Analysis Of Genes Related To Flowering In Chinese Narcissus Based On Transcriptome Sequencing

Chinese narcissus(Narcissus tazetta var.chinensis)is one of the ten most popular traditional flowers in China.It is famous for its unique form and aroma,with high ornamental,cultural and economic value.Due to triploid and complex genetic backgroun,researches of breeding and related theories of Chinese narcissus were limited.For ornamental plants,flowering is the most important process.However,there are few reportes on the molecular mechanism of flowering in Chinese narcissus.In this study,transcriptome of Chinese narcissus 'Jinzhanyintai' flower bud was sequenced by using Illumina technology,a high-throughput transcriptome method.A large number of genes closely related to bud differentiation of Chinese Narcissus were discovered through bioinformatics analysis of the data.Based on the results,some of the genes were selected and their expression patterns in different tissues,organs and flower bud differentiation periods were analyzed.SOC1 and AGL6,key genes related to flowering process in Chinese Narcissus,were cloned.The expression patterns and functional identification of SOC1 and AGL6 were analized by qPCR and genetic transformation.The main results are as follows.1.Screening of genes related to flowering in Chinese narcissusTranscriptome of Chinese narcissus flower bud was sequenced by using Illumina technology,a high-throughput transcriptome method.289,544 Transcripts and 123,544 Unigenes were obtained.36,059 Unigenes could be compared to 7 databases,and 90 were screened related to flowering of Chinese narcissus.2.Expression Analysis of genes related to flowering in Chinese narcissusEleven genes closely related to flowering regulation,which screened from the flower bud transcriptome database,were analyzed by real-time fluorescence qPCR.During the induction stage of Chinese narcissus flower formation,the expression levels of DELLA,GA2ox,SPY and TOE2 were higher in leaf bud stage,and decreased gradually with differentiation.The change of EMF2 expression was first decreased,then increased,then decreased.FT1 gradually increased and then decreased again during flower bud differentiation.The change of API gene expression in the early stage of differentiation was not significantly and reached a peak at the end of July.The SVP gene showed a tendency of decreasing firstly and then increasing.SPL3 and SPL9 decreased at the early stage of differentiation,and gradually increased when the differentiation began,and both of them reached the peak in July 30th.The highest expression level of DELLA was detected in leaves and the lowest in bulbs.GA2ox is mainly expressed in roots and buds.The expressions of two SPY homologous genes in different tissues were relatively consistent,and higher in leaves,buds,and flowers developed.The expression of EMF2 in open flowers,leaves,buds,roots and bulbs decreased in turn.The highest expression of FT was in leaves.API has a relatively high expression level in buds.SVP gene expression is higher in roots and buds.SPL3 has the highest expression level in open flowers.SPL9 has the highest expression in leaves.TOE2 has higher expression in roots,leaves,buds and flowers,especially buds.3.Cloning,expression and functional analysis of SOC1 gene in Chinese narcissusThe full length of NtSOC1,a SOC1 homolog gene,was obtained by RACE and RT-PCR from Chinese narcissus.After analyzed by real-time fluorescence qPCR,it was found that the expression of NtSOC1 was higher in leaves and roots,the expression level of NtSOCl first increased and then decreased during flower bud differentiation,the expression level of NtSOCl peaked at the end of July.Subcellular localization of NtSOC1 was analyzed by agrobacterium-mediated transformation using onion inner epidermis.The results indicate that protein encoded by NtSOCl located in the nucleus.NtSOCl was transferred into tobacco and 7 transgenic plants were obtained.They all showed different degrees of early flowering.The expression analysis of transgenic plants showed that the expression of AP1 and LEAFY in tobacco was higher than that in the control group,indicating that NtSOCl promoted the expression of API and LEAFY and then promoted flower formation.In addition,significant changes in phenotypes such as flower type and flower color were observed in the transgenic plants.In one word,SOC1 gene in Chinese narcissus is related to flowering regulation and flower development.4.Cloning,expression and function analysis of AGL6 gene in Chinese narcissusAGL6 plays an important role in flower induction and floral organ development.In this study,NtAGL61,a homologous gene of AGL6,was obtained by RT-PCR from Chinese narcissus.Clustering results showed that NtAGL6 was closely related to several monocotyledonous AGL6 genes.The expression level of NtAGL61 gene was higher in flower buds and open flowers.The expression level of NtAGL61 increased first and then decreased in the process of bud differentiation,reached a peak in late July.Subcellular localization analysis revealed that the protein encoded by NtAGL61 was located in the nucleus.The transgenic plants were found with an early flowering phenomenon.The degree of early flowering was positively correlated with the expression of NtAGL61 gene in transgenic tobacco.Fluorescence quantitative analysis showed that NtAGL61 gene promoted the expression of AP1,SOC1 and LEAFY in tobacco and made tobacco bloom earlier.In addition,morphological changes of petal,abnormal development of stamens and stamens were found in transgenic tobacco plants.This indicates that NtAGL61 gene was associated with flowering and floral organ development.