环境pH及少孢节从孢产生的NH3在其形成捕食器官中的作用机理

Nematode-trapping fungi are ideal agents for controlling parasitic nematodes of plants and animals, but little is known about the molecular mechanism underlying trap formation or nematode-fungi interaction. Some reports and our previous study indicated that ambient pH and ammonia production by Arthrobotrys oligospora （one of the best-studied nematode-trapping fungi）affected its trap formation when A. oligospora was cultivated in the presence of nematode extract. Many fungi are reported to secrete ammonia. Four genes including areA、Ato、MEP and the global pH regulator pacC are reported as the key genes related to ammonia metabolism. The mechanisms behind ammonia production are mostly unknown. In this project, we will use RNA-Seq and real time PCR technology to determine the transcriptional profile of A. oligospora growing in nematode extract under different pH and identify key genes involved in ammonia metabolism or trap formation. These genes identified and reported will be knocked out. Phenotypes of mutant strains and wild-type strain will be compared. Phenotypes include hyphae or colony morphology, capability of infecting nematode, number of traps, dynamic change of medium pH, dynamic change of ammonia concentration in the growth medium and inside the fungal mycelium, change of mycelium pH. Based on these, the role and mechanism of ambient pH, ammonia secretion from A. oligospora on trap formation in fungi by nematode extract will be elucidated. The results will be helpful to further study the mechanisms of trap formation in A. oligospora, and be advantageous to control nematode.

食线虫真菌是一类潜在的植物寄生线虫的生防资源，然而其捕食器官形成的机制不清楚。文献及前期研究表明，在线虫提取液诱导少孢节丛孢（食线虫真菌的模式种）形成捕食器官过程中，培养基pH及少孢节丛孢产生的NH3对其捕食器官形成有影响。很多真菌都会产生氨气，文献报道与氨气代谢相关的基因有areA、Ato、MEP和pH信号途径的pacC，但是氨气代谢的机理尚不清楚，本项目拟用RNA-seq、real time PCR技术，获得在不同pH条件下线虫提取液诱导少孢节从孢形成捕食器官的转录图谱，鉴定出与氨气代谢或捕食器官形成相关的关键基因，并敲除文献报道的和鉴定出的关键基因，通过比较敲除株与野生株的表型，阐明环境pH及少孢节从孢产生的NH3在其捕食器官形成过程中的作用及机理。为研究少孢节丛孢捕食器官形成的分子机制提供有效途径，为提高这类生防菌的生物防效奠定基础。

植物病原线虫对农作物造成了很大的危害，食线虫真菌是一类潜在的植物寄生线虫的生防剂，然而其捕食器官形成的机制不清楚。我们研究表明环境的pH能影响线虫提取液诱导少孢节丛孢形成捕食器官，随着pH的升高，捕食器官的数量也在增加；而且发现在此过程中，培养基中氨的浓度也增加，是由少孢节丛孢产生的。我们用RNA-seq技术对在pH5和pH7时线虫提取液诱导少孢节丛孢形成捕食器官过程中的相关基因进行了转录水平的分析。在此基础上确定了可能参与真菌氨气代谢、调控pH和捕食器官形成相关的六个关键基因：NAD+特异性的谷氨酰胺脱氢酶GDH2（编码氨气合成的基因）、调控氨气分泌的基因Amet、谷氨酰胺合成基因GS1、总氮源调控转录因子areA、铵盐转运基因MEPA，MEPB，MEPC、pH调控系统基因PacC。我们用实时荧光定量PCR研究了在环境pH为7时，此过程中这些基因的表达量，发现少孢节丛孢的GDH2、areA和GS1三个基因上调，余下基因下调。对这六个关键基因进行了敲除。三个表达量上调的基因GDH2、areA和GS1的突变株在线虫提取液诱导下，捕食器官数量较野生性降低，同时分泌出的氨气也减少，结果暗示了在线虫提取液诱导少孢节丛孢过程中，真菌能利用线虫提取液中的氮源产生氨气，一部分输送到环境中，改变环境pH，一部分参与了真菌捕食器官的形成。三个表达量下调的areA、MEP(MEPA，MEPB，MEPC)、PacC的敲除株在线虫提取液诱导条件下，捕食器官数量较野生性降低，同时分泌出的氨气也减少，结果暗示环境的pH影响线虫提取液诱导少孢节丛孢形成捕食器官；而且尽管在pH7时这些基因表达量下调，但是它们可能在其它条件下参与了真菌捕食器官的形成。研究结果阐明了外界pH和少孢节丛孢产生的氨气在线虫提取液诱导其形成捕食器官的作用和机制，为生防制剂的开发奠定了坚实的基础。