黄檗（Phellodendron amurense Rupr.），芸香科黄檗属植物，一般被认为是严格的雌雄异株植物，但我们首次发现多株雌雄同株植株（两性植株）。多数两性植株在较矮处进行分杈，主干较矮，不同的分枝形成不同性别。通过4年观察发现两性植株性别表达稳定，没有出现性别转化现象。在此之前没有关于黄檗性别可塑性的报道，也没有发现类似黄檗两性植株性别表达方式的植株。黄檗两性植株既有雄性，也有雌性，或可以克服远距离传粉障碍，将有利于黄檗有性繁殖；两性植株的雌雄枝有相同的遗传背景，但又有不同且稳定的性别表达，是性别分化相关研究良好的研究材料。因此我们对黄檗两性植株有性繁殖能力和黄檗性别形成和分化机制进行研究。

黄檗两性植株是首次发现，形态是植株首要特征，因此首先对黄檗两性植株形态进行了观察。通过观察发现，两性植株除主干较矮、性别表达特殊外，其茎、叶、花、果实和种子形态与雌雄异株植株没有差异。

黄檗在自然条件下只能通过有性繁殖方式进行繁殖，因此研究黄檗两性植株能否有性繁殖以及繁殖能力如何具有重要意义。我们分别对黄檗两性植株雄性繁殖能力、雌性繁殖能力、自交繁殖能力和种子形成幼苗能力进行研究。在雄性方面，以雄株为对照，对两性植株雄性繁殖能力进行研究。对两性植株花粉形态和花粉活力进行研究，发现两性植株花粉形态、大小和萌发率与雄株花粉没有差异。将两性植株花粉授到正常雌株上，共授51个花序，1785朵花，两性植株花粉得果率平均为86.69%，与雄株花粉没有差异，单个果实种子数量平均为3.46，略少于雄株花粉。在雌性方面，以雌株为对照，对两性植株雌性繁殖能力进行研究。对两性植株柱头形态进行观察，发现两性植株柱头形态和发育过程与雌株柱头没有差异。在人工授粉和自然受粉两种条件下对两性植株雌蕊繁殖能力进行研究，在人工授粉条件下，两性植株雌蕊接受正常雄株花粉，共授19个花序，762朵花，得果率平均为91.30%，单个果实种子数平均为4.01，种子数略多于雌株；在自然受粉条件下，采集两性植株自然受粉条件下产生的果实，共19个果序，736个果实，得果率平均为95.69%，单个果实种子数平均为3.97，结果趋势与人工授粉条件下相同。在自交繁殖能力方面，以两性植株接受雄株花粉为异交，接受自株花粉为自交，自交得果率平均为88.01%，单个果实种子数平均为3.94，与异交没有差异。在种子形成幼苗能力方面，以雌株种子对照，对两性植株种子萌发率进行研究，两性植株种子萌发率平均为61.50%，略低于雌株。综上两性植株雄性和雌性都可以正常繁殖，后代可以形成幼苗，繁殖能力与雄株和雌株基本处于同一水平，并且两性植株可以自交繁殖后代。

在转录组水平上对黄檗性别形成与分化机制进行初探。根据247篇文献中查找到的391个性别相关基因，在成年雌雄异株植株和两性植株的雌雄差异基因中筛选出性别相关基因，并筛选出雌雄差异基因中雄性特异表达和雌性特异表达但功能未知的基因，结合两者结果作为鉴别黄檗植株雌雄基因，分别称为雄性鉴别基因和雌性鉴别基因。在本实验中共筛选出69个雄性鉴别基因和143个雌性鉴别基因。通过比对，在幼龄植株中共筛选出21个雄性鉴别基因和69个雌性鉴别基因。多数雄性鉴别基因有一定表达量，但表达量不高，GPAT3（Glycerol-3-phosphate acyltransferase）和一个功能未知基因在一年生植株中表达量较高，且两个基因表达趋势相同，推测黄檗在幼龄时表达为雄性。多数雌性鉴别基因基本不表达，但在六年生植株中，相对六年生1号植株和3号植株，六年生2号植株中52个雌性鉴别基因出现较高表达，其中有6个为已知功能基因，分别是NDH（NADH dehydrogenase）、NDUFA12（NADH:ubiquinone oxidoreductase）、60s ribosomal protein、PGDH（D-3-phosphoglycerate dehydrogenase）、TCP-1（T-complex protein 1）和CCR4-NOT（glucose-repressible alcohol dehydrogenase transcriptional effector），多数与雌性繁殖相关，推测六年生2号植株分化为雌性。在2021年的观察中，该植株开雌花，确为正常雌株。 综上，黄檗性别形成与分化与树龄有关，在幼时性别表达为雄性，随树龄增长，雌性才开始表达，有六年生植株表达为雌性，七年生时开雌花，性别确定的表达是在开花前。

两性植株能够正常生存和繁殖，且能够自交繁殖，有利于黄檗植株生存与繁殖，或将为黄檗保护提供新的思路；黄檗植株性别形成与分化与树龄的相关性为以后性别相关研究提供一定的基础和方向。

Phellodendron amurense Rupr. is a plant of the genus Phellodendron in the Rutaceae. It is generally considered to be a strict dioecious plant, but we discovered the existence of mutiple monoecious plants of P.amurense. The trunk of most monoecious plants is short and usually branch in the lower part of the trunk. The sex expression differentiate with different branches. With observation for 4 consecutive years, it has been found the sex expression of these monoecious plants was stable with no sex transformation. Previously, there was neighter report on gender plasticity of P.amurense, nor monocious palnts of P.amurense with similar sex expression. With both male and female plants, the monocious plants might overcome obstacles of long-distance pollen transmission, which is thought to be benefitial to sexual reproduction of P.amurense. The male and female branches of monoecious plants have same genetic background but with different stable sex expression, the monoecious plants would be a good material for research on sex differentiation. Therefore, the author studied sexual reproduction capability of monoecious plants of P.amurense and its sex formation and differentiation mechanism.

