| Rice is a very important cereal crops in China,even in the world, but the pest is very serious. Transgenic technology has provide a new way for the prevention and control of rice pests. At present, selectable maker gene is needed to filter transformant in the plant transgenic technology, but it is redundant after obtaining transgenic plants, It is very imperative to delete the marker gene with the appropriate way.This experiment is using the chemical-inducible site-specific recombination system to build four plant expression vectors pUXSHCLF-Ab, pUXSHCLF Ah(m4), pUXSHCLF-Ca and pUXSHCLF-Fa which respectively carry cryl Ab, cryl Ah(m4) and cry1Ca of lepidoptera pests resistant genes, and cry30Fal who is wings mesh pest resistance gene. They were introduced into indica rice "Minghui 86" callus,then the resistant callus are induced with (3-estradiol to activate the site-specific recombination system. we obtain To generation rice plants after differentiation.The result analyses of To generation clone of rice plants reveal that:27 deleting plants of 39 pUXSHCLF-Ab positive plants, deleting percentage is 69.23%,48 deleting plants of 68 pUXSHCLF-Ah(m4) positive plants, deleting frequency is 70.59%; 12 deleting plants of 30 pUXSHCLF-Ca positive plants, deleting frequency is 40%; 47 deleting plants of 70 pUXSHCLF-Fa positive plants, deleting frequency is 67.14%; Then through DNA sequencing, reverse transcription RT-PCR method and so on,we verify that it has delete select maker gene, and Bt gene has been express in the plant, we aslo verify that crylAb protein is expressed stably throught crylAb strip tests.This study results show that using the improved induced expression system can efficiently cultivate mark-free transgenic rice materials, the mark-free transgenic rice materials with a variety of different insect-resistant gene play the important foundation on cultivating different spectrum of transgenic insect-resistant rice varieties. |
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