根寄生杂草中一个与感受独脚金内酯相关基因的克隆及功能分析

Strigolactones (SLs) are germination signals for root parasitic plants, but molecular mechanisms of seeds in perceiving SLs are poorly understood. MAX2 is a putative receptor for SLs in shoot branching inhibition via SLs signaling pathway, however, whether it functions in seeds perceiving SLs is unclear. This study would use the investigation on signal transduction pathway of SLs inhibiting shoot branching for reference. In this study, the broomrape (Orobanche aegyptiaca) MAX2 (OaMAX2), which is an essential gene involving SLs signal transduction, will be cloned and the full length cDNA be obtained using rapid-amplification of cDNA ends (RACE) technique. The sequence and physicochemical properties of amino acid encoded by OaMAX2 will be analyzed using bioinformatics methods. Then, a BLAST search of the amino acid sequence will be performed to predict the gene function. Both plant over-expression vector and RNAi-mediated silencing expression vector of OaMAX2 will be constructed. Subsequently, the over-expression vector is to be introduced into Arabidopsis MAX2 mutant mediated by Agrobacterium rhizogenes to examine gene function in shoot branching inhibition via SLs signaling pathway; while the RNAi-mediated silencing expression vector will be used to transform O. aegyptiaca to analyze gene function in SLs perception during seed germination. This research will provide an alternative way to dissect molecular mechanisms of strigolactones-regulated recognition of parasitic weed and its host. It is also expected to provide theory evidence for developing novel parasitic weed control strategies.

独脚金内酯是根寄生植物种子的萌发信号，但对种子识别其信号的分子机制知之甚少。MAX2被认为是独脚金内酯抑制植物分枝信号途径中的受体，但是否在种子识别独脚金内酯的过程中起作用并不清楚。本项目借鉴独脚金内酯抑制植物分枝信号途径的研究，以根寄生杂草埃及列当为材料，从中克隆参与独脚金内酯信号感受的关键基因MAX2，采用RACE技术获得该基因全长cDNA；利用生物信息学技术分析该基因编码的氨基酸序列及理化性质，然后进行序列比对预测基因功能；分别构建该基因的植物过表达载体和RNAi载体，通过农杆菌介导的方法将过表达载体转化拟南芥MAX2突变体，将RNAi载体转化列当，明确该基因在独脚金内酯抑制植物分枝信号途径及列当种子萌发过程中的作用，综合分析该基因的功能。这将为揭示根寄生植物识别寄主分泌的萌发刺激物的分子机制提供一条新的路径，同时为研究控制列当等恶性杂草的新策略提供理论依据，具有重要的科学意义。

项目采用RACE等技术，以根寄生植物向日葵列当（Orobanche cumana Wallr.）为材料，克隆得到了向日葵列当中独脚金内酯信号传导途径中相关基因DAD2全长及MAX2部分序列，分别命名为OcDAD2（GenBank accession number KY399918）和OcMAX2。实验对列当在种子调节（seed conditioning）及GR24诱导过程中DAD2的表达模式进行了分析，同时还进行了作为实时荧光定量PCR内参基因（GAPC2, PP2A, RLI, TUB5, UBQ1）的筛选。利用geNorm和NormFinder软件进行分析，最终发现UBQ1的稳定性最好，可作为列当中实时荧光定量PCR内参基因使用。在开始种子调节的24 h中，列当中DAD2的表达逐渐升高，说明DAD2的表达受到种子调节的诱导。但有意思的是，转移到GR24处理的种子中DAD2的表达与处理前的种子相比没有发生明显的变化，这表明前期DAD2的表达量提高可能与列当种子的休眠被打破有关，而在感受独脚金内酯的动态过程中DAD2转录水平的调节作用可能有限。KAI2基因是DAD2的旁系同源基因，在拟南芥中，KAI2介导karrikin特异的种子萌发响应。实验从向日葵列当中扩增出一条与AtKAI2相似性较高的序列，命名为OcKAI2；并对其表达模式进行了分析，发现在种子调节及随后GR24处理列当种子24 h后该基因表达量显著的上升，这表明该基因可能与列当种子萌发时感受独脚金内酯有关。此外，我们还构建了DAD2的过表达载体和Crispr/Cas9敲除载体，研究了寄生杂草与寄主作物互作的共培养方法，发表了相关论文。本项目主要从反向遗传学出发，通过基因克隆及分子生物学方法对根寄生杂草识别独脚金内酯的受体基因进行了研究，更好地明确根寄生植物识别寄主萌发刺激物的机制，并为寄生杂草防控提供了理论依据和技术支撑。