低温诱导的位点特异性重组植物表达载体构建及小麦转化

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Abstract The excision of selectable marker gene is an important foundation for transgenic crop to release into the environment or commercialized planting. In order to establish a low-temperature induced site-specific marker gene deletion system in wheat (Triticum aestivum), low-temperature induced promoter wcs120 was cloned from wheat Xinong 928 genome DNA. The sequencing result showed that the whole wcs120 promoter sequence was successfully cloned , which was the same as AY493570.1 and a insertion of 21 bp with AF031235. Using the plasmid pXL5513 which contained the CinH/RS2 system as basal vector, the final vector pXL5513-fwcs-RDA was constructed, in which the CinH recombinase gene was under the control of cold-induced promoter wcs120 and the interest gene pyrabactin-resistance like gene 5 (PYL5) was inserted outside the RS sites near the left border. All of CinH recombinase gene, selectable gene bar and reporter gene green fluorescent protein (GFP) were flanked by 2 recombination sites RS2, which would be expected to be excised after the expression of CinH recombinase. pXL5513-fwcs-RDA was transferred into wheat immature embryo calli of Mianyang 19 (M19) by particle bombardment. After selection and generation, 9 transformed plantlets were gained from 1 745 calli and 6 positive transgenic seedlings were confirmed by PCR of PYL5 gene. Induced by low temperature, all of these positive transgenic seedlings showed selectable gene deletion, which were initially confirmed by PCR. Because of the features of particle bombardment, the patterns of gene insertion were complex, the subsequent experiments were planned to identify the deletion pattern. This investigation will lay a foundation for the application of low-temperature induced site-specific recombination system in marker-free transformation on wheat.

Key words： Low-temperature induce wcs120 promoter CinH/RS2 site-specific recombinant Selectable marker gene

Received: 16 November 2015 Published: 24 February 2016

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	YU Mei
	XU An-Bo
	DIAO Hui-Xian
	LIU Xiang-Li

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YU Mei,XU An-Bo,DIAO Hui-Xian, et al. Construction of Low-temperature Induced Site-specific Recombinant Plant Expression Vector and Transformation of Wheat (Triticum aestivum)[J]. , 2016, 24(4): 478-487.

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http://journal05.magtech.org.cn/Jwk_ny/EN/ OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2016/V24/I4/478

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