疯草内生真菌中苦马豆素合成调控基因的筛选与功能研究

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, Undifilim oxytropis, are isolated frequently from locoweeds. There is a strong relationship between the presence of endophytes and swainsonine concentration of their host plants. The endophytes are believed to be responsible mainly for the toxicity of locoweeds. The toxicity of locoweed is likely to be reduced and eliminated by controlling the U. oxytropis within the locoweeds not to produce swainsonine. In this project, the gene expressions of endophytes treated in control and experimental conditions will be assessed by suppression subtractive hybridization(SSH). The SSH cDNA library will be constructed and differential expression genes will be isolated and cloned. The RNA interference(RNAi) vectors are also going to be developed to inhibit or interrupt the biosynthesis of swainsonine in the fungal cells according to the structures of differential expression genes. Some regulatory genes that related to the biosynthesis of swainsonine will be isolated from the differential expression genes and theirs functions will be knew. The controlling mechanism related to the biosynthesis of swainsonine will be tried to elucidate based on the above research. The results will lay a foundation to explain further the biosynthesis of swainsonine in the Undifilim fungi. The project will also offer a reference that control of locosim to prevent the Undifilum fungi from producing swainsonine by gene knockout.

疯草是严重危害草原畜牧业的毒草之一，其主要毒性成分为苦马豆素（Swainsonine, SW）。疯草内普遍存在能产生SW的内生真菌（Undifilim oxytropis），该真菌与疯草的毒性紧密相关，是疯草内SW产生的主要因素。抑制或阻断内生真菌合成SW可降低或消除疯草的毒性。本项目拟应用抑制差减杂交技术研究正常状态和诱导因子作用下内生真菌基因表达情况，构建抑制差减cDNA文库，筛选并克隆差异表达基因；根据差异表达基因构建RNA干扰载体，应用RNA干扰技术抑制或阻断内生真菌合成SW，从差异表达基因中筛选出与内生真菌合成SW相关的调控基因，分析并确定其功能，从分子水平探讨疯草内生真菌合成SW的调控机制。研究结果将为进一步阐释疯草内生真菌合成SW的机理奠定基础，为最终敲除内生真菌中SW合成调控基因，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。

经过3年研究，完成了疯草内生真菌U. oxytropis分离鉴定，研究了pH、不同前体、诱导剂等对U. oxytropis合成SW性能的影响。选择显著影响SW合成的条件诱导培养真菌，提取真菌RNA，构建了cDNA文库。采用二代测序技术对转录组进行了高通量测序，将获得的Unigene序列与NR、Swiss-Prot、GO、COG、KOG、KEGG数据库比对，对其进行了分类注释；对1kb以上Unigene进行了SSR分析，对SSR标记进行引物设计、CDS预测和SNP分析。通过表达量分析，从Unigene中筛选了差异表达基因，并对其进行了功能注释。建立了U. oxytropis原生质体转化系统，并对L-amino acid oxidase(LAAO)基因全长cDNA序列进行了克隆，根据LAAO基因序列和pDL2中间载体序列构建了基因敲除载体，并应用U. oxytropis原生质体转化系统对LAAO基因进行了同源重组转化，研究了该基因在真菌SW合成中的功能。.主要成果如下：（1）确定了低pH、PEG、L-哌可酸、L-赖氨酸、α-酮戊二酸均对U. oxytropis中SW合成产生显著影响。初步确定L-哌可酸、L-赖氨酸、α-酮戊二酸是U. oxytropis合成SW的重要前体物质，U. oxytropis可能通过赖氨酸代谢途径合成SW。确定了导致U. oxytropis合成SW产量与对照组差异显著的培养条件，在该条件下培养真菌可为cDNA文库构建提供样本材料。（2）转录组测序共获得25.23Gb Clean Data，测得的Reads经组装后获得23,492条Unigene，与各数据库比对后，获得17,627条Unigene注释信息。SSR分析获得了3,725个SSR标记。基因表达量分析筛选出了66个差异表达基因，层次聚类分析后，获得了54个差异表达基因，该类基因主要与氨基酸的代谢、转运、合成等有关。该研究为后续SW合成相关基因的功能验证提供了候选基因序列信息。（3）制备了用于候选基因功能研究的原生质体转化系统，克隆了LAAO基因的全长cDNA序列，构建了LAAO基因敲除载体，成功将其转化原生质体，筛选获得了阳性转化子，初步确定LAAO基因参与U. oxytropis中SW的合成的调控，当LAAO基因表达受到抑制后，真菌中SW的含量显著降低。