基于镰刀菌诱导的木霉菌Vel1&Lae1调控抗菌肽合成机制研究

In filamentous fungi, the global conserved regulatory velvet complex composed of VeA, VelB, and LaeA involved in the regulation of diverse cellular processes including control of asexual and sexual development as well as secondary metabolism. In this study, both VeA and LaeA homologs vel1 and lae1 have been cloned in Trichoderma spp., regulation mechanism of Vel1&Lae1 on secondary metabolites will be verified under the interaction of Trichoderma-Fusarium. Firstly，Vel1&lae1gene knockout mutant and their over-expression stain of T.atroviride were obtained, the phenotypes of growth and reproduction were analyzed and compared，and then RNA-seq transcriptional sequence was carried out to analyze the up-regulated and down-regulated genes of NPRS&PKS coded the secondary metabolites synthesis mediated by vel1&lae1 in interaction with Fusarium. Silica gel column chromatography and metabonomics (LC/ESI-MS/MS) were used to screen and indentify the Peptaibols compound response to vel1& lae1's common regulation. One or two specific compound belongs to NPRS was obtained using NMR and spectral data analyses. Finally, genomic mining the gene clusters of targeted NPRS and the regulation mode of both vel1and lae1 on the gene expression coded NPRSs was explored. The specific gene regulatory mode of Vel1& lae1 were revealed on their regulation of NPRS and its coded gene clusters.

丝状真菌中Velvet三聚体(veA,veB和laeA)全局性调控菌丝生长发育和次级代谢合成。木霉菌中克隆到同源蛋白Vel1和Lae1，研究基于木霉菌-镰刀菌互作，Vel1&Lae1调控抗菌肽类产物合成机制。首先，构建深绿木vel1和lae1敲除株和过表达株，表型分析二者对木霉菌生长和发育影响。其次，RNA-seq分析互作vel1和lae1基因调控多肽类次生代谢产物合成的关键酶，NPRSs和PKSs表达差异，筛选出受调控的NRRSs以及其合成的抗菌肽(NPRS)类产物。然后柱层析和液质LC/ESI-MS/MS对代谢产物分离纯化，确定vel1和lae1基因共同调控的抗菌肽，NMR和波谱分析鉴定1-2种目标化合物。最后，基因组查找目标NRRS合成基因簇的结构基因，考察vel1&lae1调控特定的NRRS合成基因表达的相互关系，揭示木霉菌vel1和lae1调控抗菌肽合成及其基因表达的特定模式。

抗菌肽（Peptaibols）是由非核糖体肽合成酶（Non-Ribosomal Peptide Synthetases, NRPS）合成的线性多肽，在木霉抗植物病原菌活性中发挥重要作用。本项目研究深绿木霉甲基化转移酶LaeA的同源物lae1和VeA蛋白的同源物vel1对抗菌肽生物合成的调控作用，共发现17种受调控明显的抗菌肽，探索木霉抗菌肽抑制禾谷镰孢菌的抑菌活性及可能的机制。本研究成果丰富了抗菌肽数据库，有助于发展抗菌肽为主要成分的生防产品，应用于植物病害的防治领域。.1、深绿木霉负责长链抗菌肽合成基因簇为NRPS1蛋白（ID为XP_013944039.1），含有19个氨基酸残基模块、为酸性但不稳定的蛋白质。lae1有效激活NRPS1及其侧翼基因的表达；vel1部分激活NRPS1及其侧翼基因的表达。.2、基于超高效液相色谱四极杆飞行时间质谱（UPLC-QTOF-MS）的结果表明，深绿木霉T23、lae1敲除菌株Mlae1和vel1敲除菌株Mvel1共产生了16种含量较高的新型抗菌肽，丰富了抗菌肽数据库。新型抗菌肽为18、19和20个残基抗菌肽，并且在lae1敲除后，81.5%的抗菌肽下降2～200倍；vel1敲除后，所有抗菌肽表达均下降，说明转录因子lae1和vel1在抗菌肽的生物合成中起正调控作用。.3、深绿木霉菌株lae1和vel1过表达菌株固体发酵和液体发酵的代谢物差异发现：在固体发酵时，OElae1菌株的总肽量显著高于T23（P<0.05），OEvel1菌株总肽量与T23无显著性差异；OElae1菌株较T23菌株有14种抗菌肽上调表达，而OEvel1菌株有8种上调表达。在液体发酵时，OElae1菌株和OEvel1菌株的总肽量显著低于T23（P<0.01）；OElae1菌株较T23菌株有9种抗菌肽下调表达，而OEvel1菌株有10种下调表达，说明二者对抗菌体合成调控受培养基及培养条件的影响。.4、木霉与禾谷镰孢菌对峙后检测抗菌肽种类和数量的变化，结果表明，五种18个残基或19个残基的抗菌肽含量明显提高，Pept-1781b、pept-1781a不仅受vel1和lae1显著诱导，也受禾谷镰孢菌诱导，Velvet蛋白过表达后使得细胞膜透性增大；抗菌肽粗提物可以降低禾谷镰孢菌中的呕吐毒素产量，为木霉菌抗菌肽生防菌剂研发提供理论依据。