耐干苔藓脱落酸信号关键因子ABI3调控机理研究

ABI3 is a key transcriptional factor of the ABA signal pathway, which is involved in seed dormancy, plastid development, and desiccation tolerance of bryophytes; therefore, it plays an important role in stress tolerance of plants. This project is based on the foundation of cloning of ABI3 gene from Syntrichia caninervis, this gene will be studied on three levels, namely: the upstream regulatory elements and signal transduction pathway, transcriptional regulation and the down-stream target genes, and stress resistance analysis of transgenic moss. The promoter of ScABI3 genes is cloned firstly, and the cis-elements involved in stress responsive will be identified by analyzing the activities of serial deletion mutants of promoters. Based on the identified cis-elements, yeast one-hybrid is employed for investigation of the upstream regulators and signal transduction pathway of ScABI3 genes. Yeast two-hybrid is employed in studying the interacting proteins by ScABI3, elucidating its regulatory downstream target genes. The ScABI3 gene is transformed into Physcomitrella patens generating the overexpression transgenic P. patens. The transcriptomes of the transgenic, mutant and WT P. patens are built using the high throughput sequencing method. The target genes of ScABI3 will be determined by comparing the differentially expressed genes between overexpression, mutant, and WT P. patens. These results may be contributed to the development and utilization of genetic resources of desert plants, and provide the theoretical and practical base for molecular breeding.

ABI3是ABA信号通路中关键转录调控因子，参与种子休眠、质体发育、苔藓耐干等多个重要生理过程，在植物抗逆中起到关键作用。本项目以荒漠耐干苔藓——齿肋赤藓为材料，在鉴定出其抗逆基因ScABI3的基础上，进行三个层次的研究，即：ScABI3上游调控基因和信号通路、ScABI3转录调控及下游靶基因、ScABI3转基因苔藓抗逆分析，旨在系统揭示ScABI3基因响应逆境的分子机理。通过分离ScABI3基因启动子，构建启动子系列缺失载体来鉴定响应逆境胁迫的顺式元件，以这些元件为基础，利用酵母单杂交鉴定ScABI3上游调控因子。利用酵母双杂交研究其转录激活和互作蛋白，阐明其调控的下游靶基因。将ScABI3基因转入小立碗藓，过表达后建立转录组，通过比较过表达、突变体及野生型小立碗藓的差异表达基因，探讨ScABI3基因抗逆分子机理。进而为开发利用荒漠植物基因资源，培育抗逆作物品种奠定基础。

ABI3是ABA信号通路中关键转录因子，参与种子休眠、质体发育、非生物胁迫等重要生物学过程。本项目以荒漠耐干苔藓齿肋赤藓为材料，系统开展非生物胁迫下ScABI3调控机理研究。取得了以下研究结果：(1) 利用UPLC-MS法测定出齿肋赤藓存在内源ABA，且在干旱胁迫下含量明显发生变化；通过qRT-PCR方法分析了齿肋赤藓ABA合成及信号转导基因在非生物胁迫下的表达模式。(2) 齿肋赤藓沙培和组培的两种再生模式具有不同的表型可塑性，在干旱条件下，沙培苔藓叶面呈疣状和刺状，其内源ABA和海藻糖含量显著高于组培苔藓；但是，两种模式培养的苔藓在干燥和复水过程中对水分的响应是相同的，表明两种再生苔藓具有相似的耐干特性。(3) 齿肋赤藓ScABI3转基因拟南芥在种子萌发、幼苗生长及成苗阶段均可显著增加抗盐、抗旱及减少ABA敏感性，分析发现其抗非生物胁迫能力的提高是通过增加ROS清除酶活性、积累渗调物质、增强光合作用以及调节叶片气孔大小而共同实现。(4) 比较ScABI3基因过表达小立碗藓在100mM、200mM和300mM 甘露醇处理15d和25d的表型；初步发现与野生型相比，ScABI3转基因植株具有更高的存活率和生长速率。(5) 将ScABI3基因导入紫花苜蓿，发现ScABI3基因在紫花苜蓿中过量表达能够提高植株ABA含量，增加叶面积、叶茎比和分枝数，提高光合效率和水分利用率，减少茎秆木质素含量，最终提高转基因植株的粗蛋白含量。(6) 利用齿肋赤藓酵母cDNA文库中，筛选出一个RING型E3泛素连接酶ScPRT1，证实ScPRT1能与ScABI3发生直接互作，在外源ABA处理下，ScPRT1基因的表达会影响ScABI3基因的转录；ScPRT1蛋白在进化上与小立碗藓亲缘关系最近，与被子植物相距较远；在原核体系中表达ScPRT1重组蛋白，检测ScPRT1与已知的E1、E2和泛素蛋白的结合能力，通过泛素抗体检测证实ScPRT1能够进行自身泛素化修饰。上述研究成果系统揭示了齿肋赤藓内源ABA及其ABI3转录因子的逆境响应机制，对深入理解ABA在植物进化过程中发挥的作用具有重要意义。此外，ScABI3抗逆功能的明晰以及深入挖掘，对于日益激烈竞争的农作物分子改良具有重要价值。

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