黄檗是芸香科黄檗属植物，被记载是雌雄异株植物，前期研究发现了雌雄同株黄檗的存在。雌雄同株黄檗幼年时顶端死亡，侧芽发育为不同性别的分枝。这表明黄檗的性别形成可能发生在分枝形成之后，并且在相同遗传背景之下可以稳定表达为不同的性别，推测表观遗传可能参与了黄檗性别分化。本研究对黄檗性别相关基因的表达特征进行分析，并且对其表观遗传调控方式进行了初步探究。

植物作为一个整体，其性别形成应是各器官之间信号相互传递，协调配合的结果，了解黄檗整体的性别特征可以更好地了解其性别形成过程，我们分析了黄檗雌、雄株一年生枝皮、叶片、花器官这三个部位的性别相关基因表达特征，结果显示：（1）黄檗各个部位所表达的性别相关基因的数目不同，无论雌株还是雄株，花器官中所表达的性别相关基因最多，一年生枝皮次之，叶片最少。花器官特异表达的基因最多，这些基因大多与花发育相关；一年生枝皮特异表达的基因多与胚胎发育和植物性别分化相关；叶片特异表达的基因极少，只有雌株叶片存在2个特异表达的基因，分别是激素相关基因和雌性育性基因。（2）在黄檗雌株中共获取611条性别相关基因，雄株中共获取612条性别相关基因，雌株特异表达的基因有CCR4-NOT、60s ribosomal protein、ESR2、ERF114，雄株特异表达的基因有ARR17、ABORTED MICROSPORES、X10A、CDPKO、V-type proton ATPase subunit G1。（3）雌、雄株花器官差异基因最多，一年生枝皮差异基因数目次之，叶片差异基因最少。雌、雄株花器官差异基因涉及黄檗性别发育关键基因以及大量和花发育相关的基因；雌、雄株一年生枝皮差异基因涉及部分植物性别决定候选基因MCM6、SRS5，花发育相关基因相对较少；叶片中雌、雄差异基因数目极少，且不涉及黄檗雌花发育的关键基因。性别相关基因在一年生枝皮中的表达特征表明其可作为黄檗性别相关研究的材料。此外，通过对黄檗不同部位差异基因进行分析发现，一年生枝皮相比主干树皮有大量性别相关基因差异表达，其中大量性别决定候选基因上调。

在黄檗性别的表观遗传调控方面，本研究试图从DNA甲基化方面取得突破。DNA甲基化是一种最常见也是研究最为深入的表观遗传调控方式，本研究通过去甲基化手段研究DNA甲基化对黄檗性别的影响，以期证明表观遗传参与黄檗性别分化。使用去甲基化试剂5-azaC处理黄檗雄花，部分雄花转变为雌花，没有中间变异类型。对诱导形成的雌花进行形态和功能研究发现，诱导雌花在形态结构及功能上和正常雌花完全一致，表现为雄蕊不同程度的败育，子房发育，具有结实性，并且果实内有种子。通过去甲基化，黄檗雄花可转化为形态结构和功能完整的雌花，表明表观遗传参与了黄檗的性别分化，DNA甲基化是其调控方式之一。

黄檗雄花去甲基化转变为雌花的过程必然涉及雌花发育和性别调控的关键基因，本文利用诱导雌花较为精准地对黄檗雌花发育相关基因进行了研究。为获取黄檗雌雄株中性别相关基因的差异特征，首先对黄檗雌、雄花进行差异基因分析，获得332条雌雄花差异基因，包括性别调控基因RPP0W、PAL3、MCM6、MCM2、ABA2、GPAT3、GAST1、TOZ19；性器官建成发育相关基因PIN1、PHB、AGL11、AINTEGUMENTA、AIL6、SUP、ESR2、SRS5、YABBY2、SEUSS、RL1、RL2、BAM2、FD、ZAT3等；生化途径相关基因MET1、AGO16、BASS2等；激素相关基因LAX3、RGA1、GI等。为了探究雌花发育相关基因，对诱导雌花和同株雄花进行差异分析，得到296条差异基因，其中有216条在黄檗雌、雄花中同样表现出差异，这些基因相较雄花，在雌花和诱导雌花中都表现出差异，与黄檗雌花发育相关性较大；去甲基化后未发生性别改变的雄花和同株雄花的差异基因与雌花发育相关性极小，剔除这部分基因后，最终筛选得到188条黄檗雌花发育候选基因。结合文献中报道的功能及qRT-PCR分析结果，性别调控基因和花原基起始因子RPP0W、PAL3、MCM2、MCM6、SUP、PIN1、ANT、AIL6、AGL11、SEUSS、SRS5、ESR2被初步确认为黄檗雌花发育关键基因。

此外，本文还利用黄檗雌、雄花的差异基因对雄花发育相关基因进行了初步分析，通过分析雌、雄花差异基因特征，结合文献中报道的基因功能，初步推测性别决定基因TDF1、ARR17及雄花发育早期基因PI、AP3、AG、FD、TGA10、MS1可能对黄檗雄花发育具有较为关键的作用。

本研究建立了适合黄檗雌花性别决定研究的实验平台，确认了黄檗性别分化受到表观遗传的调控，明确了部分与雌、雄花发育相关的基因，为黄檗性别形成过程和性别分化机制的研究奠定了初步的基础，对植物性别分化的研究具有参考价值。

Phellodendron amurense Rupr., a species of Rutaceae, is a dioecious plant as recorded. However, monoecious P. amurense was first discovered during previous research. The top of monoecious P. amurense died when it was young, and the lateral buds grew and developed into branches of different sexes, which suggested that the sex formation of P. amurense occurred after the formation of branches, and there was different sex expression in the same genetic background. This phenomenon has led the sex differentiation mechanism of P. amurense to epigenetic. DNA methylation is one of the most common and intensively studied modes of epigenetic regulation and we attempted to make a breakthrough in DNA methylation in this study. The expression characteristics of sex-related genes in P. amurense was analyzed, and its epigenetic sexual regulation mode was preliminarily explored in this study.

