海南龙血树（Dracaena cambodiana）是我国珍稀药用植物，其树脂（龙血竭）富含黄酮、酚酸等活性成分，具有重要药用价值。然而，龙血竭形成的分子机制尚不清晰，制约了其资源高效利用与龙血竭的药用开发。内生菌可以促进药用植物的生长发育，帮助药用植物抵抗外界胁迫，也可以促进药用次生代谢物的合成。据报道，内生菌可以促进龙血树结脂，且其品质与野生龙血竭接近。但海南龙血树内生菌尚未有基因组数据，海南龙血树与真菌互作过程的代谢和基因表达变化缺乏系统记录，在一定程度上限制了海南龙血树和内生菌互作分子机制研究及结脂机制解析，同时对利用真菌促进龙血竭合成的技术开发造成影响。

本研究以海南龙血树为研究对象，通过整合多组学手段，探索其响应内生菌（Fusarium oxysporum）侵染与物理伤害的分子机制差异，探究海南龙血树如何响应内生菌并与之互作，发掘互作过程中转录组和代谢组的调控变化。具体实验及结果如下：

接种尖孢镰刀菌（F. oxysporum）后显著促进海南龙血树红褐色树脂的形成。相较于物理损伤处理，接种F. oxysporum处理组结脂进程显著提前且效率更高，在3天即出现淡红色树脂。显微观察显示接种F. oxysporum处理组3天维管束便出现红褐色树脂沉积，30天时维管束和薄壁细胞基本被红褐色树脂填充；而物理损伤处理组（CK）7天才开始缓慢沉积。生理指标检测发现，真菌接种导致活性氧爆发，同时伴随超氧阴离子（O₂⁻）产生。接种F. oxysporum处理组的防御酶系统呈现特异性响应，酶活显著高于CK组。

2. 成功构建了尖孢镰刀菌的较高质量基因组（46.21 Mb，N50=4.77 Mb），注释到大量致病基因，其中GHs（糖苷水解酶）和AAs（辅助活性酶）占CAZy酶(碳水化合物活性酶）的主要比例。系统发育进化分析显示， F. oxysporum存在 407个基因家族扩张 与 197个基因家族收缩，扩张基因家族功能涉及氧化应激与次生代谢， MAPK信号通路等途径；F. oxysporum特有基因家族显著富集于 DNA模板转录调控与碳代谢。通过PHI数据库（Pathogen Host Interactions Database）与CAZy、DFVF（Database of Virulence Factors in Fungal Pathogens）等多数据库交叉筛选，预测出95个分泌蛋白、78个CAZy酶及54个DFVF相关基因，基因功能富集分析暗示F. oxysporum具有较强的碳代谢与次生代谢能力。

3. 2种处理组的代谢组数据的样品相关性分析表明， F. oxysporum组聚类模式与CK组不同，真菌侵染改变了海南龙血树代谢响应的时间动态。两处理组共有代谢物中，黄酮类占比随时间递增，氨基酸及其衍生物早期占比高，后期下降。CK组特有代谢物：早期以黄酮、生物碱为主，后期转向氨基酸、脂类。F. oxysporum组特有代谢物中，早期富集氨基酸、脂类，中后期萜类、木脂素，类黄酮增加。此外，两种处理在某些独特化合物的含量和大量合成时间上也有很大差异，比如真菌与海南龙血树互作后，龙血素与血竭素类化合物的含量提高。转录组分析显示，海南龙血树对物理损伤与尖孢镰刀菌侵染的响应差异显著，表现在DEGs（差异表达基因）数量和动态变化模式：真菌处理组（T36组）的差异表达基因数多于物理损伤组；前期12 h时，真菌处理组的海南龙血树DEGs显著富集在MAPK信号、真核的核糖体生物起源、植物-真菌互作通路；物理处理组组则主要富集激活内质网蛋白加工、光合相关基因。后期15-30d时，代谢组数据显示两组均通过次生代谢物（如龙血素、血竭素）合成抵御胁迫，但转录组数据发现物理处理组可能依赖氧化磷酸化提供能量，而真菌处理组可能通过剪接体调控、泛素介导蛋白水解等增强抗氧化能力。真菌转录组中，12 h，1 d，3 d，7 d这四个时间点，真菌具有较高侵染率，同时致病基因也多在这几个时间点高表达。

