地涌金莲中苯基非那烯酮生物合成[4+2]环加成关键酶的发现与功能验证

苯基非那烯酮（PhPN）是香蕉植保素，二芳基庚烷酮（DAHO）[4+2]环加成反应是形成其母核的关键，但催化该[4+2]反应的环化酶仍不清楚。因[4+2]环化酶基因常不具有保守序列，其底物也难以获得等，未知[4+2]酶的鉴定很具挑战性。本申请拟在前期发现地涌金莲种子中PhPN合成具有时间特异性及种子粗酶可加速DAHO[4+2]反应的基础上，分析不同发育期地涌金莲种子转录组和代谢组差异，筛选出环化相关基因；运用化学蛋白组学技术，合成以DAHO为底物的小分子探针，标记并富集种子中与探针相结合的蛋白，鉴定与DAHO互作的环化相关酶；将两者获得的交叉蛋白作为候选蛋白进行原核表达和纯化，验证其催化DAHO环加成反应的活性；对转入候选基因的拟南芥进行PhPN分析和抗病性测定，进一步验证功能，从而鉴定出DAHO[4+2]环加成酶，以期为阐明PhPN生物合成奠定基础，为其他[4+2]环化酶的发现提供参考。

Phenylphenalenone (PhPN)-type secondary metabolites are important phytoalexins and phytoanticipins in banana plants. [4 + 2] cycloaddition of diarylheptane ketone (DAHO) is the critical step during the formation of PhPN skeleton. However, the enzymes involved in [4 + 2] cycloaddition of DAHO have not been identified yet. For lacking of common sequence in known [4 + 2] cyclases, together with the difficulties in preparation of putative substrates, the identification of unknown [4 + 2] cyclase remains a challenging task. . During our preliminary study, the temporal specificity of PhPN biosynthesis in the seeds of Musella lasiocarpa was revealed. Furthermore, we found that [4 + 2] cycloaddition of DAHO could be accelerated by the cell-free extract of the seeds of M. lasiocarpa. To identify the unknown [4 + 2] cyclase in PhPN biosynthesis, integrative analysis of metabolome and transcriptome on the seeds of M. lasiocarpa at different stages will be conducted to screen candidate genes involved in cycloaddition of DAHO; small-molecule probes of DAHO will be designed and synthesized to capture the interaction proteins from the seeds of M. lasiocarpa via a chemoproteomic strategy, and the cyclization-related enzymes interacted with DAHO will be identified by using mass spectrum; the overlap candidate proteins obtained by the above two strategies will be subsequently expressed in Escherichia coli and purified to identify their [4 + 2] cycloaddition catalytic activity in vitro; after that, the genes encoding candidate enzymes will be introduced in Arabidopsis thaliana. Then, the PhPNs analysis and the disease resistance test on the transgenic plants will be performed to further confirm the function of candidate genes in vivo. The results of this study will lead to the identification of critical enzymes catalyzing [4 + 2] cycloaddition of DAHO, which may shed light on the biosynthesis of PhPN and provide a reference for identifying other plant-derived [4 + 2] cyclase.