苎麻韧皮部特异/优势启动子克隆与功能验证

Ramie is one of the most important bast fiber crops in China. There are few reports on molecular improvements and developmental mechanism of ramie fiber up to date. We established three transcriptome databases of drought-resistance, organogenesis and fiber development in ramie in 2014 and 2015 respectively. The phloem-specific/preferentially expressed genes will be chosen from these three transcriptome databases after evaluating by RT-PCR. The promoters of these candidate genes will be cloned using universal fast walking method. Then, the GUS reporter gene vectors containing the candidate promoters will be constructed to transform Arabidopsis, tobacco or ramie. The phloem-specific/preferentially expressed promoters will be screened based on the GUS expression analysis. We will choose one or two phloem-specific/preferentially expressed promoters to construct vectors which will be used to transform ramie. The differences of the fiber quality between the transformed ramie and the control will be detected to confirm the potential of the phloem-specific/preferentially expressed promoters. According to the results of the online analysis of the cis-acting elements, the promoter deletion vectors will be constructed and used to transform Arabidopsis. GUS expression detection will be conducted to confirm the necessary elements of the promoters. Furthermore, CRISPR/Cas9 gene editing method will be used to analysis the necessary elements. And the electrophoretic mobility shift assay will be used to illuminate the regulation mechanism of the promoters. The development and the application of the phloem-specific/preferentially expressed promoters are very important to the improvement of the fiber quality and the further research on the fiber development mechanism.

苎麻是我国重要的韧皮纤维作物，其纤维分子改良以及纤维发育机制研究还少有报道。本研究拟从3个苎麻转录组数据库中筛选出韧皮部特异/优势表达基因，采用Universal Fast Walking方法克隆候选基因的启动子。将获得的候选启动子与报告基因（GUS）融合转化拟南芥、烟草或者苎麻，对启动子表达模式进行鉴定，筛选得到韧皮部特异/优势启动子。选取1-2个韧皮部特异/优势启动子，与目标基因构建融合表达载体，转化苎麻，比较阳性植株和对照间纤维品质上的差异，验证挖掘出的启动子的应用前景和潜力。根据启动子顺式作用元件在线分析结果，依据元件分布密度将启动子进行截断并与GUS报告基因融合构建植物表达载体，利用CRISPR/Cas9基因组编辑技术对顺式作用元件进行编辑，转化拟南芥和和苎麻，观察GUS基因表达差异，获得启动子执行功能的必须元件，并结合凝胶阻滞等技术阐明单个启动子的调控机理。

以课题组建立的转录组数据库为依托，筛选出377个苎麻茎皮优势表达的候选基因。采用qRT-PCR验证，确定9个候选基因（P-49、P-167、BnPSP-1、BnPSP-2、BnPSP-3、BnPSP-4、BnPSP-5、BnPSP-6和BnPSP-7），并根据其他作物中已明确同源序列从数据库中找到了苎麻蔗糖合酶1基因（BnSUS1）和苎麻蔗糖转运蛋白2基因（BnSUT2）。通过qRT-PCR对以上11个基因进行表达模式分析，最终筛选到BnGDS、BnSTM、BnSUS1、BnSUT2、BnPSP-1、BnPSP-2以及BnPSP-4等7个基因作为后续研究对象。采用改良的UFW侧翼序列克隆方法得到7个基因的启动子区域，依据关键元件位置或者元件分布密度对以上启动子进行合适截短，并将这些启动子序列与GUS报告基因融合构建载体转入拟南芥。通过GUS组织化学染色定性分析启动子的组织特异性，通过GUS活性定量检测确定启动子功能强弱及验证顺式作用元件位点，结果表明BnSUS1pro及BnPSP-1pro具有一定的韧皮部特异性，可作为韧皮部特异型启动子用于后续研究。为了评估候选启动子的活性强弱以及其应对外界刺激的响应方式，对GA，IAA，ABA，PEG，CuSO4和6-BA处理下的GUS活性进行了定量检测，结果表明候选启动子不仅在组织特异性上存在特殊性而且对不同外界刺激也有不同的响应。