4. 系统鉴定了海南龙血树NBS-LRR (Nucleotide Binding-site Leucine-rich Repeat)基因家族。共筛选到95个基因，发现NBS-LRR蛋白在分子量、等电点和疏水性等理化性质方面都有所不同，大部分NBS-LRR序列结构域较为保守。顺式作用元件中光响应元件占比较高，同时富含激素响应元件及逆境胁迫响应元件。值得注意的是，在真菌致病基因表达量高的时候，NBS-LRR家族的表达量较低。

5. 利用WGCNA构建基因共表达网络，筛选出与龙血素类化合物（龙血素A、B、C、D及剑叶龙血素A）显著相关的基因模块（MEpink、MEblue、MEyellow、MEcyan），并揭示了类黄酮生物合成途径中关键结构基因与转录因子的调控网络。其中结构基因（如PAL、4CL、C4H）在多个模块中重复出现，而下游分支酶（如FLS、LAR、ANS）的模块特异性分布。转录因子（MYB、bHLH、NAC）呈现跨模块富集，推测其广泛参与结构基因的调控网络。

综上，本研究对海南龙血树与内生真菌互作的宏观和微观进行了统计观察，测定相关生理指标；完成了内生真菌F. oxysporum的全基因组测序，挖掘其潜在致病因子；代谢组和转录组分析揭示了海南龙血树对物理损伤和接种真菌两种处理的差异性响应特征；系统鉴定了植物抗病基因家族NBS-LRR，为后续开展海南龙血树的抗病机制研究提供基础，同时通过WGCNA构建龙血素类化合物的生物合成调控网络，为后续接种真菌促结脂技术开发与分子机制研究提供了数据基础。

Dracaena cambodiana is a rare medicinal plant in China. Its resin (Dragon's blood) is rich in active components such as flavonoids and phenolic acids, and has important medicinal value. However, the molecular mechanism of Dragon's blood formation is still unclear, which restricts the efficient utilization of its resources and the medicinal development of Dragon's blood. Endophytes can promote the growth and development of medicinal plants, help them resist external stresses, and promote the synthesis of secondary metabolites for medicinal use.It has been reported that endophytes can promote the resin formation of Dracaena trees, and the quality of resin is similar to that of wild dragon's blood. However, there are no genomic data of the endophytes of D. cambodiana yet. The metabolic and gene expression changes during the interaction process between D. cambodiana and fungi are not systematically recorded, which to a certain extent limits the research on the molecular mechanism of the interaction between D. cambodiana and endophytes and the analysis of the resin formation mechanism. At the same time, it has an impact on the development of technologies that use fungi to promote the synthesis of Dragon's blood.

In this study, by integrating multi-omics methods, the differences in the molecular mechanisms of D. cambodiana in response to infection by Fusarium oxysporum and physical injury were studied, exploring how D. cambodiana responds to and interacts with F. oxysporum, and  the regulatory changes of transcriptomes and metabolomes during the interaction. Specific experiments and results are as follows:

1. Inoculation with F. oxysporum significantly promoted the formation of reddish-brown resin in D. cambodiana. Compared with the physical damage treatment, the process of resin formation in the F. oxysporum inoculation group was significantly earlier and more efficient, with light red resin appearing in 3 days. Microscopic observation showed that reddish-brown resin deposition occurred in the vascular bundles of the F. oxysporum inoculation treatment group 3 days later, and by 30 days later, the vascular bundles and parenchyma cells were basically filled with reddish-brown resin. However, in the physical damage group (CK), deposition began slowly at 7 days. Physiological measurements showed that fungal inoculation caused a burst of reactive oxygen species (ROS), accompanied by the production of superoxide anion. The defense enzyme system of F. oxysporum treated group showed a specific response, and the enzyme activity was significantly higher than that of the CK group.

2. A high-quality genome of F. oxysporum was successfully constructed (46.21 Mb, N50=4.77 Mb). Functional annotation revealed that a large number of pathogenic genes were annotated, among which GHs (glycoside hydrolase) and AAs (auxiliary active enzyme) accounted for the main proportion of CAZy (carbohydrate active enzyme). Phylogenetic analysis showed that 407 gene families were expanded and 197 gene families were contracted in F. oxysporum. The functions of expanded gene families were involved in oxidative stress, secondary metabolism, MAPK signaling pathway and other pathways. F. oxysporum unique gene family was significantly enriched in DNA template transcription regulation and carbon metabolism. By cross-screening PHI database with CAZy, DFVF and other databases, 95 secreted proteins, 78 CAZy enzymes and 54 DFVF related genes were predicted. Gene function enrichment analysis suggested that F. oxysporum had strong carbon metabolism and secondary metabolism ability.