For the first time, the monoecious plants of P.amurense was found. Considering morphology is the primary characteristic of palnts, the morphology of monoecious plants was firstly ovserved. Apart from shorter trunk and special sex expression, there is no difference in the stem, leaf, flower, fruit and seed of monoecious plants and those of dioecious plants through observation.

Under natural condition, P.amurense reproduce only through sexual propagation. It is of great importance to study whether the the monoecious plants of P.amurense is able to reproduce sexually and its reproductive capacity. The reproductive capacity of male, female，selfing and the capacity of seeds to form seedlings were studies respectively in this research. In term of male, compared to male plant, this paper studied the reproductive capacity of male of the monoecious plants of P.amurense. Through research on the morphology and vitality of monoecious plants pollen, there was no difference in pollen morphology, size and germination rate between monoecious and male plants. In this research, a total of 51 inflorescences and 1785 flowers of normal female plants were pollinated by pollen of the monoecious plants of P.amurense. The result showed that the average fruit yield of monoecious plants pollen was 86.69%, which was not different from that of male pollen. The average number of seeds per fruit was 3.46, which was slightly less than that of male pollen. In term of female, compared to female plant, this paper also studied the reproductive capacity of female of the monoecious plants of P.amurense. Through observation of the monoecious plants stigma, there was no difference in morphology and development process and those of female plant stigma. Studying the reproductive capacity of female of monoecious plants of P.amurense, under artificial and natural pollination conditions respectively, it was found that the monoecious plants pistils accepted the male plants pollen with a total of 19 inflorescences and 762 flowers and the fruit rate of 91.30% under artificial pollination condition. In addition, the seed number of per fruit is 4.01, slightly higer than that of the female plants. Collecting 19 infructescences and 736 fruits of monoecious plants under natural pollination condition, the fruit rate is 95.69%, the seed number of per fruit is 3.97, similar to the result under artificial pollination. In term of selfing capacity, monoecious plants accepting the male plants pollen is outcrossing, while accepting monoecious plants pollen is selfing. The fruit rate of selfing is 88.01%, the seed number of per fruit is 3.94, not different from that of outcrossing. In term of capacity of seeds to form seedlings, compared to female plants to studying the seed germination rate, the germination rate of monoecious seeds is 61.50%, slightly lower than that of the female plants. In conclusion, both male and female of monoecious plants of P.amurense can reproduce normally, and are able to form seedlings, and the reproductive ability is basically at the same level with male and female plants. The monoecious plants of P.amurense can selfing to reproduce offsprings.

The exploration of the mechanism of sex formation and differentiation of P.amurense was on the basis of transcriptome level. Base on 391 sex related genes from 247 literaures, this paper screened sex related genes in the sex differential genes of adult dioecious plants and monoecious plants, and filter out male-specific gene expressed and femalt- specific gene expressed with unknown functions in the sex differetial genes. Combining the results, it was taken as the gene to identify the gender of P.amurense; namely, the male discriminant gene and female discriminant genes. In the research, 69 male discriminant genes and 143 female discriminant genes were screened out. Through comparison, a total of 21 male and 69 female discriminant genes were screened out from young plants. Most of the male discriminant genes were expressed to some extent, but the expression level was not high. GPAT3 (Glycerol-3-phosphate acyltransferase) and a gene with unknown function were highly expressed in annual plants, with same gene expression trend. It could be inferred that the expression of P.amurense was likely to be male at young age. Most female discriminant gene should not be expressed, but in six-year plants, 52 female discriminant genes presented higher expression in plant No. 2 of six-year plants relative plant No. 1 and plant No. 3 of the six-year plants, among which, NDH (NADH dehydrogenase), NDUFA12 (NADH: ubiquinone oxidoreductase), 60s ribosomal protein, PGDH (D-3-phosphoglycerate dehydrogenase), TCP-1 (T-complex protein 1) and CCR4-NOT (glucose-repressible alcohol dehydrogenase transcriptional effector) were genes with known functions and most genes related to female reproduction. It could be inferred that the expression of No. 2 of six-year plants was female. In this year's observation, the flowers of this plant was female and this plant was indeed a normal female plant. In conclusion, the gender formation and differentiation of P.amurense is related to the age of the plant. The gender expression is male at the young age, and the female expression begins with the growth of the plant. Some six-year plant expression are female, the seven-year plant blossom female flowers, and the expression of gender determination begins before flowering.

The normal growth, reproduction and selfing of monoecious plants of P.amurense were beneficial to the survival and reproduction of P.amurense. It is also conducive to providing new thought on the conservation of P.amurense. The relevance of sex formation and differentiation and the plant age would provide foundation and direction for further sex related research.