Molecular Mechanisms Of Flowering In Catalpa Bungei Mutant ‘bairihua’

Flowering is a very complex process in the life of plants.However,the research on the molecular mechanism of flowering of woody plants is slow because of the long juvenile period,high genome heterozygosity and other objective factors.Catalpa bungei(Catalpa bungei C.A.Mey.)is a perennial woody plant of the genus Catalpa of the bignoniaceae.It is a unique timber tree species in China.In nature,Catalpa bungei has a longer period of infancy.It blooms more than five years after planting,and the whole flowering period is 10-15 days.‘bairihua’,which is a new variety of C.bungei developed by the research group.It blooms for the year after planting and the accumulative flowering period of ‘bairihua’ reaches 100 days.‘bairihua’ provides valuable materials for studying the molecular mechanism of flowering in C.bungei.This paper takes “bairihua” as the research object,and 9-1 as the control,to studies the flowering mechanism of C.bungei.The buds were categorized into three consecutive developmental stages(Vegetative growth period,Floral transition period and Reproductive growth period).The full length sequence of gene were get based on SMRT,and hub flowering related genes were got by WGCNA analysis,and the differences between these genes in different periods were revealed from the perspective of selective splicing.By high-throughput sequencing technology,differentially expressed m RNAs and lnc RNAs,lnc RNA with precursors of mi RNA and flowering related lnc RNA-m RNA pairs were screened out.The key flowering related gene Cbu SPL9 was obtained by molecular biology technique,and the potential function of Cbu SPL9 and its interaction protein in the flowering process of C.bungei was analyzed.The main results are as follows:The SAM of “bairihua” underwent floral transition and began to reproduce growth in middle of March and the inflorescence primordia appeared in late March.At the same time,the SAM of 9-1 showed no floral transition,always maintained vegetative growth.The RNA samples were pooled according to flowering buds from “bairihua” and leaf buds from 9-1 for SMRT sequencing.The full-length c DNAs of these samples were sequenced and constructed using the Pac Bio RS II platform.In contrast,83,638 High-quality full-length transcript from flowering buds and 82,191 High-quality full-length transcripts from leaf buds were detected.Functional enrichment of the differentially expressed genes resulted in 15 KEGG pathways related to flowering.58 TF families were significantly differentially expressed in EF compared to NF during floral transition,and 13 TFs might related with the flowering.61 hub genes associated with flowering were screened out by WGCNA.Interestingly,7 out of the 61 hub genes had a close connection with Cbu.gene.18280,and the 7 genes were from different pathways.To verify the intersection results of GASA,we performed the protein-protein interaction analysis online.The genes in C.bungei exhibit divergent structures of isoform splicing in different bud stages.In this study,we categorized C.bungei buds from “bairihua” and 9-1 into three consecutive developmental stages(Vegetative growth period,Floral transition period and Reproductive growth period).In total,12,532 lnc RNAs and 26,936 m RNAs(m RNAs)were detected using high-throughput sequencing based on Illumina Hi Seq 4000 platform.A total of 680 differentially expressed genes(DEGs)and 817 differentially expressed lnc RNAs(DELs)were identified during the initiation of floral transition.The 817 DELs were distributed in 437 families,including 34 known mi RNA families.Some lnc RNAs were selected as potential precursors of the corresponding mi RNAs,and seven lnc RNAs were identified.Of these,three were known lnc RNAs and four were unknown lnc RNAs.In total,119 lnc RNA-m RNA pairs were identified using this method,and seven lnc RNA-m RNA pairs involved in floral transition were identified.The SPL homologous genes in C.bungei were isolated by RACE.Phylogenetic analysis showed that the isolated sequence was clustered with At SPL9.The isolated sequences encoded a protein with typical SPL protein structure,which included a highly conserved SBP-box domain bearing two zinc-binding sites(C3H-type and C2HC-type)and one bipartite nuclear localization signal(NLS).The expression changes of Cbu SPL9 during the flowering process(Dormant period,Germination period,Floral transition period and Reproductive growth period)was significantly evaluated in the flowering buds than in the leaf buds,especially during the dormant period and the reproductive growth period.Overexpression Cbu SPL9 in Arabidopsis showed two phenotypes of floral organ structure change and early flowering.A total of 11 functional proteins with potential interactions with Cbu SPL9 protein were obtained by screening yeast c DNA library.HMGA is identified as a structural transcription factor.The interaction between Cbu SPL9 protein and Cbu HMGA protein in the nucleus was verified by subcellular localization,yeast two-hybrid assay,BLI and Bi FC.The HMGA homologous genes in C.bungei were isolated by PCR of C.bungei genome.Phylogenetic analysis showed that the isolated sequence was clustered with At HMGA.The isolated sequences encoded a protein with typical HMGA protein structure,which encoded a H15 domian at 5’ end and 4 AT-hook at 3’end.The expression changes of Cbu HMGA during the flowering process(Dormant period,Germination period,Floral transition period and Reproductive growth period)was elevated in the dormant period and the highest expression level was presented in the reproductive growth period.Overexpression Cbu HMGA in Arabidopsis showed mutant phenotypes of floral organ structure and flowering time have no obvious change.