SGER: Identification of Eukaryotic Genes Required for Nitrogen Fixation

在研究杨树内的固氮内生菌时，人们深刻地发现，其中一些菌株是真核生物酵母菌。由于人们认为只有原核生物才能利用空气中的氮，这是一个令人兴奋的发现。在这笔用于探索性研究的小额拨款中，PI将从这些固氮酵母菌中鉴定出能够在无氮条件下生长的真核基因，并试图分离出不再在无氮条件下生长的突变株。NIF基因簇将被分析并与已知的重氮菌进行比较，他们将确定这些基因是否具有真核转录信号。确定酵母菌株基因组中固氮所需的已知基因将为这种真核生物的固氮提供令人信服的证据。验证已知的与固氮有关的基因在酵母菌株中的表达也将为真核固氮提供进一步的支持。如果能够分离出不再固定氮的nif突变体，如果重新添加固氮酶基因可以在无氮条件下恢复生长，这将为真核细胞固氮提供明确的证据。拟议活动产生的更广泛影响包括强调女性的科学培训。Doty博士将继续主持研究生级别的研讨会课程，即植物-微生物相互作用，在该课程中，她将分享她关于杨树内生固氮细菌的研究结果。目标是在一流的期刊上发表研究结果，并举办研讨会广泛传播这一消息。如果成功，这项研究将导致目前对固氮的理解发生范式转变，因为它将是第一个固氮真核生物的例子。

While studying the nitrogen-fixing endophytes within poplar trees, the profound discovery was made that some of the isolates were yeast, a eukaryotic organism. Since it is believed that only prokaryotic organisms can utilize nitrogen from the air, this was an exciting discovery. In this small grant for exploratory research, the PIs will identify the eukaryotic genes from these nitrogen-fixing yeasts that allow growth on nitrogen-free medium, and attempt to isolate mutants that no longer grow in nitrogen-free medium. The nif gene cluster will be analyzed and compared with known diazotrophic bacteria and they will determine whether or not the genes have eukaryotic transcription signals. Identification of the known genes required for nitrogen fixation within the genome of the yeast strain will provide compelling evidence for nitrogen fixation by this eukaryotic organism. Verification that the genes known to be involved in nitrogen fixation are being expressed in the yeast strains will also provide further support for eukaryotic nitrogen fixation. If nif mutants can be isolated that no longer fix nitrogen, and if adding back the nitrogenase genes restores growth on nitrogen-free medium, it would provide unequivocal evidence of eukaryotic nitrogen fixation.The broader impacts resulting from the proposed activity include an emphasis on training of women in science. Dr. Doty will continue to lead a graduate level seminar course, Plant-Microbe Interactions in which she shares her results about the nitrogen fixing endophytes of poplar. The goal is to publish the results in a first-rate journal, and give seminars to disseminate the news widely. If successful, this research would cause a paradigm shift in current understanding of nitrogen fixation since it would be the first example of a nitrogen-fixing eukaryotic organism.