利用FOX hunting system解析油菜耐旱的分子机制

Rape is an important oil crop. As the global climate changes, perennial drought stress is serious constraint on rape production. How to improve the ability of drought tolerance and water use efficiency(WUE) of rapeseed is an urgent scientific issues. The Full-length cDNA Over-eXpressing (FOX) gene hunting system is an alternative gain-of-function approach that uses full length cDNAs (fl-cDNA), which enables a quick gene isolation and sequencing, paralelly with functional studies. This system have been developed to systematically generate gain-of function mutants using fl-cDNAs. The utilization of fl-cDNAs can eliminate the problem of truncated cDNAs that lead to cosuppression rather than overexpression. In this study we plan to do the following research:.(1) Construct drought stress induced full-length cDNA library of oilseed rape：.On the basis of preliminary studies, take the selected drought-tolerance cultivar as material, extract total RNA in different degree of drought-induced, purify mRNA, transcript to fl-cDNA. Then, construct cDNA library of oilseed rape..(2) construct rape FOX Arabidopsis mutant library and select drought-tolerance mutants.Using high-throughput Gateway technology, clone rape transcriptome full-length cDNA into Gateway-compatible vector to construct rape fl-cDNA expression library. Then introduce the expression library into Agrobacterium. Transform Arabidopsis by Agrobacterium library to form rape FOX Arabidopsis mutant library. Select drought-tolerance mutants by far-infrared thermal imaging technology..(3) Isolation of drought responding genes.Isolate candidate genes related drought stress from rape FOX Arabidopsis by PCR and sequence. Analyze these genes expression pattern by RT-PCR in different drought stress treatment. By bioinformatics analysis and according to Arabidopsis drought signal transduction network, primarily explain drought tolerance mechanism of rape and explore key genes for drought stress responding in Brassica napus L..

油菜是重要的油料作物。随着全球气候变化，常年受到干旱的影响，严重制约其发展。如何通过提高油菜的耐旱能力和水分利用效率来增加其产量，是当前油菜生产迫切需要解决的科学问题。本研究拟在油菜抗旱生物学过程研究的基础上，以筛选出的耐旱品种为材料，运用高通量的Gateway技术构建干旱胁迫处理诱导的油菜转录组全长cDNA表达文库。利用FOX hunting system将cDNA文库转入拟南芥，获得油菜FOX拟南芥突变体库。进而运用高通量的远红外热成像技术筛选抗旱的FOX突变株，克隆获得相应的油菜干旱响应相关基因。通过表达谱分析并借助油菜的近缘模式植物拟南芥的干旱信号转导网络，揭示油菜对干旱信号的传递过程，初步阐明油菜的耐旱机制，克隆水分高效利用的关键基因，从而为最大限度地挖掘油菜的耐旱潜力、培育耐旱的油菜新品种奠定基础。

油菜作为重要的油料作物，同时也是抗逆性较差的作物。干旱，冷害，盐渍等胁迫因素严重制约油菜生产的发展。本研究利用FOX hunting技术，结合高通量的Gateway技术，建立了一个油菜抗逆相关基因筛选鉴定及功能研究的方法。. 根据拟南芥抗逆研究机制及油菜抗旱转录组分析数据，筛选克隆了70条具有完整ORF的油菜潜在抗逆相关基因。利用Gateway体系构建35S强启动子驱动的70个基因的表达载体文库。农杆菌介导的拟南芥花浸染技术转化1000多株Col-0生态型拟南芥，收获T0代种子约50000粒。PPT筛选和分子鉴定得到727株T1代阳性植株。通过模拟胁迫条件筛选，在Mannitol、NaCl和ABA板上筛选获得抗性植株229株，敏感植株684株。表型明显的植株测序比对分析后得到具有胁迫响应的油菜基因21个。这21个基因转化油菜，获得其中19个基因的转基因油菜株系69个。同时在T1代阳性植株中发现一些形态表型异常的植株（包括叶片卷曲，植株矮小等），对这些植株插入片段进行了鉴定。这些插入基因包括FAB1，CAT1，RD26，LOS2，COL1，P5CS1，NAC102。本研究为最大限度地挖掘油菜的耐旱潜力、培育耐旱的油菜新品种奠定理论基础和基因资源。