| Litsea cubeba（Lour.）Pers.is a shrub or small tree in the genus Litsea in Lauraceae.It mainly distributes in South-east Asia and the southern provinces of China.The peel contains volatile oil,also referred as L.cubeba essential oil,which can be widely used as a fragrance in the fields of manufacture of.food,tobacco,toothpaste,soap,perfume,cosmetics,fungicides,etc.With the demands for natural chemical plant extract,L.cubeba essential oil is popular and in the great demand for the world market.China is the main producer of L.cubeba essential oil,which not only has strong domestic sales,but also has a rising export volume year by year.However,the pheasant pepper is mainly based on wild resources at present,the yield of which is limited,and the content of essential oil is not uniform,which greatly limits the development of L.cubeba essential oil industry.Therefore,it is urgent to breed varieties with high yield and essential oil content for cultivation and promotion.During the collection,preservation and evaluation of germplasm resources in the early stage,we found that the elite female plants with higher essential oil content showed lower yield（mainly caused by smaller crown）and earlier flowering,while male plants with higher crown width and other excellent growth traits flowered later,and the flowering period of the two germplasms did not meet which leads to hybridization difficulties.In order to obtain genetically improved offspring with high yield and essential oil content,it is urgent to explore the flower bud differentiation and florescence regulation to provide theoretical basis and technical support for the development of hybrid breeding between superior germplasms of L.cubeba.In addition,the quantity and quality of flower bud differentiation largely affect the yield and quality of the fruit,thus affecting the yield and quality of the essential oil.Through the study of flower bud differentiation physiology and molecular mechanism of L.cubeba,it also lays a theoretical foundation for molecular breeding of L.cubeba in the future.In this study,based on the morphological characteristics of flower bud differentiation of L.cubeba,the changes of hormones and gene expression during flower bud differentiation were analyzed.The application of exogenous hormones regulated the florescence of L.cubeba and explored the changes of related gene expression level.Finally,the function of target genes capable of effectively regulating flowering was analyzed by Arabidopsis transgenic and mutant-related experiments.The main findings are as follows:1.The flower bud differentiation process of L.cubeba was closely correlated with gibberellin content.Based on the morphological and anatomical characteristics of flower bud differentiation,we developed the change rules of endogenous carbon and nitrogen nutrition,main mineral elements and main endogenous hormones in different differentiation stages of L.cubeba.The results showed that the increase of soluble sugar,mineral elements such as Ca,Mg,Mn,B,Zn and gibberellin（GA3）played an important role in the flower bud differentiation of L.cubeba.The size of female flowers and male flower buds of L.cubeba were significantly positively correlated with the content of GA3.In addition,we found that different concentrations of exogenous gibberellin（GA3）spraying treatment of flower buds could promote flowering of L.cubeba.Among them,200 mg·L-1spraying concentration had the best effect on promoting early flowering,which was about one week earlier than the control plant.The result indicated that gibberellin（GA3）is one of the main factors affecting the flower bud differentiation and florescence in L.cubeba.2.Differentially expressed genes during flower bud differentiation were significantly enriched in“Plant hormone signal transduction”pathway.and“Starch and sucrose metabolism”pathway.According to the morphological characteristics of the flower buds,the comparative transcriptome analysis of the five differentiation stages of male and female flower buds of L.cubeba was carried out.Through the KEGG enrichment analysis of DEGs in different stages of flower bud differentiation,it was found that they were significantly enriched in the pathways of“Plant hormone signal transduction”and“Starch and sucrose metabolism”,indicating that plant hormone and carbohydrate play an important regulatory role in flower bud differentiation of L.cubeba.3.79 gibberellin-related differentially expressed genes were screened out.The differentially expressed genes of different stages of male and female flower bud differentiation were analyzed by k-means clustering and divided into 20 groups.GO enrichment analysis was performed on 15 clusters with obvious trend,and 79 genes related to gibberellin were screened from differentially expressed genes with significant enrichment（P<0.05）.4.Response genes for exogenous gibberellin regulating florescence of L.cubeba were screened.By analyzing the comparative transcriptome data of four stages of flower bud before and after gibberellin application,it was found that they were again significantly enriched in“plant hormone signal transduction”pathway which was similar to the analysis of differentially expressed genes at different stages of flower bud differentiation.According to the analysis of differentially expressed genes before and after the application of exogenous gibberellin,the expression of the above 79 genes were analyzed,and three transcription factors of Lc NAC1,Lc CBF3 and Lc RAV2 which were highly expressed in response to exogenous gibberellin were further screened out.The regulatory network constructed by WGCNA showed that Lc RAV2was the central gene in the flower bud differentiation process of L.cubeba.It has a co-expression relationship with gibberellin-related genes such as GA2OX8-1,GA2OX8-2 in the regulatory network.The expression patterns of the three gibberellin-responsive genes were verified by real-time quantitative PCR experiments.5.Transgenic Arabidopsis thaliana preliminarily verified the regulation of three gibberellin responsive genes on florescence.Through the over-expression transgenic experiment in A.thaliana,it was found that A.thaliana overexpressing Lc NAC1 flowered about 3 days earlier than the control plant,while A.thaliana overexpressed Lc CBF3 and Lc RAV2 delayed flowering about 4 days and 5 days,respectively.The florescence of mutants nac1,cbf3 and rav2 with three homologous genes in A.thaliana was observed.We found that the mutant nac1 delayed flowering for about 7 days compared with the control plant,and the mutant cbf3 and rav2 flowered about 5 days and 3 days earlier than the control plant.These results indicated that Lc NAC1 could promote flowering,while Lc CBF3 and Lc RAV2 could inhibit flowering. |
