宁夏苦豆子氧化苦参碱合成关键酶SaLDC基因功能研究

Oxymatrine (OMA) is the main medicinal components of Sophora alopecuroides L., which is one of the most important medicinal resources plant in Ningxia with local characteristics. While previous evidences have indicated that drought stress could affect the content of oxymatrine and the genes expression in oxymatrine biosynthesis, the biosynthetic pathway of oxymatrine is not clear yet. With the Support of National Natural Science Foundation of China (No.30660017, 2007-2009) and Ningxia Natural Science Foundation (No.NZ14033, 2014-2015), the genetic diversity and the content of oxymatrine in different S.alopecuroides populations has been studied in our lab. The cDNA of SaLDC of an S.alopecuroides population with identical genetic background and stable content of active components has also been cloned. Based on the results of existing, this project aims to illustrate the role of SaLDC and its promoter in oxymatrine biosynthetic pathways and the relationship between the content of oxymatrine and gene expression. Firstly, real-time PCR will be used to verify the spatio-temporal expression patterns of SaLDC, followed by GUS gene expression and GFP tracer technique be used to study the subcellular location of SaLDC in S. alopecuroides L.. Then, the genetic complementation test and the technique of target gene inhibition of expression (RNAi) will be applied to confirm the functions of SaLDC gene. Finally, the sequence of promoter of SaLDC gene will be cloned to reveal its main functions. PEG 6000 will be applied to induce differential expression of the key enzyme genes in oxymatrine biosynthesis which directly promote the accumulation of oxymatrine. An expressed genes sequence database and a whole-genomic gene expression profiles on oxymatrine in S. alopecuroides will be constructed using De novo transcriptome sequencing by performing Solexa deep sequencing and screening differential expression genes to predict their main biological function and metabolic pathways. The molecular mechanism of oxymatrine biosynthesis is expected to be demonstrated to provide molecular-level base for the medicinal quality formation of S. alopecuroides.

氧化苦参碱(OMA)是宁夏重要药用植物苦豆子的主要有效成份，干旱胁迫对OMA含量积累有影响。在国家自然科学基金(30660017)和宁夏自然科学基金(NZ14033)的支持下，已对来源不同的苦豆子种群的遗传多样性、OMA含量等作了较系统的基础性研究，并选择遗传背景一致、有效成份含量稳定的种群，克隆得到OMA生物合成关键酶基因SaLDC全长cDNA。本项目拟在现有基础上：对SaLDC开展基因时空表达特性和亚细胞定位分析；利用遗传互补分析鉴定SaLDC的功能；克隆SaLDC的启动子序列并进行相应功能验证，明确SaLDC及其启动子在OMA生物合成中的重要作用。以PEG模拟干旱胁迫，诱导OMA生物合成关键基因的差异表达，采用高通量转录组测序(RNA-seq)和qRT-PCR技术，探索OMA合成过程中差异表达基因与SaLDC的关系，初步揭示OMA生物合成的分子机制，为苦豆子药材品质形成奠定基础。

氧化苦参碱是宁夏药用植物苦豆子的主要有效成份，赖氨酸脱羧酶是合成此生物碱的关键酶。本项目在前期已经克隆获得苦豆子赖氨酸脱羧酶基因（SaLDC）的基础上，开展以下工作：⑴对该基因开展基因表达模式与生物碱含量关系分析；⑵克隆SaLDC启动子基因，通过干旱和光照等因子的诱导，明确启动子的类型和功能；⑶尝试建立苦豆子遗传转化体系，利用基因超表达和沉默技术进行SaLDC功能互补验证；⑷对用150 mM NaCl胁迫的苦豆子开展转录组和蛋白组学分析，探索盐胁迫对苦豆子主要生物碱代谢通路与差异基因/蛋白的关系。结果显示：苦豆子SaLDC既是一个组成型基因，也是一个诱导型基因。SaLDC直接影响尸胺的生成，其相对表达量与尸胺含量极显著正相关。盐胁迫也影响苦豆子根、茎、叶中氧化苦参碱含量，氧化苦参碱含量与SaLDC相对表达量、尸胺含量均极显著正相关。克隆SaLDC启动子基因，生信分析表明该启动子序列有多个增强子、光响应和干旱响应元件，通过光诱导和干旱胁迫转SaLDC启动子基因的拟南芥幼苗，表明该启动子基因既有组织表达特异性又有时空表达特异性，是一个诱导型启动子。初步建立了苦豆子子叶节遗传转化体系。异源表达结果显示，SaLDC在转基因拟南芥株系中表现出较高的耐盐性和耐冷性。亚细胞定位显示SaLDC蛋白位于线粒体上；SaLDC原核表达产生的重组蛋白经LC-MS/MS鉴定，分子量是48 990.51 Da，GO注释表明该蛋白主要生物学功能是催化赖氨酸脱羧生成尸胺。苦豆子转录组测序和TMT蛋白组学分析发现，3d的盐胁迫主要影响与信号转导相关的通路和转运基因/蛋白的表达，7 d盐胁迫则可通过调节与次生代谢产物黄酮类、生物碱合成和氨基酸代谢相关基因/蛋白的表达以响应盐胁迫。筛选出几个与盐胁迫相关的潜在基因，研究结果为苦豆子药材品质形成提供了基础。