microRNA在疯草内生真菌苦马豆素合成中的分子调控机制研究

Locoweeds are poisonous plants containing the toxic alkaloid swainsonine that causes significant economic losses to the livestock industry on western grasslands.The swainsonine-producing fungal endophytes, Undifilum sp，are believed to be responsible mainly for the biosynthesis of swainsonine. If the regulation mechanism related with the biosynthesis of swainsonine could be elucidated, the toxicity of locoweed should be reduced and eliminated by controlling the endophytes within the locoweeds not to produce swainsonine. By base pairing binding with complementary sequences in target mRNAs, microRNAs induce degration of mRNAs or inhibititon of protein translation and regulate gene expression in eukaryocyte post-transcriptional level. In this project, the libraries of microRNAs and mRNAs would be constructed respectively. The genes sequences of libraries of microRNAs would be sequenced by high-throughput sequencing. The function of microRNAs and mRNAs could be known preliminary, and some target sites of microRNAs could be also predicted within partial mRNAs. The microRNA expression vectors are also going to be developed to transform endophytes. By assessing the biosynthesis of swainsonine and gene expression of microRNAs or target mRNAs in transformed fungal cells, the microRNAs and mRNAs related with the biosynthesis of swainsonine would be picked up, and theirs function could be known. The molecular regulation mechanism related with the biosynthesis of swainsonine could be partially elucidated based on the analysing genes regulatory networks in fungal cells. The project would also offer a reference that control of locosim by inhibiting the locoweed endophytes from producing swainsonine based on microRNAs induced gene silencing.

疯草是严重危害西部草原畜牧业的毒草，主要毒性成分为苦马豆素（SW）。疯草内生真菌是疯草中SW产生的主要因素。阐明内生真菌合成SW的分子调控机制，可为阻断内生真菌合成SW，消除疯草的毒性奠定基础。microRNA在真菌转录后基因表达调控中发挥着重要作用，它可降解与之互补的靶mRNA，抑制蛋白质编码基因表达。本项目拟构建内生真菌microRNA文库，对其进行高通量测序，并对microRNA及其靶mRNA进行功能解析和靶位点预测；构建microRNA表达载体，将microRNA转化内生真菌，比较转化子SW产量、microRNA和靶mRNA表达水平的变化，筛选出参与SW合成调控的microRNA，并确定其功能；探讨SW合成相关调控网络，阐释疯草内生真菌SW合成中的分子调控机制。研究成果将为进一步应用基因沉默技术阻断SW生成，从控制内生真菌途径防除动物疯草中毒病提供依据。

疯草严重危害西部草原畜牧业，主要毒性成分为生物碱苦马豆素（SW）。疯草内生真菌是疯草中SW产生的主要因素。阐明内生真菌合成SW的分子调控机制，可为阻断内生真菌合成SW，消除疯草毒性奠定基础。microRNA可降解与之互补的靶基因，在真菌转录后基因表达调控中发挥着重要作用。本研究主要通过对疯草内生真菌基因组、转录组和microRNA组的高通量测序，获得相关基因序列及其功能注释信息，为筛选SW合成相关基因及microRNA提供基因资源。对候选基因表达模式及真菌SW合成关联分析、microRNA及其靶基因表达和互作预测分析，筛选SW合成相关基因。构建基因敲除载体，敲除候选基因，分析其对真菌SW合成及相关基因表达的影响，验证候选基因功能，确定与SW合成相关基因及其microRNA。.获得了疯草内生真菌A.oxytropis UA003菌株基因组精细图、转录组和microRNA组的序列与功能注释信息。基因组全长为77,083,474 bp，组装为102个Contig，10,569个基因，分别在nr、SwissProt、KEGG等12个数据库获得功能注释信息，预测到次生代谢产物合成基因簇33个。转录组分析获得差异表达基因243个，可能参与经P6C途径合成L-哌可酸及SW的基因8个，生物碱合成相关基因4个，萜类物质合成相关基因6个。其中6个差异基因为同一个T1型-PKS次生代谢产物合成基因簇（swn）的不同基因，swn基因簇由氨基酸转运样蛋白基因、swnN、swnR、swnH1、swnH2、swnK等基因组成，其中swnK是一个多功能基因，由A、T、KS、AT、SDR和SDRe1组成。候选基因表达模式及其与SW合成的关联分析结果表明，swnK基因中各区域的表达模式与SW合成正相关，其他基因负相关。将真菌swnK基因KS区域敲除后，真菌不产生SW。表明swn基因簇在SW合成中发挥着重要作用，可能参与从L-哌可酸到SW这一合成途径中相关酶的合成。microRNA组及其靶基因互作的预测分析结果表明，获得的15个microRNA中10个预测到靶基因，其中novel_18的靶基因为swn基因簇中的氨基酸转运样蛋白基因，novel_18可能参与swn基因簇基因表达和SW合成的转录后调控。本研究为进一步阐明疯草内生真菌合成SW的转录后调控机理奠定了基础。