Identification Of Target Genes And The Functional Specificity Of LEAFY And NEEDLY In Pinus Tabuliformis Carr.

The LEAFY(LFY)gene encodes a plant-specific transcription factor Which is found in algae and all land plants.LFY homologous genes exert roles in regulating cell division and the formation of floral meristem and flower organs,which acts as a key player in floral induction genes regulatory network.Priviously studies have shown that the amino acid sequences of LFY are very conserved between different seed plants,and their functions are also similar.While existing primarily as a single copy gene in most angiosperms,gymnosperms possess two LFY homologous genes(LEAFY/NEEDLY),indicating that the LFY gene may have other functions in plant development,especially gymnosperms.While,the specific roles of LFY and NLY,two highly conserved homologous genes,in the reproductive development of Pinus tabuliformis,are still unclear.In the present study,we analyzed the expression patterns of LFY and NLY genes in P.tabuliformis,functional specificity and the screening of target genes in model plants,differential response genes in P.tabuliformis and the difference of binding sites in genome.We revealed the functions specificity of Pt LFY and Pt NLY in the reproductive development of P.tabuliformis,and provided new insights into the roles and functions of conifer LFY homologous genes in reproductive development and evolution.The main results and conclusions obtained in this study are shown as follows:(1)Pt LFY and Pt NLY genes play an important role in regulating the reproductive development of P.tabuliformis and Pt NLY might play a core regulatory role in coniferous trees.There were significant differences in the expression of Pt LFY and Pt NLY genes in thedifferent tissues,with the highest expression level in vegetative buds and flower buds,but did not expressed in roots,stems and needles;The expression level decreased in the later stage of male and female cones,and the decline was more significant in the male cones than female cones,and trace expressed in the later stage of pollen maturity;Pt LFY and Pt NLY were similarly accumulated in different tissues,however,Pt NLY had a higher expression level among the various tissue samples of P.tabuliformis.(2)More solitary flowers and flower variations were obtained by overexpressing Pt LFY and Pt NLY genes in wild-type Arabidopsis,indicating that Pt LFY and Pt NLY had different functions with LFY homologous genes in Arabidopsis.The plant expression vectors of p35S::LFY-GFP and p35S::NLY-GFP were constructed and ectopic expressed in wild-type Arabidopsis,resulted in some obvious changes in plant morphology,the apical inflorescence meristem transformed into two or three flowers and converted lateral branch to solitary flower,in addition,the Pt NLY transgenic plants also showed phenotypes of solitary flowers from rosette leaves and floral variations,which showed an increase the number of carpels.However,no early flowering phenotype was observed in the Pt LFY and Pt NLY transgenic Arabidopsis.(3)Pt LFY and Pt NLY genes could not completely replace At LFY gene,and Pt NLY gene function was more similar to At LFY gene.Overexpression of Pt LFY and Pt NLY genes under the lfy-1 mutant background showed that Pt LFY and Pt NLY could partially rescued the mutant phenotype,and Pt NLY had a stronger regulation function in floral organ development in Arabidopsis.RNA-seq analysis were performed and the results revealed that compared with Pt LFY,more genes of DEGs(Differentially Expressed Genes)responsed to Pt NLY overlaped with At LFY target genes,of which 54.8%(890/1624)DEGs of Pt NLY/lfy-1 overlaped with At LFY target genes,while Pt LFY/lfy-1 was 39.7%(658/1658).Meanwile,Pt NLY and Pt LFY only shared 20-25% of the regulatory genes,and Pt LFY can independently regulate a part of At LFY target genes.(4)We established a highly efficient Agrobacterium-mediated transient gene expressionsystem for P.tabuliformis callus and ontogenetic transformation.Under the conditions of 0.8OD of Agrobacterium,120 ?mol/L of AS(Acetosyringone),transformed for 30 min and co-cultured for 3 days,the highest transformation efficiency(70.1%)was obsatined.This system can be easily transfer to different tissues in various coniferous trees.By overexpressing Pt LFY and Pt NLY in P.tabuliformis,the DEGs responsed to Pt LFY and Pt NLY were obtained,and indicating that their function did have redundancy in P.tabuliformis,but they also had their own unique response genes.(5)Preliminarily explains of the binding sites of Pt LFY and Pt NLY in the whole genome and the functions of related genes.Using DAP-Seq technology,Pt LFY and Pt NLY had 5666 and 2021 binding sites on the whole genome,respectively,of which 765 directly bound target sites in Pt LFY,and 425 directly bound target sites in Pt NLY are in the promoter region;GO enrichment revealed that differentially binding sites of Pt LFY and Pt NLY were mainly involved in vitamin synthesis,metabolic pathways and multiple response-related pathways,and in the development of the reproductive organs,such as petals,carpels and ovules.