As a whole, the sex formation of plants should be the result of signal transmission and coordination among various organs. In order to get the information of the overall sex characteristics and better understand its sexual formation process, we analyzed the expression characteristics of sex-related genes in the annual branch bark, leaves, and flower organs of both female and male plants of P. amurense. The results are as follows: (1) The number of sex-related genes expressed in each part of P. amurense was different, and regardless of the female or male, the most sex-related genes were expressed in flower organs, followed by annual branch bark and the least in leaves. The specifically expressed genes were most in flower organs, most of which were related to flower development; the genes specifically expressed in annual branch bark are mostly related to embryonic development and plant sexual differentiation; there are very few genes specifically expressed in leaves (only two genes: a hormone-related gene and a female fertility gene). (2) A total of 611 sex-related genes were obtained from the female, while 612 sex-related genes were obtained from the male. The genes specifically expressed in the female were CCR4-NOT, 60s ribosomal protein, ESR2, ERF114, while the genes specifically expressed in the male plants were ARR17, ABORTED MICROSPORES, X10A, CDPKO, V-type proton ATPase subunit G1; (3) There are most differentially expressed genes (DEGs) in floral organs between female and male, followed by annual branch bark, and fewest differentially expressed genes in leaves. The DEGs in floral organs between male and female included sex-determining candidate genes and a large number of flower development genes. The DEGs in the annual branch bark between female and male involved some sex-determining candidate genes, such as SRS5 and MCM6, and relatively few genes related to flower development. The number of DEGs between male and female leaves is extremely small, and key genes for female flowers development were not involved. We speculate that annual branch bark might be used as a good material for studying P. amurense sex according to its expression characteristics of sex-related genes. In addition, through analysis of the DEGs between the bark of different stages, it was found that there were a large number of sex-related genes differentially expressed in annual branch bark compared to trunk bark, and many sex-determining candidate genes are upregulated.

As to sexual epigenetic regulation in P. amurense, we attempted to make a breakthrough in DNA methylation. DNA methylation is one of the most common and intensively studied modes of epigenetic regulation. The effect of DNA methylation on P. amurense was studied by a precise demethylation method, in order to prove epigenetic involvement in sex differentiation of P. amurense. After treatment with 5-azaC, some male flowers were induced to transform into female flowers with complete structure and function, and there were no other variation types. Studies on the morphology and function of induced female flowers have found that they are completely consistent with natural female flowers in terms of morphology, structure, and function, characterized in that stamens degenerated to different degrees and ovaries could develop into fruits with seeds. Male flowers of P. amurense could be induced into female flowers with complete structure and function after 5-azaC treatment, demonstrating that epigenetics is involved in the sex regulation of P. amurense, with DNA methylation as one of its regulatory modes.

Some male flowers could be induced into female flowers with no other variation types after treatment with 5-azaC, during which key genes related to female flower development were involved. Genes related to the development of P. amurense female flowers were explored in this study using the induced female flowers. Differential expression analysis was conducted between female and male flowers to acquire information on DEGs between the two samples. A total of 332 sex-related DEGs were identified between female flowers and male flowers, which involved sex regulatory genes (including RPP0W, PAL3, MCM6, MCM2, ABA2, GPAT3, GAST1, TOZ19), genes related to floral initiation and development (including PIN1, PHB, AGL11, AINTEGUMENTA, AIL6, SUP, ESR2, SRS5, YABBY2, SEUSS, RL1, RL2, BAM2, FD, ZAT3 etc.), biochemical pathway related genes (including MET1, AGO16, BASS2 etc.) and hormone related genes (including LAX3, RGA1, GI etc.). In order to explore genes related to the development of female flowers, 296 DEGs were obtained by the differential analysis between the induced female flowers and male flowers, among which 216 genes also showed differences in female and male flowers. Compared with male flowers, these genes showed differences in both the female and the induced female flowers, which were more likely to be the genes related to the development of female flowers. DEGs between male flowers with no change after demethylation and male flowers were less likely to be key genes and could be regarded as not linked with sex formation. After removing such genes, 188 candidate genes were screened out finally. Combining with their functions, genes associated with sex regulation and floral initiation, including RPP0W, PAL3, MCM2, MCM6, SUP, ANT, AIL6, AGL11, SEUSS, SRS5, and ESR2, were preliminarily considered to be the key genes for female flower development.

Moreover, genes related to male flower development were explored using DEGs between female and male flowers of P. amurense. Through the differential characteristics between female and male flowers and combining with gene functions reported in literature, it was preliminarily speculated that the sex-determining genes TDF1 and ARR17, and early male flower development genes PI, AP3, AG, FD, TGA10, and MS1, may play a crucial role in the development of male flowers in P. amurense.

This study has established an experimental platform suitable for studying sex determination of P. amurense female flowers, confirmed the sexual epigenetic regulation of P. amurense, and identified some genes related to female and male flower development. It has laid a preliminary foundation for understanding P. amurense sex formation process and studying sex differentiation mechanism, and has reference value for the study of plant sex differentiation.