Digital Gene Expression Proifling And Cloning And Expression Analysis Of Key Gene During Chrysanthemum ’Jinba’ Flower Bud Differentiation

Chrysanthemum morifolium Ramat. is a chrysanthemum plant species in the Asteraceaefamily. This plant is one of four major cut flowers worldwide, and it has more than3000cultivated varieties. Cut flowers are typically obtained from short-day (SD) plant species, thephotoperiod regulation technique primarily controls flowering through light receptor geneproducts which receive a light signal and transmit the signal to circadian gene products. Thecircadian rhythm clock output gene activates the expression of the transcription factor andthen enhances the expression of the flowering-promoting gene in the leaves. Then transportedto the shoot-tip and functions together with flowering-integrating factors, regulation ofdownstream flowering-promoting factors in the photoperiod pathway.This study with white cut-flower chrysanthemum‘Jinba’as test materials, because of itstypical characteristics of short day, can be accurately controlled flower bud differentiationprocess, is the study of molecular mechanism of chrysanthemum flower bud differentiationmodel material. By high-throughput EST sequencing technique, the data compare with theNCBI nucleic acid protein database, through bioinformatics analysis, determine the functionclassification and metabolic pathways involved, into chrysanthemum flowering related geneexpression information, explore different in chrysanthemum flower bud differentiation phasedifferentially expressed enrichment pattern and expression of related genes，the photoperiodpathway related the key gene cloning and expression analysis, at the molecular level ofchrysanthemum flowering time of network way to have a preliminary understanding,So the information might be the base of system research the chrysanthemum into themolecular regulation mechanism and its biological function and sellecting flowering relatedgenes and using for transgenes.The main research results are as follows:1. Studies on transcriptomic express sequence tags (ESTs) of chrysanthemum duringfloral differentiation. sellected54,488unigenes which of moleculor functions, cell conpenentsand bioprocesses. All of the above unigenes involved12of molecular functions and11of cellcomponents, and18of bioprocesses. Sequences of nucleic acids and amino acids of54,644 encoding protein boxes (CDS) were sellected from all of the unigenes of chrysanthemumduring floral differentiation by BLAST software. Also8,506unigene sequences of nucleicacids and amino acids of CDS by ESTscan software. Comparison to the GenBanknon-redundant (nr) database in COG database revealed that22,871unigenes sequences weresimilar to other known plant homologous genes which classfied to23homologys and780unigenes sequences maybe new genes in chrysanthemum. Also25,001unigenes weresellected which of participating in metabolism pathway using KEGG database. From there269unigenes also sellected that related to participating circation rhythm metablism pathways,and also sellected20homologous genes which related to photoperiod pathways includingphotoreceptor genes, circadian rhthm clock genes, circadian rhythm import and export genes.2. Digital gene expression of chrysanthemum bud differentiation period involving plantrhythm pathways：Chrysanthemum light receptors PHYB orthologous genes expressed peaked at buddifferentiation initiation stage, is nearly five times more the pre-treatment level, then a slightdecline, until the involucre scales middle differentiation stage, nearly three times more thepre-treatment level，at bud differentiation completion stage，nearly two times the amount ofexpression.Circadian clock component APRS, TOC1expressed peaked at involucre scaledifferentiation mid-term stage.PIF3expressed peaked at bud differentiation initiation stage.FKF1express the change of the peak, dark treatment decreased expression a day after，the expression peaked at bud differentiation initiation stage and the floret primordiumdifferentiation and bud differentiation completion stages.Circadian clock output gene expression GI dark treatment after a day’s slight decline,expressed in bud differentiation initiation periods reaches peak, in the involucre scales middledifferentiation are down a bit, at bud differentiation completion stages have rebounded.Expression of CO dark treatment after a day’s slight decline, expressed in involucrescales differentiation medium quantity reaches peak, is nearly two