水稻细菌性基腐病菌zeamine毒素基因簇的克隆与功能分析

Dickeya zeae is the causal agent of the rice foot rot disease. Compared with other closely related species, strain EC1 can produce toxins that inhibit the germination of rice seeds. Through collaborative research, active compounds were purified from D. zeae EC1 and were structurally characterized, and the results have unveiled two polyamino compounds, i.e., zeamine (C49H105O4N6) and zeamine II (C40H88ON5), accounting for 60% and 40% of the total toxins produced, respectively. Recently, zmsA that encodes a novel polyketide synthase has been cloned through Tn5 transposon mutagenesis and TAIL-PCR analysis. Deletion mutagenesis confirmed that ZmsA is essential for zeamine biosynthesis and bacterial virulence. In addition, we have also obtained about 20 Tn5 inserted mutants which were defective in toxin production. In this study, we aim to identify and characterize the mutated genes and understand their roles in biosynthesis of zeamines and in bacterial pathogenesis through gene knockout, complementation and metabolite analysis. The results would facilitate understanding the roles of various enzymes in zeamine synthesis pathway and in pathogenesis, and may also provide useful clues for synthetic biology in engineering of high potency and safe antibiotics with low side-effects.

Dickeya zeae菌株EC1是自广东水稻基腐病组织分离的一株致病菌，可产生抑制水稻种子萌发的毒素。研究证明，该毒素为聚酮多胺类抗生素，其中zeamine（C49H105O4N6）占EC1产毒量的60%，zeamineⅡ（C40H88ON5）占40%。在前期研究中，我们通过Tn5转座子插入突变、TAIL-PCR扩增及测序，克隆分析了一个聚酮合成酶编码基因zmsA，基因敲除证明ZmsA参与zeamine的生物合成，另外还获得了近20个毒素产生明显减少的Tn5插入突变体。本课题拟用hiTAIL-PCR方法结合基因组测序结果，对突变体进行分子鉴定，用生物信息学方法对克隆的基因进行功能预测，用基因敲除、功能互补及中间产物分析手段进行功能验证，以揭示这些基因在zeamine生物合成及致病过程中的作用。研究结果可为合成生物学提供线索，通过对结构基因等的改造提高zeamine产量或产生新的衍生物。

本项目筛选获得了zeamines毒素的高产发酵培养方法，有利于后续对zeamines的分离和化学结构鉴定，为合成生物学和明确水稻基腐菌Dickeya zeae EC1的毒素致病调控机制提供线索。通过基因组测序和生物信息学分析获得了D. zeae EC1的全基因组序列，为研究其重要致病因子及致病调控机理提供目标基因。通过hiTAIL-PCR方法结合基因组测序结果，获得了zeamines的生物合成基因簇，该基因簇全长51,228 bp，含18个ORF，其中zmsA到zmsK的10个基因为zeamines生物合成相关基因，zmsL、zmsM、zmsP、zmsQ和zmsR的5个基因为zeamines运输相关基因，zmsO为zeamine合成的辅助基因，zmsN和zmsS的功能尚未明确。在已发现的所有Dickeya zeae的多个菌株中，该基因簇只存在于分离自水稻的菌株中，且该基因簇的GC含量为48.76%，显著低于EC1菌株的基因组GC含量53.43%，表明该基因簇很可能是细菌在进化过程后期通过水平基因转移进入到D. zeae基因组中，与菌株的寄主专化性相关。