龙血竭是龙血树属（Dracaena）植物含脂木材经提取而成的树脂类物质。龙血竭是我国传统名贵中药，具有活血散瘀、定痛止痛、生肌敛疮的功效，距今已有上千年的应用历史。国产龙血竭的基原植物主要为剑叶龙血树（Dracaena cochinchinensis (Lour.) S. C. Chen）和海南龙血树（ D. cambodiana Pierre ex Gagnep）。自然条件下，龙血树伤害后产生龙血竭的过程非常缓慢，近年来龙血竭的需求量持续增加，传统的“毁树取材”的生产方法对龙血树资源破坏十分严重，龙血树植物资源在全球日益匮乏。由于龙血树的结脂机制尚不明确，迄今尚无高效的人工诱导龙血树生产龙血竭的技术，制约了龙血竭的可持续利用。

本课题以剑叶龙血树和海南龙血树为材料，首先利用组织化学染色和质谱成像技术，观察伤害诱导龙血树结脂的物理防御特征。然后通过化学显色法和超高效液相色谱-质谱联用方法，确定伤害诱导龙血树结脂的化学防御特点。最后结合基因组、转录组、代谢组等多组学技术及生物信息分析方法，从龙血树防御反应的遗传基础、伤害后龙血树的基因表达谱及代谢特征、伤害信号分子调控机制、miRNA调控等多个角度，全面解析伤害诱导龙血树防御反应产生树脂的机制。主要研究结果如下：

1. 伤害诱导龙血树结脂的结构特征和物质基础

健康的龙血树茎皮层中有大量草酸钙针晶束，起到重要物理防御作用，生长区中维管束与周围薄壁细胞通过大量小孔紧密相连。伤害至皮层后，龙血树茎不结脂，皮层受伤表面栓化形成新的栓化层。当伤害突破皮层至生长区后，受伤的生长区出现明显分层特征：由外而内分别是腐烂层、结脂层、健康组织。伤害后树脂最先从维管束韧皮部中出现，随后填充木质部，再扩散至周围薄壁细胞。伤害后随时间迁移，树脂、黄酮类成分和糖类物质呈现出类似的变化。伤害后维管束韧皮部内苯丙氨酸含量明显下降，两种黄酮类化合物含量明显上升，维管束是龙血树茎的主要结脂场所，糖类物质可能是伤害诱导龙血树结脂的物质基础。

2. 伤害诱导龙血树结脂过程中信号分子的变化

利用化学显色法和高效液相色谱-质谱联用法测定伤害后海南龙血树茎中多种信号分子的含量变化，发现伤害后过氧化氢与茉莉酸浓度呈现上升，施加外源过氧化氢和茉莉酸可明显提高海南龙血树茎中的黄酮类化合物含量。

3. 剑叶龙血树基因组特征

结合二代测序、三代测序及Hi-C测序，得到了剑叶龙血树染色体水平的高

质量基因组。剑叶龙血树基因组大小约为1.26Gb。剑叶龙血树基因组内DNA损伤修复相关基因和黄酮合成相关基因被正向选择，这是龙血树防御反应的遗传基础。

4. 剑叶龙血树伤害后通过调节以黄酮生物合成为主的多种代谢过程形成树脂从而进行物理和化学防御

联合转录组和代谢组对伤害后不同时间剑叶龙血树茎进行分析，发现剑叶龙血树茎在伤害后从基因表达谱和代谢谱都表现出了阶段性的特征。伤害初期包括三羧酸循环、糖酵解、淀粉和蔗糖代谢等多种代谢通路的多个基因上调表达，促进次生代谢进程；伤害中后期黄酮生物合成通路上的多个基因包括CHS、DFR、LAR、OMT等基因持续高表达，黄酮类化合物是主要富集的化合物类型。MYBs和bHLH转录因子可能是调节龙血树黄酮生物合成的主要转录因子。此外，伤害后与活性氧产生和清除相关基因表达量发生明显变化，多种激素（茉莉酸、油菜素内酯）合成相关基因及感受蛋白（茉莉酸感受器、赤霉素感受器、脱落酸感受器）基因上调表达。对海南龙血树茎施加外源过氧化氢可提高茎中茉莉酸和赤霉素含量，过氧化氢和茉莉酸处理可上调CHS、OPP、OMT等基因的表达。已知上述代谢途径和信号分子与植物伤害防御密切相关，产生的黄酮类化合物和黄酮糖苷形成对外界伤害的物理和化学防御。

5. miRNA通过调节多种通路广泛参与了伤害诱导的龙血树防御反应和树脂形成

通过miRNA测序和降解组测序，发现伤害后海南龙血树miRNA谱发生显著变化，并预测了一个新miRNA靶向海南龙血树CHS基因，该miRNA伤害后表达量明显降低且其CHS靶基因伤害后表达量明显升高。

本研究首次使用质谱成像技术直观展示了高良姜素等化合物在龙血树受到伤害后主要在维管束细胞中合成和积累。首次报道了剑叶龙血树染色体水平的基因组，可为龙血树属植物生物学特点的后续研究提供有力数据支持。首次提出龙血树受到伤害后的代谢进程由短期到长期表现出阶段性的特点，黄酮生物合成是伤害诱导龙血树防御反应的主要进程，发现并验证了过氧化氢与茉莉酸是其防御反应的重要信号分子，首次报道了海南龙血树的miRNA谱，并分析了其对龙血树防御反应相关基因的调控模式，较为全面地揭示了龙血树的结脂机制。本研究阐释了伤害胁迫与龙血树结脂的关系，确定了龙血树结脂场所，挖掘了决定龙血竭质量的潜在关键基因，可为调控龙血树次生代谢，建立高效人工结脂技术提供理论依据。

