摘要
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)。
In this study, a platform was set up for function analysis of oilseed rape stress-related genes using FOX hunting system and high throughput Gateway technology. We con structed a rape-FOX-Arabidopsis library overexpressed the Brassica napus stress-related genes, designated as rape-FOX-Arobidopsis librar y. 80 Arabidopsis stress-related genes were selected based on stress signal transduction and aligned with rape genome sequence. 70 rape EST sequences with complete ORF were screened out. 70 genes were all amplifi ed by PCR, and an overexpression library were constructed driven by Ca MV 35S（ca ulifl ower mosaic virus 35S） promoter using Gateway technology. Then the library with 70 genes was introduced into Col-0 ecotypes Arabidopsis thaliana（more than 600 plants） by fl oral-dip method, and harvested about 30 000 seeds. 355 transformants were screened out by PPT and confi rmed by PCR, and 328 transformants completed growth period and set seeds. According root growing, 68 stress-resistant plants and 65 sensitive p lants were screened out on MS medium with Mannitol, Na Cl or ABA, and based on cotyledon greening, 11 plants were screened out on MS medium with Mannitol or Na Cl. 5 plants were found with abnormal phe n otypes in the FOX library, which were leaf curl, dwarf, etc.
出处
《植物生理学报》
CAS
CSCD
北大核心
2015年第8期1257-1264,共8页
Plant Physiology Journal
基金
国家自然科学基金(31470085和31201148)
