SGER: Identification of Eukaryotic Genes Required for Nitrogen Fixation

在对杨树固氮内生菌的研究中，有一个深刻的发现，即一些分离物是真核生物酵母。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。在这项探索性研究的小额拨款中，pi将从这些允许在无氮培养基上生长的固氮酵母中鉴定真核基因，并试图分离出不再在无氮培养基中生长的突变体。nif基因簇将被分析并与已知的重氮营养细菌进行比较，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。证实已知参与固氮的基因正在酵母株中表达，也将为真核固氮提供进一步的支持。如果可以分离出不再固氮的nif突变体，并且如果添加回氮酶基因可以恢复在无氮培养基上的生长，这将为真核生物固氮提供明确的证据。拟议的活动所产生的更广泛的影响包括强调对妇女的科学培训。多蒂博士将继续领导研究生水平的研讨会课程，植物与微生物的相互作用，在这门课程中，她将分享她关于杨树固氮内生菌的研究成果。目标是在一流的期刊上发表研究结果，并举办研讨会以广泛传播这一消息。如果成功，这项研究将导致目前对固氮的理解的范式转变，因为它将是固氮真核生物的第一个例子。

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.