The Molecular Mechanism Of Bud Dormancy In Narcissus Tazetta Var.chinensis

Narcissus tazetta var.chinensis is one kind of the most favorite summer dormancy plant because of its ornamental value,commercial benefit and medical advantage.Temperature has an important effect on its growth and development,dormancy induction and release during the life cycle.In this experiment,on the basis of determination of the dormant stage,three-year-old bulbs were used as materials for establishing the transcriptome in different stages using the Illumium RNA-Seq method,further studying the cloning and expression of dormancy-related genes and the relation between temperature and dormancy,and preliminarily studying the transformation of dormancy-related genes mediated by Agrobacterium tumefaciens.The main results were described as follows:1 Determination of the dormant stageIn these studies,dormant stage was analysized from morphological,anatomical and amyloplast changes in the apical meristem of main buds,amyloplast changes in scale layers(interior bulb),soluble sugar content changes in the main buds.The results show that bud dormancy lasts from early-July(mid-July)to early-July(mid-July)between bulb swelling stage and flower differeniatiation stage,which contains early-dormancy stage and late-dormancy stage.Differences of dormancy stage were that the apical meristem of bulb swelling stage with blue leaves and stronger root growth was active,amyloplasts in scale cell became bigger and more,soluble sugar content decreased.The typical characteristic of the dormant phase is that bulb weight and size reached a maximum,leaves sensed the irrigated soil;the apical meristem was inactive,with the highest number of amyloplasts and then decreasing,soluble sugar content increased first and then decreased.The apical meristem of flower differentiation without leaves and sensed root differentiated flower,amyloplasts became small;soluble sugar content was more than 3.00%.2 Analysis of the effect of temperature on bud dormancyThe results from temperature analysis in Zhangzhou in 2011 and 2012 in accompany with morphological observation of dormant buds shows that dormant buds were inducted while average temperature of consist five days was more than 25?;when efficient acumulative temperature(T?25 ?)reached from 5 to 150?:buds were in dormant stage;dormant buds released and started flower differentiation while efficient acumulative temperature reached 150?.Temperature is an environment cue for dormant induction and release.3 Establishment and analysis of transcriptome during different stagesFive cDNA libraries were generated from RNA obtained from apical meristem at bulb swelling(2012.4.29),dormant stage I(2012.5.5),dormant stage II(2012.6.2),dormant stage III(2012.6.9),and flower differentiation stage(2012.7.7).After cleaning raw reads,data were assembled de novo into 142,866 All-unigenes.Using the BLASTx and BLASTn methods(E value<10-5),the rate of 70,313 All-unigenes annotated in five public databases(NCBI non-redundant(nr)database,nucleotide database(nt),Swiss-Prot protein database,Kyoto Encyclopedia of Genes and Genomes(KEGG),and Gene Ontology(GO)database)were up to 93.98%?66.08%?33.43%?59.11%?55.50%.According to FDR?0.001 and log2Ratio ?1,18 common differentially-expressed genes responsive to temperature was obtained from neighboring comparative,which contained triosephosphate isomerase gene,temperature-induced lipocalin gene and so on;6 of 19 unigenes annotated FT genes were differentially-expressed genes.4 Cloning and bioinformatics analysis of dormancy-related genesOn the basis of five cDNA libraries analysis,8 dormancy-related genes were cloned and obtained with RT-PCRor RACE method and bioinformatics analysis shows that(1)4 NtFTs genes belonged to PEBPs genes family.Protein encoded by NtFT1,encoded 174 amino acids,was a kind of stable and acidic protein without transmembrane domain;protein encoded by NtFT2,encoded,176 amino acids,was a kind of unstable and neutral protein without transmembrane domain;protein encoded by NtFT3,encoded 181 amino acids,was a kind of stable and alkaline protein with a transmembrane domain;N(FT4 gene was cloned only 256 bp length.(2)NtTIL gene,belonging to lipocalin gene family,encoded 188 amino acids.Protein was a kind of stable,acidic and hydrophilic protein with I transmembrane domain.(3)NtTIM gene,belonging to TIM-phosphate-binding protein gene family,encoded 254 amino acids.Protein was a kind of stable,acidic and hydrophobic protein with I transmembrane domain.