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利用FOX hunting system技术,结合高通量的Gateway技术,以模式植物拟南芥为载体,建立了一个油菜抗逆相关基因功能研究体系,构建了一个超表达油菜抗逆相关基因的拟南芥文库,并将其命名为油菜-FOX-拟南芥文库。根据拟南芥抗逆信号转导研究基础,筛选出80个拟南芥抗逆相关基因并与油菜基因组信息进行同源性比对,最终获得70条具有完整ORF的EST序列。PCR扩增上述70个候选基因。利用Gateway技术构建35S强启动子驱动的70个候选基因的植物表达文库。农杆菌介导的拟南芥Flower-dip法转化600多株Col-0生态型拟南芥,收获种子约30 000粒。PPT筛选和PCR鉴定得到355个转化株,其中328个单株收获到种子。经Mannitol、Na Cl和ABA平板培养基上筛选,获得根长不敏感植株68株,根长敏感植株65株。在Mannitol和Na Cl平板培养基上筛选到子叶变绿不敏感植株11株。同时在FOX库中发现5株形态表型异常的植株(包括叶片卷曲,植株矮小等)。
Using FOX hunting system technology and high-throughput Gateway technology, a model plant Arabidopsis thaliana was established as a vector to study the function of resistance-related genes in rapeseed. A Arabidopsis library , And named it rape-FOX-Arabidopsis library. Based on the research of Arabidopsis antidepressant signal transduction, 80 Arabidopsis thaliana related genes were screened and compared with the genome information of rapeseed to obtain 70 EST sequences with complete ORF. The above 70 candidate genes were amplified by PCR. Using Gateway technology, a 35S strong promoter-driven plant expression library of 70 candidate genes was constructed. Agrobacterium-mediated Arabidopsis Flower-dip method transformed more than 600 strains of Col-0 ecotype Arabidopsis, about 30,000 seeds were harvested. 355 transformants were obtained by PPT screening and PCR identification, of which 328 were harvested to seed. After screening on Mannitol, NaCl and ABA plate medium, 68 plants with root length insensitivity and 65 plants with root length sensitivity were obtained. 11 cotyledons insensitive plants were screened on Mannitol and Na Cl plates. At the same time, five plants with abnormal morphological phenotypes (including leaf curling, dwarf plants, etc.) were found in FOX bank.