高通量测序技术筛选真菌紫杉醇合成相关基因及其功能研究

Molecular cloning and functional research of genes related to fungal taxol biosynthesis, is a key aspect for clarification of molecular mechanism of fungal taxol biosynthesis and for increasing taxol production in taxol-producing fungi with genetic engineering technology. However, only a few of genes related to fungal taxol biosynthesis were reported and their function was scarce. In this project, Cladosporium cladosporioides MD2, a taxol-producing fungus, was used as a research subject. Differentially expressed genes (DEGs) in C. cladosporioides MD2 responded to Methyl jasmonate (MeJA) were analyzed by RNA-seq technology. The candidate genes related to fungal taxol biosynthesis were screened from DEGs, and the full-length cDNA and DNA sequences of the candidate genes were cloned by RACE technology. The biological functions of these candidate genes in C. cladosporioides MD2 were in vivo clarified by gene over-expression, targeted gene replacement (TGR) technology and RNA interference (RNAi) technology. The catalytic functions of the expression product of these candidate genes were clarified by heterologous expression analysis in E.coli or yeast. By these above research, we would illuminate five to eight key genes related to taxol biosynthesis in C. cladosporioides MD2. The research results can not only lay the foundations for clarification of the molecular mechanism of fungal taxol biosynthesis, also can provide the evidence for the genetic engineering improvement of taxol-producing fungi.

克隆真菌中紫杉醇合成相关基因并明晰其功能，是研究真菌紫杉醇生物合成的分子机理，以及利用基因工程技术提高真菌紫杉醇产量的关键环节。目前相关研究甚少。本项目以产紫杉醇内生真菌枝状枝孢霉MD2为研究材料，采用RNA-seq技术对枝状枝孢霉MD2受茉莉酸甲酯诱导前后的差异表达基因进行全面分析, 筛选与紫杉醇合成相关的侯选基因，采用基因敲除、RNA干扰、基因超表达技术在宿主体内（in vivo）研究候选基因的生物学功能，并通过在大肠杆菌或酵母中异源表达候选基因以及检测其表达产物的催化底物和产物，体外（in vitro）分析候选基因的催化作用，最终确定5-8个枝状枝孢霉MD2的紫杉醇合成相关基因及其功能，初步揭示枝状枝孢霉MD2中紫杉醇生物合成的分子机制，为高产紫杉醇基因工程菌株的构建提供理论依据。

筛选真菌紫杉醇合成相关基因资源，并克隆和功能分析相关候选基因，是研究真菌紫杉醇合成的分子机理以及利用基因工程技术提高真菌紫杉醇产量的关键。针对枝状枝孢霉（简称：枝霉）MD2继代过程中紫杉醇产量下降的现象，本项目采用RNA-seq技术首先分析了枝霉MD2的T0转录组（不添加MeJA），通过注释该转录组信息首次预测了枝霉MD2的紫杉醇合成途径及其基因信息，根据同源基因注释结果初步揭示了枝霉MD2继代过程中紫杉醇产量降低的可能原因。针对MeJA可以诱导提高枝霉MD2紫杉醇产量的现象，进一步完成了MeJA诱导不同时间点（0h、6h、12h、18h、24h、30h）的枝霉MD2的转录组测序以及枝霉MD2的基因组测序与分析工作，初步揭示了响应MeJA诱导的基因表达特征，进一步揭示了枝霉MD2紫杉醇合成的潜在途径及其相关候选基因。克隆了35个候选基因的DNA和cDNA全长序列，并完成其拷贝数和表达特征分析。构建了16个候选基因的过表达载体，并获得其转基因菌株，完成了16个候选基因过表达菌株的检测，初步分析了这些候选基因对枝霉MD2紫杉烷类合成的影响，一定程度上揭示了枝霉MD2紫杉醇合成相关的基因资源。分别采用同源重组方法以及Talen和CRISPR/Cas9系统构建了10个Unigenes的敲除载体，并进行了转基因分析，为后期相关研究提供了基础。分别构建了8个Unigenes的原核表达菌株，完成了6个Unigenes的诱导表达条件的优化工作，纯化了3个Unigenes的表达蛋白，为后期的酶学检测奠定了基础。另外，为了增加药用植物内生菌研究资源，开展了七叶一枝花和蛇足石杉内生菌研究，获得多株产重楼皂苷和石杉碱甲（乙）的内生菌资源，这是本项目的延伸，为后期研究扩展了新方向。本项目已发表论文16篇，已投SCI期刊3篇；授权发明专利1项，申请发明专利7项。本项目的完成为深入研究枝霉MD2的紫杉醇合成和调控机理奠定了基础，也为其它植物内生菌研究提供了参考。