Separation And Function Analysis On Flower Coloration Genes In Narcissus Tazetta

Narcissus is one of the ten traditional flowers in China and a famous bulb flower in theworld, which has more than1000years of cultivation history in China. Single cultivar andspecies recession have severely restricted the development of domestic Narcissus industry.Due to the nature of triploid, it is difficult for Narcissus to breed new varieties via sexualhybridization. Narcissus flower color molecular breeding has not yet been established in ourcountry and few researches on physiology and biochemistry have been reported. In thisresearch, changes of flavonoids and carotenoids content in Narcissus tazetta flower weretested through high performance liquid chromatography (HPLC), and the suppressionsubtractive hybridization (SSH) cDNA libraries between yellow and white petal wereconstructed for the isolation of flower color related genes. In order to select the key gene offlower pigmentation formation, the relative expression of candidate genes were tested viafluorescence quantitative PCR (qRT-PCR) at different flowering stages. And the PSY genepromoter of HUANGHUASHUIXIAN Ⅱ was cloned using chromosome walkingtechnology. This study provided a certain theoretical basis and gene resources for furtherresearch on genetic engineering. The main results were as follows:1. Analysis of flavonoids and carotenoids content in Narcissus tazetta flowerThe contents of carotenoids and flavonoids in petal and corona of JINZHANYENTAIand HUANGHUASHUIXIANⅡ at flowering phase were tested. The results showed thatthere were a large number of various pigmentations accumulated in petal and corona. Rutinand phytoxanthin were the main pigmentations in HUANGHUASHUIXIANⅡflower, andrutin, naringin and phytoxanthin were main pigmentations in JINZHANYENTAI flower.The dynamics of rutin and naringin contents in two varieties above are consistent atflowering phase, showing that the pigmentation contents both in petal and corona were thehighest at flower budding period, decreased slightly at early flowering stage and full-bloomstage, and increased at decline stage. However, the changes of phytoxanthin content showeda different pattern. T test analysis results showed that the color differences of Narcissustazetta flower may be affected mainly by the phytoxanthin content. It is speculated that thephytoxanthin plays a key role for the formation of orange and yellow, and rutin and naringinmay be important supplementary factors for pigmentation.2. Construction and analysis of the SSH cDNA libraries between yellow and white petalThe forward and reverse SSH cDNA libraries were constructed between yellow and white petal, and were tested to be of reliable quality. Nine hundred and thirty-two cloneswere randomly selected and sequenced. Gene Ontology (GO) annotation functionclassification results showed that the flower color difference of Narcissus might beinfluenced by multiple metabolic pathways. The uniESTs involved in the color formationwas verified by fluorescence quantitative PCR, and thirteen uniESTs were obtained forfurther investigation of their roles in the flower color formation of Narcissus.3. Cloning of the full length cDNA of NtIPI, NtCRTISO, NtNCED, NtF3’MO-like genes(1) Two genes, named NtIPI-H (accession number: KC841854.1) and NtIPI-J(accession number: KC841855.1), were cloned from Narcissus tazetta var. by SSH andRACE. The ORF is858bp encoding285amino acids, and there are eight amino acidsdifferent between NtIPI-H and NtIPI-J. Sequence analysis showed that the amino acidsequence of NtIPI-H was87%,86%,77%and75%homologous with that of IPI genes fromNicotiana tabacum, Zea mays, Vitis vinifera, Ipomoea batatas, respectively.(2) Two genes, named NtCRTISO-H (accession number: KC207079.1) andNtCRTISO-J (accession number: JX469116.1), were cloned from Narcissus tazetta var. bySSH and RACE. The ORF is1767bp encoding588amino acids, and there are two aminoacids different between NtCRTISO-H and NtCRTISO-J. Sequence analysis showed that theamino acid sequence of NtCRTISO-H was84%,84%,84%and83%homologous with thatof CRTISO genes from Arabidopsis thaliana, Calendula officinalis, Oryza sativa Japonica,Zea may, respectively.(3) Two genes, named NtNCED-H (accession number: KC841856.1) and NtNCED-J(accession number: KF017562.1), were cloned from Narcissus tazetta var. by SSH andRACE. The ORF is1803bp encoding600amino acids, and there are nine amino acidsdifferent between NtNCED-H and NtNCED-J. Sequence analysis showed that the aminoacid sequence of NtNCED-H was80%,80%,77%,77%homologous with that of NCEDgenes from Gladiolus hybrid cultivar, Lilium formosanum, Crocus sativus, Solanumtuberosum, respectively.(4) Two genes, named NtF3’MO-like-H (accession number: KC841857.1) andNtF3’MO-like-J (accession number: KC841858.1), were cloned from Narcissus tazetta var.by SSH and RACE. The ORF is1518bp encoding505amino acids, and there are six aminoacids different between NtF3’MO-like-H and NtF3’MO-like-J. Sequence analysis showedthat the amino acid sequence of NtF3’MO-like-H was65%,65%,64%,64%homologouswith that of F3’MO-like genes from Solanum lycopersicum, Vitis vinifera, Cucumis sativusv, Fragaria vesca, respectively.4. qRT-PCR of the differential expression genes during flowering phaseThe expression analysis of NtPSY, NtCRTISO, NtIPI, NtNCED, NtF3’MO-like genes inthe alabastrum stage, early flowering stage, full-bloom stage, and faded stage wereconducted by qRT-PCR with NtActin as reference gene. The qRT-PCR results showed thatthere were three genes, NtPSY, NtIPI, and NtNCED, of which the differences of relativeexpression level at four flowering phase were significantly differential betweenJINAHANYINTAI white petal and JINAHANYINTAI orange coronal, also betweenJINAHANYINTAI white petal and HUANGHUASHUIXIAN II yellow petal. It isspeculated that NtPSY, NtIPI and NtNCED gene might play important role during the colorformation of Narcissus tazetta flower.5. Cloning of PSY promoter of Narcissus tazetta HUANGHUASHUIXIAN IIIn order to study NtPSY gene expression and regulation, a2522bp sequence of the5’flanking region of NtPSY gene was isolated via genome-walking method. One198bp intronwas found in NtPSY gene5’ UTR region. Neural Network Promoter Prediction resultsshowed that the transcription start site might be at305bp upstream from the start codon ofPSY and the transcription initiation site was the base C. Cis-regulatory element predictionresults by plantCARE showed that NtPSY5’ flanking sequence contained a lot of lightresponse elements, adversity stress and hormone signal response elements.