黄酮类化合物在罗布麻适应盐胁迫环境中的作用机制研究

As an perennial plant, Apocynum venetum Linn. is well known for its high resistance to salinity,drought,heat and leanness as well as being wildly used as medicine,fiber,ornamental plant, forage and nectariferous plant.To prepare two sequence libraries from control and NaCl-treated samples ,which are sequenced using short reads sequencing technology to abtain the transcriptome information of Apocynum venetum Linn.,and to investigate changes in the transcriptome of the species in response to salt stress,to discuss the role of flavonoids in Apocynum venetum Linn.’adaptation to salt stress.To discuss the regulation mechanism of flavonoid biosynthetic pathway under salt stress by analyze the physio-chemical mechanism of antioxidant protection in adapting salt stress.To clone flavonols synthetase gene(AvFLS),and use real-time RT-PCR to quantitative analysis the transcript abundance of AvFLS gene under salt stress,to elucidate the gene characteristics and stress expression and the adapting regulation at metabolite level of flavonoid biosynthetic pathway.Desert plants formed special salt tolerance mechanism in the long process of evolution . In the aspect of cultivation of crop genetic improvement of salt resistance has a broad application prospects,at the same time to studies on the biosynthetic pathways of flavonoids in Apocynum, at molecular and biochemical levels.For using bioengineering Apocynum fiavonoids for human health in the future has important theoretical significance and practical value.

罗布麻为多年生植物，具有耐盐碱、干旱、酷暑、寒冷、贫瘠等优良抗性。罗布麻用途广泛，可药用、也是良好的饲用、纤维、绿化和蜜源植物。采用Illumina测序技术对罗布麻进行转录组深度测序，获得该植物大量的表达基因信息，分析其响应盐胁迫的转录组水平变化，探讨罗布麻黄酮类化合物适应盐胁迫的抗氧化保护生理生化机制。克隆黄酮醇合成酶基因AvFLS，利用real-time RT-PCR技术，定量分析盐胁迫下AvFLS基因的转录丰度，阐明AvFLS基因特征与胁迫表达以及黄酮类代谢终产物响应环境胁迫的变化特征。荒漠植物在漫长进化过程中形成的特殊耐盐机制，在栽培作物耐盐性的遗传改良方面有广阔的应用前景，同时对罗布麻黄酮类化合物生物合成的分子生物学、遗传学及生物化学机理进行深入研究，为利用代谢工程生产对人体健康有益的罗布麻黄酮具有重要的理论意义和实践价值。

黄酮类化合物作为罗布麻中的一类重要次生代谢物，毫无疑问在罗布麻耐逆过程中发挥着无可代替的作用，因此，有必要对其与罗布麻逆境胁迫应答之间的关系进行深入探讨。本项目首先开展代谢组分析，对代谢物进行盐处理与对照组差异分析，挖掘差异代谢物；开展空白对照及盐胁迫下罗布麻转录组分析，完成测序数据过滤，De novo组装，Unigene功能注释；黄酮类化合物合成代谢途径差异基因检测、GO功能分析、Pathway功能分析；通过关联分析构建转录组-代谢组调控网络，挖掘盐胁迫下参与黄酮类代谢物合成的关键基因。其次，通过研究盐胁迫下罗布麻植株黄酮类化合物的种类和含量，比较分析在植株整体水平代谢途径表达的代谢产物的适应性变化，确定代谢途径特定关键基因在植物盐胁迫下的响应及适应性调控；其次通过研究罗布麻叶片抗氧化系统酶活性变化、黄酮类化合物含量和抗氧化能力等变化特征，分析罗布麻黄酮类化合物在盐胁迫过程中抗氧化的生理生化机制以及罗布麻黄酮类化合物代谢途径关键酶响应盐胁迫的适应调控机制。最后，克隆关键差异基因AvFLS和AvANS，分析其亚细胞定位、基因特征及其在盐胁迫条件下组织特异性和表达水平的响应分析，开展罗布麻黄酮醇合成酶基因AvFLS转拟南芥及适应盐逆境的功能验证生理及分子实验。本项目实现在生理生化层面及分子和代谢水平阐明了罗布麻黄酮类化合物的调控机制及抗逆功能，为罗布麻的分子育种及盐碱化土壤改良提供了理论基础。