The Key Genes Screening And Function Analysis Of Vernalization And Photoperiodic Flowering Pathway In Lilium

Lilium is an important commercial flower,and flowering time is its important character.At present,studies on the regulation of Lilium flowering mainly focus on vernalization or photoperiod pathway.However,there are many species of Lilium and flowering time is different,and there are few studies on whether these two main flowering pathways interact with each other and how they are related to the environment.Therefore,transcriptomic data were used to screen the key genes of vernalization and photoperiod and the key genes of proline synthase(P5CSs)in different flowering characteristics Lilium.On this basis,the expression patterns of these genes in different Lilium were compared,and studied the the expression patterns of vernalization and photoperiod key genes in different sizes bulbs of Lilium longiflorum under different vernalization and light conditions,and screened out the key gene members that regulate different flowering types.The correlation between LP5 CSs gene and key genes in photoperiodic flowering regulation was analyzed.This study provides a theoretical basis for the study of the evolutionary mechanism of Lilium flowering and the establishment of cultivation techniques.The main results are as follows:(1)Based on transcriptomic data and NCBI online database,we analyzed the phylogenetic relationship,conserved protein domain sequence,primary structure and physicochemical properties of the gene family sequences from different Lilium species,included vernalization genes of 20 LFRI,3 LSVP,14 LVIN3,13 LVRN,photoperiod related genes of 10 LCRYs,11 LGIs,5 LFKF1,and 42 LCOs,flowering integration genes of 5 LLFY,6 LSOC1.It is speculated that LFRI3-1,LFRI3-2,LVIN3-1,LVRV1,LCRY1 and LGI2 genes may be unique genes related to flowering in monocotyledons.(2)Analysis of LFTs gene family expression patterns in potted Lilium during flowering process showed that LFT3 was highly expressed in stem during flowering transition stage,which may be involved in flowering transition in Lilium cultivars with high vernalization requirements.We analyzed the expression patterns of LFTs in the leaves and scales of potted lily after anthesis,the result showed that the expression of LFT1 in the scales increased at the early stage after anthesis,which may be involved in the development of bulblets;LFT2 increased first and then decreased in both leaves and scales,which may be related to the meristem and proliferation of bulblets,LFT3 was highly expressed in later stage,which may be involved in the accumulation of plant nutrients after anthesis.(3)Three-generation-transcriptome sequencing and analysis were carried out in the bud stage of Lilium longiflorum and L × formolongi.As a result,we respectively obtained the Unigene data amount was 12.39 G and 10.67 G at the bud stage of Lilium longiflorum and L × formolongi,get 124001 and145038 nucleotide sequences.After the obtained gene sequences were functionally annotated in NR,Swiss Prot,KEGG,KOG,GO,NT and Pfam databases,we get a large amount of gene functional annotation information;GRAS and FAR1 transcription factors related to photochrome signal were found in both Lilium species,and GARP-ARR-B transcription factors related to circadian dimension were unique in L × formolongi,suggesting that the flowering of both Lilium species was closely related to photoperiod,while the flowering of L × formolongi was closely related to circadian gene expression.(4)We analyzed the flowering characteristics and expression patterns of key genes in vernalization and photoperiod pathway during flower formation in different sizes Lilium longiflorum bulbs that under different vernalization and photoperiod conditions.We found that the flower formation of Lilium longiflorum was both influenced by vernalization and photoperiod pathways.The vernalization pathway had a great influence on the flowering transition of Lilium longiflorum large bulbs,and the flowering of Lilium longiflorum small bulbs was greatly affected by photoperiod.In addition,low temperature treatment may increase the sensitivity of small bulbs to LDs.Ll VRN1 in vernalization pathway,Ll FKF1,Ll GI,Ll CO5,Ll CO7,Ll CO16,Ll FT1,Ll FT3 in photoperiod pathway,and integration factor Ll SOC1 might be directly involved in the floral transformation process of Lilium longiflorum.(5)To research the expression patterns of flowering related genes in vernalization and photoperiod pathway of Lilium henryi and OT-hybrids‘ Black beauty’,the results showed that Lh VRN1、Lh VIN3-2、Lh CO5、Lh CO6、Lh CO7、Lh CO9、Lh CO11、Lh CO16、Lh FT1、Lh FT2、Lh SOC1、Lh FKF1、Lh GIs may be involved in the flowering transformation of Lilium henryi,while the Lbb VRN1、Lbb VIN3-2、Lbb CO5、Lbb CO6、Lbb CO9、Lbb CO11、Lbb CO14、Lbb FT1、Lbb FT3、Lbb FKF1、Lbb GIs may be involved to the flowering transformation of OT-hybrids‘ Black beauty’.According to the expression pattern analysis,both Lilium henryi and OT-hybrids ‘Black beauty’ bloom after planting,and the photoperiod pathway is the main regulation of flower formation,but vernalization can promote flower formation.The common key genes of Lilium henryi and OT-hybrids ‘Black beauty’ were LFKF1,Lh GIs,LCO5,LCO6,LCO9,Lh CO11 and LFT1.(6)We cloned 2 LP5 CSs homologous genes from Lilium longiflorum,L × formolongi and‘Sorbonne’,3 LP5 CSs homologous genes from Lilium henryi and OT-hybrids ‘Black beauty’,bioinformatics analysis showed that LP5 CS gene is more conservative.The expression patterns of LP5 CSs during flowering process of Lilium longiflorum,L × formolongi,Lilium henryi and OT-hybrids‘Black beauty’ showed that LP5CS1 is closely related to all growth stages of Lilium.The involvement of LP5CS2 in plant vegetative growth may be related to LD conditions,and LP5CS2 may also be involved in the flowering transition of Lilium.LP5CS3 was speculated to be related to floral induction of plants,well it has little relationship with light conditions.(7)We did the tests of drought and saline-alkali resistance to trans Va P5CS-F129 A gene ‘Sorbonne’.Under stress,the content of proline in leaves of transgenic plants was higher than that of ‘Sorbonne’,while the content of malondialdehyde was lower than that of ‘Sorbonne’.Both Va P5 CS and Lo P5CS1 are significantly up-regulated by drought and saline-alkali treatment,the up-regulation-expression of Lo P5CS2 is relatively low.And the transgenic plants had higher drought tolerance and saline-alkali tolerance than the un-transgenic plant.In addition,the transgenic plants grew better under the same environmental conditions,at the same time,the transgenic plants had longer bottle insertion life.