(4)2 NtCOBs gene,belonging to COBRAs gene family,encoded 451 amino acids.Both of their proteins were a kind of stable,alkaline and hydrophilic protein with signal peptide.The differences was that protein eccoded by NtCOB1 gene contained 2 transmembrane domain;protein eccoded by NtCOB2 gene contained 3 transmembrane domain.5 Analysis of dormancy-related genes expressionThe expression of 8 dormancy-related genes at bulb swelling,dormancy and flower differentiation stage shows that(1)The NtFT1 gene expression was inconsistent with the other NtFTs gene expression at bulb swelling and dormancy stage;NtFT1 genes expression was more than the others at flower differentiation stage.(2)The NtTIL expression increased at bulb swelling stage;its expression increased in accopamy with rising ambiance temperature and then decreased at dormancy stage;finally increased at flower differentiation stage(3)The NtTIM expression increased at bulb swelling stage,then decreased at deep-dormancy stage,finally increased.(4)Both of 2 genes expression increased at bulb swelling stage;but NtCOBI expression,showing " W",was inconsistent with NtCOB2 during dormancy stage;their expression at flower differentiation stage was lower.The dormancy regulation of 8 dormancy-related genes shows that environmental cues changed(temperature ? 25?),NtCOBs genes were conductive genes of temperature-signal pathway,accepted and transmited signals,the expression of NtTIL gene in the cell changed which was responsive and cumulative to temperature and transmit it;at the same time,the expression of NtTIM gene also changed,finally NtFTs genes expression changed which were target genes of temperature-signal pathway and key genes of dormanct induction and release,NtFT1 inducted late-deep dormancy,NtFT2 promoted flower differentiation,dormancy release and flower differentiation,NtFT3 was involved in dormancy induction and release and promoted flower differentiation.NtFT4 inducted and maintained bud dormancy.When temperature changed,the expression of dormancy-related genes(NtTIM?NtTIL?NtCOBs)which took part in changes in physiological processes and signal transduction passway from metabolism,energy metabolism,temperature-signal transduction was affected,which led to changes of NtFTs genes expression,and further regulated dormancy in N.tazetta var.chinensis.6 Establishment of the plant regeneration system and o histological bservation of callusCallus induction from ovaries was up to 66.27%on the MN+4.0 mg/L 6-BA+0.4 mg/L 2,4-D+0.2 mg/L NAA;the multiplication medium for creamy-white,small,globular and friable callus culture was MN+2.0 mg/L 6-BA+0.2 mg/L NAA+0.5 mg/L 2,4-D for 60d culture interval,the rate reached 5.16 times;the differentiation rate of bulb was 94.17%on the MN+4.0 mg/L 6-BA+0.4 mg/LNAA,bulbs cultured on the 1/2 MS+0.2 mg/L NAA rooted.Callus was very sensitive to Hyg.,25.0 mg/L for Hyg.concentration which proved to be restricted callus growth was used for selecting transgenic plantlets.Four stages were observed during bulb regeneration from callus,the first stage was induction stage,creamy-white and nodular callus with thin cytoplasm and fragile cell walls were formed;the second stage was division stage,callus with condense cytoplasm and compact cell multiplied;the third stage was differentiation stage,white tissues with bigger cell and ductals were formed;the last one was bulb formation,bulb regenerated from white tissue.7 Obtainment of the induction of dormancy-related genes to callus used in Agrobacterium-mediated transformationSense expression vector of dormancy-related genes(p1301-NtFT2,p1301-NtTIM,p1301-NtTIL)were constructed by vector pCMABIA1301,all of which were transfered into Agrobacterium tumefaciens EHA105 via freeze-thaw method.The transformation of sense NtFT2 gene mediated by Agrobacterium tumefaciens was developed from callus,the best transient expression of GUS reached 64.28%under the conditions as follows:callus which were not precultured but pretreated with 0.5 mol/L mannitol for 6 h;and the Agrobacterium suspension was with OD600 value of 1.0,the samples were infected for 20 min,and then they were transferred onto the MN medium supplemented with 2.0 mg/L 6-BA+0.2 mg/L NAA+0.5 mg/L 2,4-D for co-cultivation for 6 days at 25? in the dark.After immersed in 300 mg/L Cef.for 20 min,callus was cultured on MN+2.0 mg/L 6-BA+0.2 mg/L NAA+0.5 mg/L 2,4-D+100 mg/L Cef.for some days,and then selected.This transformation system laid a foundation for studing the function verification of dormancy-related genes and breeding new varieties.This study laid a foundation for solving the effect of high temperature on dormancy and studying summer dormancy mechanism,and provided new ideas for cultivating new varieties in N.tazetta var.chinensis.