Development of Transgenic Rice (Oryza sativa) E1C9K-18 and Establishment of Its Event-specific Detection Method
XU Li-Kui1,2, DENG Li-Hua1, LI Hua1, XIAO Guo-Ying1,3,*
1 Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China; 2 University of Chinese Academy of Sciences, Beijing 100049, China; 3 Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing 100101, China
Abstract：The herbicide resistance and Lepidopteran resistance of rice (Oryza sativa) play important roles in weeds management and reduction of yield loss by insect pests, and the event-specific detection method is an essential technique for supervision of transgenic rice. In this study, the expression vector was constructed firstly, in which the Lepidopteran resistance gene Cry1Ca# (gene coding insecticidal crystal protein Cry1Ca) was driven by wound-induced promoter and the herbicide resistance gene Epsps# (gene coding 5-enolpyruvylshikimate 3-phosphate synthase) was driven by constitutive promoter, and a single copy transgenic event E1C9K-18 was developed by Agrobacterium-mediated genetic transformation from 9K19-5 (O. sativa subsp. indica), which possessed elite glyphosate resistance. Then, the hiTAIL-PCR (high-efficiency thermal asymmetric interlaced PCR) was adopted to reveal the flanking sequence of transgenic rice E1C9K-18, and the right flanking sequence with 420 bp length was discovered. Comparing the right flanking sequence with rice genome database, it was found the exogenous fragment was inserted behind the nucleotide residue No. 33 189 510 of chromosome 4. The left flanking sequence with 613 bp length was amplified using the primers that were designed according to the sequence of integration site on rice genome and left side sequence of T-DNA. Compared the left flanking sequence with rice genome database, it was found the exogenous fragment was inserted before the nucleotide residue No. 33 189 480 of chromosome 4. However, the insertion of T-DNA resulted in a deletion of 29 nucleotide residues in rice genome. Based on the left and right flanking sequences, the event-specific PCR detection method and tri-primers PCR method were developed for E1C9K-18, which will provide technical supports for identification of this transgenic event and rapidly selection of homozygote from segregation population.
徐礼逵, 邓力华, 李华, 肖国樱. 转基因水稻E1C9K-18的获得及其事件特异性检测方法的建立[J]. 农业生物技术学报, 2020, 28(12): 2250-2260.
XU Li-Kui, DENG Li-Hua, LI Hua, XIAO Guo-Ying. Development of Transgenic Rice (Oryza sativa) E1C9K-18 and Establishment of Its Event-specific Detection Method. 农业生物技术学报, 2020, 28(12): 2250-2260.
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