小花棘豆Embellisia内生真菌的酵母氨酸还原酶基因在苦马豆素代谢中的作用

The Oxyrropis glabra is a locoweed species widely distributed in the grassland and desert regions among Inner Mongolia. Many plants of this species contain poisonous swainsonine to livestock. The swainsonine inhibits the activity of mannosidase in animal cells. The relaterd matabolisms in anamals were blocked after feeding the plants, therefore the great loss in animal hasbandry is caused .The previous investigation results of the applicant discovered that the swansonine toxicity of the O.glabra was caused by the fungal endophyte belonging to Embellisia.However,the biochemical pathway of swainsonine in this fungal endophyte is still unknown.It was reported in 2012 that the contents of swainsonine and its precusor pipecolic acid in fungal endophytes isolated from other locoweed species distributed in USA increased along with lacking of saccharopine reductase,while the contents of saccharopine and lysine decreased.In this research the saccharopine reductase gene from our Embellisia fungal endophytes in Oxytropis glabra will be seperated by molecular biological and gene engineering techniques, and the lacking of saccharopine reductase mutant of the fungal endophyte will be constructed.The changes and relationship in contents of swaisonine, pipecolic acid, saccharopine and lysine will be investigated both in the mutant and the wild strain, thus the the function of saccharopine reductase gene in swainsonine metabolism will be explored. The studies are very significant to reveal the biochemical pathway and the molecular toxic mechanism of swainsonine in the fungal endophyte.

小花棘豆是内蒙古草原和荒漠区广泛分布的重要疯草物种，该物种许多植株含对牲畜有毒的吲哚里兹啶类生物碱-苦马豆素，苦马豆素可抑制动物细胞内的甘露糖苷酶，牲畜食用后体内相关代谢发生障碍而中毒，造成草原畜牧业重大损失。笔者前期研究表明小花棘豆的苦马豆素毒性由其Embellisia内生真菌引起，有关该内生真菌内苦马豆素的生化代谢途径未知。2012年有研究发现：从美国分布的疯草物种里分离的内生真菌中如果缺乏酵母氨酸还原酶，则苦马豆素及其前体吡啶2-羧酸的含量增加，而酵母氨酸和赖氨酸含量降低。本研究拟应用分子生物学与基因工程技术从小花棘豆内生真菌中分离酵母氨酸还原酶基因序列，然后构建该基因缺失的小花棘豆内生真菌突变株，研究在突变株和野生株中苦马豆素、吡啶羧酸、酵母氨酸和赖氨酸的含量变化及其相互关系，探索酵母氨酸还原酶基因在苦马豆素代谢中的作用，对揭示内生真菌中苦马豆素代谢途径及其分子机制有重要意义。

小花棘豆是内蒙古草原和荒漠区广泛分布的重要疯草物种，许多植株含对牲畜有毒的生物碱苦马豆素（SW），SW可抑制动物细胞内的甘露糖苷酶，牲畜食用后体内相关代谢发生障碍而中毒，造成草原畜牧业重大损失。笔者前期研究表明小花棘豆SW毒性由其内生真菌Alternaria oxytropis（之前归到Embellisia）引起，该内生真菌中SW生化代谢途径未知，酵母氨酸还原酶基因（sac）可能起重要作用。本研究应用分子生物学与基因工程技术从小花棘豆内生真菌中分离到sac，构建内生真菌sac缺失突变株M1，检测了在M1和野生株OW7.8中SW及前体的含量，分析了添加四种前体α-氨基己二酸、哌啶酸、赖氨酸和酵母氨酸后真菌SW的含量变化及其相互关系。构建了sac功能互补株C1。探索了光照和培养基条件对真菌的SW水平和生长的影响。进行了OW7.8和M1转录组测序的部分工作。结果表明：OW7.8的SW水平高于M1，OW7.8和M1中赖氨酸的水平变化很小。添加前体的OW7.8和M1内SW水平和sac表达量均高于对照组，随添加浓度不同，SW合成量不同。sac表达量高，SW合成量多；反之sac表达量低，则SW合成少。根据Sac cDNA序列预测的多肽含472个氨基酸，克隆到sac启动子及上游调控序列，含TATA-box、CAAT-box、G-box和7个胁迫应答元件。发现sac互补株C1的SW含量高于OW7.8和M1。暗培养利于真菌生长，间歇光照条件利于真菌SW合成。根据转录组数据注释到45634个差异表达基因（DEGs），鉴定出41个与SW生物合成相关的DEGs，5个基因注释到酵母氨酸还原酶（Sac），它们在M1中均呈下调，表明Sac促进SW合成。研究结果对深入揭示内生真菌A. oxytropis的SW毒性分子机制及SW合成代谢途径有重要意义。