长叶红砂黄酮类次生代谢产物积累与调控对逆境胁迫的响应

It is an important strategy that a vast array of the small organic molecules are synthesized and accumulated in plants to against certain biotic and abiotic stresses. Wild plants not only have ability to adapt to severe environment, but also have nutritional and medicinal value because of their abundant secondary metabolites. The current research will focuse on the wild plant Reaumuria trigyna, which is a recretohalophyte endemic to desert regions, with strong capability of salt and drought tolerance. The metabolomics analysis of Reaumuria trigyna will be performed by LC-MC and NMR technique, and the effects of the synthesis and accumulation of flavonoids will be systematiclly studied. Based on the analysis of the transcriptome data of salt stress, the differentially expressed genes of flavonoid biosynthetic pathway will be screened, and the resistant candidate genes will be obtained through detecting the changes of expression and enzyme activity. The target proteins will be obtained by prokaryotic expression, and the regulation and specific catalytic capacity would be detected. Transforming these target genes into tobacco, measuring the resistance phenotype and chemical composition of the transformed plants certaining the functions of the resistant candidate genes related to the accumulation of flavonoids, regulation of the tissue colour and, the improvement of the stress tolerance of plants the relationship between the synthesis and accumulation of flavonoids and plant resistanc to abiotic-stresses will be explored.

植物通过积累多种小分子次生代谢产物，保护其抵抗各种生物及非生物胁迫。自然界中生存着很多野生植物，不仅具有极强的抗逆能力，而且由于含有丰富的次生代谢产物，具有极好的利用价值。本项目以我国西部阿拉善荒漠区极具适应能力的强旱生泌盐盐生植物长叶红砂为研究对象，利用LC-MC与NMR技术分析长叶红砂代谢组，比较盐和UV-B胁迫前后其体内黄酮类代谢成分的变化。在深入分析长叶红砂盐胁迫前后转录组数据的基础上，筛选出黄酮合成途径中差异表达基因，通过基因表达量变化和酶活性检测，获得被诱导的抗逆候选基因。原核表达候选关键酶基因，通过检测纯化蛋白的调控和催化底物能力，发现有特殊催化活性的目的蛋白。将抗逆候选基因转化烟草，检测转化植株的抗逆表型和化学成分变化，筛选促进逆境胁迫下植物体内黄酮积累、组织颜色调控以及增强植物抗逆性的优异基因，探讨野生抗逆资源植物体内黄酮类次生代谢产物的合成和积累与植物抗逆性的内在联系。

本项目以对盐渍化荒漠环境具有极强适应性的柽柳科珍稀泌盐植物长叶红砂为研究对象，从次生代谢产物积累、基因表达调控以及生理生化特征等多个层面深入探讨了野生抗逆资源植物长叶红砂黄酮类化合物的积累与调控与植物抗逆性的内在联系以及其逆境响应机制，鉴定了9个相关基因功能，并筛选出8个可应用于植物抗逆性状优化及牧草和林木抗逆新品种培育的优良工程基因。主要结果如下：1.基于代谢组分析，鉴定出响应中性盐、碱性盐胁迫的黄酮类化合物各136种，其中有40和41种分别发生差异显著积累，黄酮和黄酮醇的生物合成通路对两种盐胁迫的响应最为积极, 中性盐对黄酮类物质积累的影响更为显著；2.基于转录组数据分析，筛选并克隆了2个黄酮合成途径关键基因RtLDOX2、RtF3’H1和7个逆境响应关键基因RtNAC100、RtNAC055、RtWRKY23、RtHKT1、RtGLX1、RtCML42、RtCDPK16，分析了基因表达特性并遗传转化拟南芥、紫花苜蓿和杨树，验证了基因功能，明确了除RtNAC100以外的8个基因均通过不同途径提高了转基因植物的抗逆性，探讨了RtLDOX2、RtF3’H1对黄酮类次生代谢产物的积累调控作用及与植物抗逆性的内在联系，解析了长叶红砂耐受环境胁迫的分子调控机制；3.RtLDOX2蛋白纯化和体外催化活性检测结果表明，重组RtLDOX2在体外将二氢山奈酚转化为山奈酚，可与类黄酮途径中的一种双加氧酶黄酮醇合成酶功能互换；4.生理特性研究表明，相比于碱性盐和干旱胁迫，长叶红砂对中性盐胁迫具有更高的耐受性，干旱、盐、碱、UV-B 等胁迫处理均显著提高其体内总黄酮和花青素含量，表明长叶红砂通过积累黄酮类次生代谢产物，提高抗氧化能力和渗透调节能力，进而抵御各种非生物胁迫；适度的NaCl和CaCl2通过ABA信号通路调控Na+和ROS积累以及PCD进程，缓解长叶红砂干旱引起的生长抑制和叶片衰老，并提高其耐旱性；5.从我国特有珍稀孑遗植物四合木叶中分离鉴定出21种化合物，4种为新化合物，发现1种苯甲醇苷类化合物对CdCl2造成的细胞损伤具有良好的保护活性；发现茎中分离的挥发油和三萜类化合物通过抑制羧酸酯酶和胆碱酯酶的活性，对菜青虫造成毒杀和生长抑制作用。