| The ephemeral early-spring plants mainly consist of the understory herb layers and are one of the main components of the forest ecosystem.The sexual reproduction of the ephemeral early-spring plants includes two stages: the underground development of floral organs in a long time and the flowering period after overwintering.Erythronium japonicum has a short period of growth cycle on the ground.And in this short early-spring period of time,it completes the process of blooming,pollinating,fertilizing and producing seeds quickly.The floral organs development in the underground corm,bulb,or rhizome doesn’t begin until the aerial parts have withered.The development lasts till the formation of flowers in autumn,and after overwintering it blooms quickly in next early-spring.The flower development of Erythronium japonicum originates under the ground without photoperiodic induction and vernalization,which is very special in the mechanism relating to molecular genetics.Therefore,it’s great significance to explore and appraise the genetic regulation factors in the differentiation of flowers.As a representative of ephemeral early-spring plants in Changbai Mountains area,Erythronium japonicum falls on the role of the subject in the research.The analysis methods combined transcriptomics and metabonomics are applied into the research to explore both the expression dynamics and changes of key candidate genes and metabolites that affect the floral organs development in their underground bulbs,and to speculate the genetic network of the ways of the floral organs development,thus revealing the molecular mechanism of the floral organs development of the ephemeral early-spring plant Erythronium japonicum.The main results are as follows:(1)High-throughput sequencing technology was used to analyze the transcriptome of the four stages of the underground development of the floral organs of Erythronium japonicum.A total of 178,951 single genes are obtained from the transcriptome,and the N50 is 880 bp.The average length of a single gene is 706 bp,and the longest sequence is ≥2000bp.Among them,28,508 are differential expressed genes(DEGs)at different stages of the flowers development.Further annotation of DEGs helps to identify 70 DEGs that are specifically targeted at flower differentiation.ELF3,PHD,cullin-1,SE14,ZSWIM3,GIGNATEA and SERPINB are set definitely as potential candidate genes involved in the regulation of flower differentiation.In addition,we explored transcription factors that have differential function of regulation at different development stages,and identified b HLH,FAR1,m TERF,MYB-related,NAC,Tify and WRKY transcription factors that may be involved in the process of underground flower differentiation.Finally,18 genes that may be related to flower differentiation of Erythronium japonicum are identified,and preliminary verification of the transcriptome data and corresponding expression of candidate genes at different floral organs development stages was completed by using q RT-PCR technology.(2)Extensive targeted metabolomics was applied to identify the metabolites of four stages of the underground development of the floral organs of Erythronium japonicum,and a total of586 metabolites were identified.These metabolites are further divided into 13 categories according to their primary structure,including lipids(19.97%),organic acids(13.31%),phenols(13.31%),amino acids and their derivatives(12.29%),and flavonoids(8.19%),alkaloids(7.51%),nucleotides and derivatives(6.14%),terpenes(2.22%),lignans and coumarins(2.05%),steroids(1.88%),tannins(0.51%),quinone(0.51%)and others(12.97%).By the way of multivariate statistical analysis,the research established the OPLS-DA model to analyze the specificity of tissues in each period.Comparative metabolomics among different stages of the flower development reveals several abundant metabolites at each stage.The accumulation of 15:0 of coumadin,scopolamine,isorhoifolin,chrysanthetin,genistein and lysophosphatidylcholine emphasizes the importance of these compounds in the process of flower organs development.(3)In the joint analysis,we used the DEGs-ELF3,FLK,cullin-1,PHD and ZSWIM3-determined by transcriptomics for flower differentiation,to depict the high correlation with lipids and flavonoids and the accumulation of phenols during the flower development.ELF3,with the closest connection with the metabolites,shows the most significant difference during the flower development.Therefore,the further analysis of biological function of ELF3 is going to be carried out for its role of the first chosen gene during the floral organs development.(4)First we,using RACE technology,extend the length of Ej ELF3 gene,and the c DNA becomes 2232 bp.Coding Polypeptide chain,made up of 743 amino acids,is hydrophilic unstable protein composing the alpha-helix and irregular structures.By using Blastn,highly homologous species are selected from NCBI database,and 20 plant homologous protein sequences are downloaded.On the basis of the analysis of the phylogenetic tree built by MEGA 7.0,the conclusion is drawn that the highest homology exists between Ej ELF3 genes and Dendrobium tosaense,which confirms the existence of Ej ELF3 cloned by us on a genetic level.Preliminary verification of Ej ELF3 gene on the basis of subcellular localization and BIMOLECULAR fluorescence complementation,shows that Ej ELF3 gene-coded protein is located in the cell nucleus and is expressed with Arabidopsis At ELF4 instantaneously.It is tentatively identified that Ej ELF3 gene cloned,having biological function,is an important gene that is able to influence the developmental regulation of the flowers.In conclusion,the results of our research have made significant progress in knowing the underground development of floral organs of Erythronium japonicum,and enhanced understanding of the underground flower differentiation and genetic regulation of Erythronium japonicum.The research reveals the molecular mechanism of the floral organ development of ephemeral early-spring plants,and provides theoretical basis and genetic resources for the study on the floral organ development mechanism and molecular breeding of Erythronium japonicum and other early-spring plants. |