3. Sample correlation analysis of metabolomic data from the two treatment groups indicated that the clustering pattern of the F. oxysporum group was different from that of the CK group, and fungal infection changed the temporal dynamics of the metabolic response of D. cambodiana. Among the common metabolites in the two treatment groups, the proportion of flavonoids increased with time, and the proportion of amino acids and their derivatives was high in the early stage and decreased in the later stage. The unique metabolites of CK group were mainly flavonoids and alkaloids in the early stage, and turned to amino acids and lipids in the later stage. In the unique metabolites of F. oxysporum group, amino acids and lipids were enriched in the early stage, while terpenoids, lignans, and flavonoids were increased in the middle and late stages. In addition, there are also significant differences between the two treatments in terms of the content of certain unique compounds and the time for large-scale synthesis. For instance, after the interaction between the fungus and D. cambodiana, the content of loureirins and dracorhodina increased. Transcriptome analysis revealed that the responses of D. cambodiana to physical damage and F. oxysporum infection were significantly different, manifested in the number and dynamic change pattern of DEGs (differentially expressed genes) : the number of differentially expressed genes in the fungal treatment group (group T36) was more than that in the physical damage group; At 12 hours in the early stage, the DEGs of D. cambodiana in the fungal treatment group were significantly enriched in MAPK signaling, the bioorigin of eukaryotic ribosomes, and the plant-fungal interaction pathway. The physical treatment group was mainly enriched in activated endoplasmic reticulum protein processing and photosynthesis related genes. At 15-30 days after stress, the metabolome data showed that both groups were resistant to stress through the synthesis of secondary metabolites (such as loureirins and dracorhodina), but the transcriptome data showed that the physical treatment group depended on oxidative phosphorylation to provide energy, while the fungal treatment group may enhance antioxidant capacity through spliceosome regulation and ubiquitin-mediated proteolysis. In the fungal transcriptome, 12 h, 1 d, 3 d, and 7 d were the four time points, the fungi had a high infection rate, and most pathogenic genes were highly expressed at these time points.

4. The NBS-LRR gene family of D. cambodiana was systematically identified. A total of 95 genes were screened, and it was found that the physical and chemical properties of NBS-LRR proteins were different in molecular weight, isoelectric point and hydrophobicity. Most of the sequence domains of NBS-LRR were conservative. The cis-acting elements accounted for a relatively high proportion of light-responsive elements, and were enriched in hormone-responsive elements and stress-responsive elements. It should be noted that the NBS-LRR family is less expressed during times of high fungal pathogenic gene expression.

5. WGCNA was used to construct gene co-expression network, and the gene modules （MEpink, MEblue, MEyellow, MEcyan） significantly related to loureirin compounds （loureirin A, B, C, D and xiphogenin A） were screened. The regulatory network of key structural genes and transcription factors in the flavonoid biosynthesis pathway was also revealed. Among them, structural genes such as PAL, 4CL, C4H are repeated in multiple modules. The module-specific distribution of downstream branch enzymes such as FLS, LAR and ANS. The presentation of transcription factor （MYB, bHLH, NAC） is enriched across modules and is widely involved in the regulatory network of structural genes.

In conclusion, in this study, macroscopic and microscopic statistical observations were made on the interaction between D. cambodiana and endophyte, and relevant physiological indicators were measured. The whole genome sequencing of endophytes was completed to explore the potential pathogenic factors. Metabolomic and transcriptomic analyses revealed the differential response characteristics of D. cambodiana to physical damage and inoculation with fungi. The plant disease resistance gene family NBS-LRR was systematically identified, which provided a good basis for the subsequent research on the disease resistance mechanism of D. cambodiana. Meanwhile, the biosynthesis regulatory network of loureirin compounds was constructed by WGCNA, which provided a data basis for the development of resin promoting technology and molecular mechanism research of subsequent fungal inoculation.