times more thepre-treatment level; CHS on dark expression quantity rise, a day after start-up of the highest innearly five times more, involucre scales differentiation medium expression quantity slightlydecreased, then in the middle of the floret primordium differentiation decline.3. Preliminary concluded that chrysanthemum flower bud differentiation periodsignificant enrichment of differentially expressed genes of the main biochemical metabolic pathways and signal transduction pathways, concentrated in photosynthesis and ribosomepathways.4. Preliminary concluded that different chrysanthemum flower bud differentiation stageenrichment of differentially expressed genes involved in biological function：At the bud un-differentiated：single stranded RNA binding，poly-pyrimidine tractbinding，protein-arginine N-methyltransferase，oxidoreductase activity，acting on paireddonors，with incorporation or reduction of molecular oxygen. At the bud differentiationinitiation：protein-arginine N-methyl transferase activity，fatty acid synthase activity，sugar-hydrogen symporter activity. At the involucre scale differentiation mid-term stages：protein transmembrane transporter activity，pyrimidine nucleotide sugar transmembranetransporter activity. At the floret primordia differentiated mid-term stage：oxidoreductaseactivity，oxidizing metal ions，NAD-NADP as acceptor，protein-arginine N-methyltransferaseactivity. At the corolla differentiation mid-term and bud differentiation completion stages：structural molecule activity，guanyl ribonucleotide binding，arsenate reductase activity.5. The cDNA sequence of GIGANTEA was cloned from chrysanthemum‘Jniba’，The sequence was submitted to GenBank, and the registration number is JQ043439. Sequenceinformation is done in the analysis. Fluorescent relative quantitative analysis shows that theexpression patterns of chrysanthemum CmGI are circadian rhythms expression, with its peakexpression occurring at16:00. At different flower bud differentiation stage, the CmGI gene inthe leaf blade mRNA level is different, two peak values were appeared in the flower buddifferentiation start-up and floret primordia middle differentiation periods. CmGI mRNA ishighly expressed in the leaves at the full-blossom stage.6. We cloned the flowering gene CmCOL (GenBank accession:KC589293) fromChrysanthemum morifolium‘Jinba’. Sequence information is done in the analysis. Relativefluorescence quantitative analysis showed that CmCOL mRNA is most highly expressed inthe leaves and demonstrates a circadian expression pattern, with its peak expression occurringat04:00. A short-day treatment elevated CmCOL mRNA expression in the leaves but did notsignificantly affect expression in the buds. The CmCOL mRNA level in the leaves changedsignificantly at various flower bud differentiation stages, and it peaked at the floret primordiadifferentiation mid-term stage. CmCOL mRNA is highly expressed in the leaves at thefull-blossom stage.7. Relative fluorescence quantitative analysis showed that CmFTL mRNA is most highly expressed in the leaves and demonstrates a circadian expression pattern, with its peakexpression occurring at00:00. A short-day treatment elevated CmFTL mRNA expression inthe leaves but did not significantly affect expression in the buds. The CmFTL mRNA level inthe leaves changed significantly at various flower bud differentiation stages, and it peaked atthe floret primordia differentiation mid-term stage. CmFTL mRNA is highly expressed in theleaves at the bud stage.8. The present study showed that Chrysanthemum morifolium leaves could sense thephotoperiod. Under SD conditions, CmGI mRNA expression began to increase one day afterthe SD treatment and reached the first peak at the bud differentiation initiation stage. CmCOLmRNA expression lagged behind that of CmGI; it increaseds lightly at the bud differentiationinitiation stage, suggesting that CmGI operates upstream of CmCOL. CmCOL expressionreached its peak at the floret primordia differentiated mid-term stage, which occurred afterCmGI expression peaked. The expression of CmFTL and CmCOL did not change significantlyunder micro-regulation. CmFTL mRNA expression peaked at the floret primordiadifferentiated mid-term stage, which occurred after CmCOL mRNA expression peaked,suggesting that CmFTL operates downstream of CmCOL. These results suggest that theGI-CO-FT regulation pattern is similar in Chrysanthemum morifolium under SD conditions,arabidopsis thaliana under LD conditions and Oryza sativa under SD conditions.