The Molecular Regulation Approach During Floral Bud Differentiation And Cloning Of Related Genes From Tung Oil Tree

Tung oil tree (Vernicia fordii) is a representative non-wood forest species especially in subtropical area in China, and is also an important woody oil and bio-energy plant. It is used as experimental material for the research on development biology science of woody oil plants as its special biological characters.Tung oil tree was used as material as its early blossom character for the study on the genes related to the flower bud differentiation as to supply the methodological basis of flower period modification and flower molecular mechanism, also to supppy the possible gene resources of molecular breeding of Tung oil tree. This research used Illumina sequencing technology to explore the transcriptome of tung oil tree’s flower bud in varied flower bud differentiation periods in August and October, respectively, and then transcriptome data was bioinformatically analyzed in which to identify a lot of genes related to flower bud differentiation. The metabolisms of sucrose, starch and nitrogenwere elucidated through metabolism pathway analysis, and the accumulation and utilization, transportation and attribution of sucroseduring the flower bud differentiation period were primarily clarified. The clone and express mode of FLC, LFY and SOC1genes were analyzed based on achieved results. The key results were showed as below.1. Sequence assembly, gene functional annotation and classification. This research performed transcriptome sequencing of tung oil tree’sflower buds at2developmental stages (August and October) using Illumina sequencing technology. Sequence assembly of allclean reads of transcriptome sequencing data obtained106,507Contigs and59,270Unigenes.Length of most contig sequences accounting to93.54percentage of all contigs were distributed between200bp and3,000bp whereas length of Unigenes were primarily distributed between300bp and3,000bp.Atotal of44,579Unigenes could be matched to gene sequences already known in public database such as NR (42,655), Swiss-Prot (25,052), KEGG (23,070) and COG (14,413). Statistics ontranscriptome sequencing data, a total of14,413Unigenes had homologous genes in COG database and could be classified into25categories according to their predicted functions. Through GO classification, all non-redundant Unigenes could be classified into62GO categories. Pathway enrichment analysis classified all non-redundant Unigenes into128metabolic pathways.Ninety-six genes involved in flowering of tung oil tree were identified following alignments of all non-redundant Unigenes against their counterparts in Arabidopsis.2. Expression profile elucidation of genes involved in2differentiation stages of tung oil tree’s flower buds. Lots of differentially expressed Unigenes (24,566) in which14,182Unigens’s expression were up-regulated and10,384Unigens’s expression were down-regulaed were identified form transcriptome data. All differentially expressed Unigeneswere classified into55GO categories and128metabolism pathways, respectively. The expression profiles of most of differentially expressed genes involved in autonomous pathway, GA pathway, iarovization pathway and photoperiod pathway were up-regulated. The mechanisms of nutrition accumulation and utilization, transportation and distribution were elucidated.3. Cloning and characterization of tung oil tree’s FLC, LFY and SOC1gene. We cloned full-length cDNA sequence of tung oil tree’s FLC, LFY and SOC1genes with916bp,1,370bp and1,036bp length, respectively. Through multiple sequence alignments, FLC, LFYand SOC1protein sequences were found to possess high homology against other plants’counterparts. This result demonstrated that the genes we cloned were definitelythe FLC, LFY and SOC1genes of tung oil tree. Phylogenetic analysis identified thattung oil tree’s LFY and SOClhad closest evolutional relationshipswith their counterparts in Jatrophacurcas whereas tung oil tree’s FLC had closest evolutional relationshipswith their counterparts in Betulaplatyphylla. The expression of FLC, LFY and SOC1genes of tung oil treedetected in different stages of flower buds and leaf buds showed significant variation in expression profiles. The expression of LFY gene was firstly down-regulated and then up-regulated while the expression of LFY gene wasfirstly dramatically up-regulated between pre-differentiation stage and mid-differentiation stage, and then down-regulated. The expression profiles of SOC1gene were exactly the same with LFYgene. The expression level of FLC gene in leaf bud was higher than in flower bud while LFYand SOC1genes showed opposite expression profiles.Previous researches indicated that flowering of tung oil tree were not only regulated by autonomous pathway, GA pathway, iarovization pathway and photoperiod pathway but also affected by starch, sucrose and nitrogen compounds. Through clone and expression profile analyses of LFY,FLC and SOC1genes, our researches preliminarily elucidated the molecular mechanism of differentiation of tung oil tree’s flower bud and further provided theoretical reference to the studies in regulation mechanism of tung oil tree’s flowering stage.