基于双分子标记法的金铁锁核心种质构建与优良种质筛选

Psammosilene tunicoides is one of the main ingredients of the “Yunnan Baiyao”. Besides as a geoherbs and endemic species in Southwest China, P. tunicoides is an endangered species under second grades protection in China Plant Red Data Book. Its natural resources can not meet the needs of pharmaceutical production. Core germplasm resources should be screened urgently for GAP cultivation. The proposed project is based on a lot of preliminary works from our group, including cultivation, phytochemical analysis, transcriptome analysis, etc. A comprehensive field investigation across P. tunicoides distribution area and wild population sampling will be conducted. The strategy of Bimolecular Marking Method (BIMM) will be adopted, including molecular genetic markers and metabonomic markers. Chloroplast gene sequences, two key synthase gene of the active ingredient (β-AS and PSS) and EST-SSR results will be joint studied for the purpose of P. tunicoides wild populations genetic diversity and phylogeographic analysis. Differently origined P. tunicoides root metabolomic fingerprints, especially the active ingredient content and variation, will be determined by conventional phytochemical techniques and LC-MS to screen for origin characteristic metabolic markers. The construction of P. tunicoides core germplasm and elite germplasm screening will be completed based on the results. Combined BIMM results and the two key gene expression analysis by real time PCR, the correlation of different P. tunicoides germplasm resources genetic variation and the medical quality will be investigated. This project will provide scientific basis for the P. tunicoides protective strategy development, elite germplasm identification and cultivation and also have significance in its conservation biology and actual pharmaceutical production.

金铁锁是“云南白药”的重要组分，为我国西南地区的道地药材和特有二级濒危保护植物。其野生资源已不能满足医药生产的需求，亟需通过优良种质筛选、引种驯化、人工栽培加以解决。本项目拟在本研究组大量前期工作基础上（包括栽培、有效成分分析、转录组等），通过全面的野外调查及取样，运用双分子标记法（分子遗传标记和代谢组学标记）：联合叶绿体基因序列、两个有效成分关键酶基因（β-AS和PSS）的多态性与EST-SSR分子标记，对不同金铁锁野生种群进行遗传多样性及分子谱系地理学分析；再利用常规植化方法结合LC-MS技术对金铁锁种群进行代谢组学分析，寻找具有产地特征的代谢标识物，并关注其有效成分含量和变化规律，进而完成金铁锁核心种质构建和优良种质的筛选。综合双分子标记的结果与关键酶基因表达差异分析，探讨金铁锁种质资源的遗传差异与药材品质的相关性。为金铁锁的保护策略制定、优良种质鉴定、筛选及人工育种提供科学依据。

金铁锁是“云南白药”的重要组分，为我国西南地区的道地药材和特有二级濒危保护植物。其野生资源已不能满足医药生产的需求，亟需通过优良种质筛选、引种驯化、人工栽培加以解决。本课题紧密结合目前本学科国内国际主流的组学研究新技术新方法，利用高通量测序技术完成了金铁锁的全叶绿体基因组测序分析工作，筛选了适合的叶绿体基因片段用于群体遗传分析；完成了金铁锁不同组织的转录组测序工作，筛选并克隆了金铁锁三萜皂苷合成途径中的六个关键酶基因，并利用RT-PCR进行了基因表达分析；选取克隆得到的β-AS基因作为核基因标记，完成了对11个野生金铁锁居群的谱系地理学分析；利用cpDNA序列及SSR分子标记分析了野生及栽培金铁锁居群的遗传多样性和遗传结构，提出了金铁锁的保护策略并探讨了栽培居群种质混杂的原因；综上，成功完成了金铁锁的核心种质构建。本项目系统全面地分析了金铁锁种质资源的遗传多样性、遗传分化及功能基因差异表达式样，并完成了不同产地的金铁锁化学指纹图谱研究。通过项目研究的实施，相关研究技术得到了完善和提高，为深入开展后续相关研究提供了可靠的技术支撑。本项目的研究结果将为金铁锁野生种质资源保存以及优良种质资源的筛选提供新的理论依据。项目基本完成了任务书规定的研究内容及预期目标。发表相关科研论文6篇，出版专著1部，培养研究生2名。