Construction Of Plant Expression Vectors And Transformation On Inducible Site-specific Recombination

In order to obtain transformants from a large number of non-transformed cells, theselectable marker gene plays a very important role in plant genetic transformation. As moreand more transformed plants released into the environment or commercialized planting, therisk of selectable marker gene on ecological environment and food safety has become thefocus of the international controversy. Therefore, how to effectively delete the marker genesafter transformation has become crucial. In the methods of the marker gene deletion, theinducible site-specific recombination can save time, streamline operations, and obtainmarker-free transgenic plants more early.In this study, the heat shock promoter hsp18.2and cold-inducible promoter wcs120wascloned, the inducible expression CinH/RS2site-specific recombinant plant expression vectorwere constructed, and wheat Mianyang19and tobacco SR1were transformed. The study hasachieved the following results:(1) The construction of heat-inducible site-specific recombinant plant expression vectorand transformation of tobaccoThe heat shock promoter hsp18.2was cloned from the Arabidopsis, and the plantexpression vector pXL5513-hsp with CinH/RS2site-specific recombination systems wasconstructed. After transformation of tobacco by Agrobacterium,12positive transgenic plantswere successfully obtained, which laid the foundation to further study on heat-inducedCinH/RS2site-specific recombination system in plant genetic transformation.(2) The construction of low-temperature induced site-specific recombination plantexpression vector and wheat transformationThe cold-inducible promoter wcs120was cloned from Xinong928, the plant expressionvector of wcs120promoter induced CinH/RS2site-specific recombination systems wasconstructed. After the subclone of expression cassette with the（PYR1-LIKE5）PYL5gene,low-temperature induced site-specific recombination vector with RD29A-PYL5-nosT wassuccessfully constructed. After transformed wheat callus by gene gun, six positive transgenicplants with the PYL5gene was obtained, which laid the foundation for marker-free wheat transformation.