Comparison Of Structure And Expression Patterns And Indirect Functional Verification Of Multi Season Flowering Candidate Genes In ’Qiugui’ And ’Sijigui’

Osmanthus fragrans has extremely high ornamental value in garden applications.According to the flowering period,Osmanthus fragrans can be divided into two major groups:’Qiugui’and’Sijigui’.’Sijigui’is a multi-season flowering plant.However,there is currently no research to prove the molecular mechanism of multi-season flowering in’Sijigui’.The obvious differences in flowering characteristics between the two types of Osmanthus fragrans varieties provides us with excellent research materials for research.This study is to clarify whether there are structural differences or isomers in the two types of Osmanthus fragrans cultivars candidate genes for multi-season flowering.Using the autumn flower buds of‘Qiugui’ and’Sijigui’ as experimental materials,using the third-generation full-length transcriptome sequencing technology,combined with morphological observations,the transcriptome data was analyzed for differentially expressed genes,and perform bioinformatics analysis and functional verification of candidate genes for multi-season flowering.The results of the study are as follows:1.The continuous observation and recording of autumn flower buds of ’Qiugui’ and’Sijigui’ showed that the overall morphological process of differentiation of autumn flower bud of’Qiugui’and ’Sijigui’was similar.The overall rate of flower bud differentiation is relatively slow in the early stage and relatively rapid in the later stage.The differentiation process of flower buds in the early stage of ’Qiugui’has always been ahead of that of ’Sijigui’ fragrans,while the differentiation rate of ’Sijigui’ accelerated in the later stage,and completed the differentiation ahead of ’Qiugui’,and then entered the flower opening stage.During the whole flower bud differentiation process,the ’Qiugui’ buds were abundant and the morphology was full,while the number of ’Sijigui’ buds are relatively sparse and the morphology was small.2.In this study,the three periods with obvious differences in bud morphology of ’Qiugui’and ’Sijigui’ were used as experimental materials for Oxford Nanopore Technology sequencing,and 2628,3573 and 3819 differential genes were screened in the ’Qiugui’ group,’Sijigui’group,and the comparison between ’Qiugui’ group and ’Sijigui’group,respectively.Through the analysis of differential genes,the candidate genes AP1 and TFL1 for multi-season flowering were mined.The GO annotation and enrichment analysis of differential genes shows that translation,redox process,and secondary metabolite biosynthesis process are the most significant enrichment items in biological processes.Enrichment analysis of the KEGG pathway found that the differential genes were significantly concentrated in pathways such as sugar metabolism pathway,plant hormone transduction,redox pathway,carotenoid biosynthesis and flavonoid synthesis pathway.3.Through sequence analysis of candidate genes for multi-season flowering,it is found that AP1 has two homologous genes in ’Qiugui’ and ’Sijigui’,namely OfAP1-a and OfAP1-b.There are two splicing isomers OfAP1-a1/OfAP1-a2,OfAP1-b1/OfAP1-b2,which are full-length splicing and intron-reserved splicing isomers in ’Qiugui’ and ’Sijigui’.There are two isoforms of TFL1 gene in ’Qiugui’ and ’Sijigui’ namely the normal transcribed isoform and the frameshift mutant isoform OfTFL1-1/OfTFL1-2 which is caused by the addition of 2bp in the first exon.We predicted the three-dimensional structure of these genes,and the results showed that OfAP1-a and OfAP1-b genes belong to the MADS family of genes;OfTFL1 gene is a PEBP family gene,and OfTFL1-2 isoforms are partially missing due to frameshift mutations.The expression pattern analysis showed that OfAP1-a1/OfAP1-a2,OfAP1-b1/OfAP1-b2 and OfTFL1-2 isoforms had different expression levels in the T4 or T6 stage of ’Qiugui’ and’Sijigui’ bud differentiation.Through the subcellular localization experiment of transformed tobacco,it is found that OfAP1-a1/OfAP1-a2 are located in the nucleus.4.Observation of the phenotype of transformed Arabidopsis plants showed that OfAP1-a1 isomers promoted the early flowering of Arabidopsis and OfAP1-a1/OfAP1-a2,OfAP1-b1/OfAP1-b2 isomers reduced wild-type Plant height of Arabidopsis.OfTFL1-1/OfTFL1-2 isomers delayed the flowering of Arabidopsis and increased the plant height of wild-type Arabidopsis.