沉香为瑞香科（Thymelaeceae）沉香属（Aquilaria spp.）或拟沉香属（Gyrinops spp.）植物含有树脂的木材，是中国、日本、印度等国家的传统珍贵药材，也是一种天然香料。白木香（Aquilaria sinensis (Lour.) Gilg）是中国产沉香的唯一法定基原物种，只有受到外界伤害时，才能产生沉香。沉香形成过程实质是其主要成分倍半萜类和2-（2-苯乙基）色酮类化合物合成和积累的缓慢过程。在倍半萜生物合成途径中，倍半萜合酶是催化法尼基焦磷酸（FPP）合成倍半萜的关键酶。通过白木香全基因组测序分析结合转录组，课题组发现两个表达模式十分特别的倍半萜合酶AsTPS13/18，它们在健康状态下不表达，在初始伤害15 d后才开始表达，且表达量上调可达数千倍，是国内外所有已分离鉴别到的白木香萜类合酶基因中伤害诱导表达最高的。课题组随后克隆了AsTPS13/18基因，获得其纯化蛋白，在体外以FPP为底物进行催化，发现它们都合成了新的沉香倍半萜。因此提出两个科学问题：能够诱导AsTPS13/18响应的有效伤害信号是什么？为什么AsTPS13/18会延迟高表达，其受信号诱导启动的调控机制是什么？基于此，本论文通过研究得到如下结果：

1. 确定能够有效诱导白木香倍半萜合酶基因AsTPS13/18表达的伤害信号：MeJA和NaCl。结合文献对白木香愈伤组织进行8种不同的处理，用qPCR 检测处理白木香愈伤组织中AsTPS13/18的表达，结果表明，MeJA和NaCl都可以有效诱导AsTPS13/18的表达，上调程度可达数千倍，且MeJA和NaCl处理下AsTPS13/18表达变化模式相同。

2. 确定AsTPS13/18的核心启动子区域。对AsTPS13/18的启动子进行5’端截断，分别构建promoter :: GUS融合表达系统，稳定转化拟南芥，获得其 T3代纯合子后用qPCR检测阳性植株GUS表达活性并进行GUS组织染色。结果表明AsTPS13上游-722~-350是其核心启动子区域，AsTPS18上游-554~-262是其核心启动子区域。AsTPS13/18的诱导表达可能是多个或多种调控因子共同作用的结果。

3. 发现AsH3.3对AsTPS13的转录抑制作用。以AsTPS13启动子构建酵母单杂诱饵质粒，通过筛选白木香健康/伤害cDNA文库发现组蛋白变体 AsH3.3与AsTPS13有强结合作用，染色质免疫共沉淀实验确认AsH3.3与AsTPS13 的启动子多个片段结合。双荧光素酶报告基因检测和RNAi转基因等发现AsH3.3抑制AsTPS13的表达，MeJA和NaCl处理可以使AsH3.3蛋白缓慢降解，逆转这种抑制作用。由此研究表观遗传调控的另两个主要方面：DNA甲基化和组蛋白修饰，对AsTPS13调控作用，发现DNA甲基化对基因表达影响不显著，而组蛋白修饰起着重要调控作用。

4. 发现AsH3.3对AsTPS18的转录抑制作用。以AsTPS18启动子进行酵母单杂筛库发现AsH3.3和AsLBD41结合AsTPS18启动子，以染色质免疫共沉淀、双荧光素酶报告基因检测等验证发现AsH3.3对AsTPS18的调控作用与对AsTPS13的调控作用相似。

5. 发现AsLBD41以二聚体形式对AsTPS18起转录抑制作用，受到伤害后被AsKIN10磷酸化解除抑制。以染色质免疫共沉淀等实验验证AsLBD41对AsTPS18的抑制作用后，通过酵母双杂交筛库发现AsLBD41与它本身和磷酸化蛋白AsKIN10互作，并通过pull-down和Co-IP进行了验证，因此AsLBD41以二聚体形式存在，通过发生磷酸化反应被降解。

综合以上研究，本论文发现健康状态下，AsTPS13/18启动子上AsH3.3过度富集，降低染色质可及性，抑制基因表达，受到伤害（MeJA或 NaCl）后，AsH3.3缓慢降解，组蛋白修饰变化，染色质可及性增加，基因开始高表达；研究还发现了AsTPS18的转录抑制因子AsLBD41，伤害处理下AsLBD41二聚体被AsKIN10磷酸化降解从而解除对AsTPS18的抑制。本论文不仅解析了白木香中一类倍半萜基因受伤害后延迟表达的分子机制，对沉香伤害诱导形成的分子机制的完善有重要意义，同时还丰富了植物表观遗传理论。