Dragon’s blood is an extract from resinous wood of Dracaena spp.. It is a traditional Chinese medicine that has been applied over thousands of years. Dragon’s blood could activate blood, dissolve stasis, kill pain and regenerate tissues to heal wounds. In China, the source plants of dragon’s blood are D. cochinchinensis and D. cambodiana. In nature conditions, the resin formation process would be time-consuming. The traditional method of manufacturing severely destroys the plant resources. As the demand of dragon’s blood keeps growing in recent years, the world plant resources become more and more deficient. As little is known about the resin formation mechanism of Dracaena spp., no efficient artificial induction technique is available, which restrict the sustainable utilization of dragon’s blood.

The present study works on the D. cochinchinensis and D. cambodiana. First of all, histochemical stain method and imaging mass spectrometry technique were used to confirm the structure characteristics and material basis of wound-induced resin formation process. Then the chemical reagent chromogenic method and liquid chromatography-mass spectrometry were used to detect the content change of mutiple signal molecules within stems of D. cambodiana. Finally, muti-omics analysis was used to comprehensively reveal the molecular mechanism of would-induced resin formation process of Dracaena spp. from multiple aspects, including the genetic basis, gene expression patterns, wounding signal regulation and miRNA regulation. The findings are as followed：

1. Structure characteristics and materials basis of wound-induced resin formation process

Cortex in healthy stems of D. cambodiana have abundant of calcium oxalate raphide that play roles in physical defenses. Vascular bundles are well connected with surrounding parenchymal cells through plenty of pits in their cell walls. When the damage only reaches cortex, the stem would not produce resin. The injured cortex would be suberized to form new bark. When the damage reaches the ground tissue, the injured ground tissue would be obviously layered: decomposed layer, resin layer and healthy stem. After being damaged, resin initially occures in phloem, then fills the xylem within vascular bundles and finally spreads to the surrounding parenchymal cells. As time goes after wounded, the resin, the flavonoid compounds and saccharides show similar variation trend. After being wounded, the content of phenylalanine within vascular bundle phloem decreased and two flavones (3,4-hydroxyflavone and galangin) are generated in the same spots. Vascular bundles are the resin formation location of dragon trees and the saccharides is speculated to be the material basis of the wound-induced resin formation process.

2. Concentration change of wounding signals during the wound-induced resin formation process.

Among mutiple plant signal molecules, the content of H₂O₂ and jasmonate dramatically increased after the stem were wounded. Exogenous H₂O₂ and jasmonate could significantly improve the flavonoid content within stems of D. cambodiana.

3. Genome characteristics of D. cochinchinensis.

Using next Generation Sequencing, Biopac sequencing and Hi-C sequencing, the high-quality genome of D. cochinchinensis at chromosomal level was obtained 1.26G. DNA damage repair related genes and flavonoid biosynthesis related genes were positively selected, which could be the genetic basis of the wound-induced defensive responses to produce resin.

4. After damaged, adjusts mutiple metabolism pathways (centered on flavonoid biosynthesis) to produce resin thus form the physical and chemical defense.

Both the gene expression pattern and metabolites accumulation pattern showed characteristics of stages after the stems of D. cochinchinensis were wounded. In the short term after damage, genes involved in vairous of pathways were up-regulated including TCA cycle, glycolysis, starch and surcrose metabolism, etc. and promote the flux from primary metabolism to secondary metabolism. During this stage, different types of metabolites were synthesized; In the middle and late stages, many genes annotated in the flavonoid biosynthesis including CHS, CHI, DFR, LAR, OPP were significantly up-regulated and the flavonoid compounds is the main enriched metabolite type. MYBs an bHLH might be the main transcriptome factor regulating the flavonoid biosynthesis in Dracaena spp.. Besides, ROS generation and scavenging related genes were differentially expressed after wounded. Biosynthesis and receptor genes of multiple hormones including jasmonate, gibberellin and abscisic acid were up-regulated. The exogenous H₂O₂ and JA could enhance the expression of CHS, OMT and PPO. It is known that the pathways and signals mentioned above is closely related to plant wounding responses. The flavonoid compounds and flavone glycoside form the physical and chemical defenses.

5. miRNAs are widely involved in the wound-induced defense responses and resin formation process through multiple pathways.

MiRNA profile significantly changed after the D. cambodiana stem were damaed. A novel miRNA was discovered to target a CHS gene. The expression of this miRNA were decreased while that of the CHS gene were increased after wounding.

The present study initially applies imaging mass spectrometry to show flavonoids were synthezied and accumulated in vascular bundles. The study first reports the chromosomal level genome of D. cochinchinensis, which could provide data foundations for the biological characteristics research on Dracaena spp..The study initially proposes that dragon trees showed stage characteristics in the metabolic process from short term to long term after wounding. Flavonoid biosyhthesis is the main process in the defensive responses of dragon trees. H2O2 and JA are important signals in the defensice responses. The study first reports the miRNA profile of D. cambodiana and analyzes the role of miRNAs in the defensive responses. The present study demonstrates the relationship between wound-stress and resin formation, confirms the resin formation spot and excavates the potent genes related to the quality of dragon’s blood, which could provide theoretical basis for the development of high efficient induction technique.