, Lacroix B, et al.2004. Agrobacterium T-DNA integration: Molecules and models[J]. Trends in Genetics, 20(8): 375-383.
[23] Wang X L, Yan Y, Li Y Z, et al.2014. GhWRKY40, a multiple stress-responsive cotton WRKY gene, plays an important role in the wounding response and enhances susceptibility to ralstonia solanacearum infection in transgenic Nicotiana benthamiana[J]. PLOS ONE, 9(4): e93577.
[24] Yan H R, Jia H H, Chen X B, et al.2014. The cotton WRKY transcription factor GhWRKY17 functions in drought and salt stress in transgenic Nicotiana benthamiana through ABA signaling and the modulation of reactive oxygen species production[J]. Plant & Cell Physiology, 55(12): 2060-2076.
[25] Zhang J, Guo D, Chang Y X, et al.2007. Non-random distribution of T-DNA insertions at various levels of the genome hierarchy as revealed by analyzing 13 804 T-DNA flanking sequences from an enhancer-trap mutant library[J]. The Plant Journal, 49(5): 947-959.
[26] Zhou Z W, Ma H Y, Qu L J, et al.2012. Establishment of an improved high-efficiency thermal asymmetric interlaced PCR for identification of genomic integration sites mediated by phiC31 integrase[J]. World Journal of Microbiology and Biotechnology, 28: 1295-1299. |
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