Agarwood is the resin-containing wood from plants of the genus Aquilaria spp. or Gyrinops spp. of the Thymelaeceae family. It is a precious traditional medicine and natural fragrance in China, Japan, India, and many other countries. Aquilaria sinensis (Lour.) Gilg is the only legally recognized source tree for agarwood in China. Agarwood formation occurs only when the tree is injured. The process of agarwood formation essentially involves the gradual synthesis and accumulation of its primary components-sesquiterpenes and 2-(2-phenylethyl) chromones. Sesquiterpene synthases (TPSs), key enzymes in sesquiterpene biosynthesis, catalyze the conversion of FPP into sesquiterpenes. Through whole-genome sequencing and transcriptomic analysis of A. sinensis, two uniquely expressed sesquiterpenesynthase genes, AsTPS13 and AsTPS18, were identified. These genes exhibit no expression under healthy conditions but show upregulation by several thousand-fold 15 days after the initial injury, representing the highest injury-induced expression among all characterized sesquiterpene synthase genes in A. sinensis worldwide. Subsequent cloning and purification of AsTPS13/18 proteins enabled in vitro catalysis using FPP as substrate, revealing their ability to synthesize novel agarwood sesquiterpenes. These raise two scientific questions: 1) What are the effective injury signals that induce AsTPS13/18 genes response? 2) What regulatory mechanisms underlie their exceptionally delayed high expression upon signal induction? Our findings are as follows:

1. Identification of effective injury signals inducing AsTPS13/18 expression: MeJA and NaCl. Eight different treatments were performed on the calli of A. sinensis. The expression of AsTPS13/18 was detected by qPCR. The results showed that both methyl jasmonate (MeJA) and sodium chloride (NaCl) could effectively induce the expression of AsTPS13/18, which could be up regulated by thousands of times. The expression patterns of AsTPS13/18 under MeJA and NaCl were the same.

2. Determination of core promoter regions for AsTPS13/18. By constructing 5'-truncated promoter :: GUS fusion systems and stable transformation in Arabidopsis, T3 homozygous lines were analyzed via qPCR and GUS histochemical staining. Results revealed the core promoter regions as -722~-350 bp upstream of AsTPS13 and -554~-262 bp upstream of AsTPS18. The induced expression likely involves synergistic effects of multiple regulatory factors.

3. Discovery of AsH3.3-mediated transcriptional repression of AsTPS13. Yeast one-hybrid screening using the AsTPS13 promoter as bait identified strong binding between histone variant AsH3.3 and the promoter, confirmed by chromatin immunoprecipitation (ChIP). Dual-luciferase assays and RNAi transgenic experiments demonstrated AsH3.3's repressive effect on AsTPS13. MeJA/NaCl treatments reversed this repression via AsH3.3 degradation. Further investigation into epigenetic regulation highlighted the important role of histone modifications.

4. Identification of AsH3.3-mediated transcriptional repression of AsTPS18. Similar screening strategies revealed AsH3.3 and AsLBD41 binding to the AsTPS18 promoter. ChIP and dual-luciferase assays confirmed that AsH3.3 represses AsTPS18 via mechanisms analogous to its regulation of AsTPS13.

5. Characterization of AsLBD41 dimer-mediated repression of AsTPS18 and its relief via AsKIN10 phosphorylation. Yeast two-hybrid screening and pull-down/Co-IP validation demonstrated that AsLBD41 forms dimers to repress AsTPS18, while injury-induced phosphorylation by AsKIN10 alleviates this inhibition through potential dimer dissociation.

In summary, under healthy conditions, excessive AsH3.3 enrichment on the AsTPS13/18 promoters reduce chromatin accessibility, suppressing gene expression. After being injured (MeJA/NaCl), AsH3.3 is degraded slowly, histone modifications change, chromatin accessibility increases, and genes begin to be highly expressed. Additionally, AsLBD41 dimers repress AsTPS18 until phosphorylation by AsKIN10 during injury. This thesis not only analyzes the molecular mechanism of delayed expression of a class of sesquiterpene genes in A. sinensis after injury, which is of great significance for the improvement of the molecular mechanism induced by injury in agarwood, but also enriches the theory of plant epigenetics.