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
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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