| Wintersweet?Chimonanthus praecox?which belongs to the Calycanthaceae,is a deciduous shrub endemic to China.Its strong fragrance,unique flowering time,beautiful appearance and elegant color make it popular.Wintersweet is widely used as potted plants,street trees,and cut flowers in China.Flowers are the most important ornamental organs of wintersweet,however,the research of molecular mechanism that regulates flower development in wintersweet is still limited.On other hand,most of the CCCH type zinc finger protein genes are reported function in biotic or abiotic stressed tolerance;few of them are involved in plant growth and development.CpCZF1 which was obtained from the flower developmental transcriptome database of wintersweet expressed differentially in the flower developmental stages,so we try to explore the fuction of CpCZF1 to figer out whether it function in flower development.The flower development regulatory network was extensively studied in the 1990s in model plants Arabidosis,six major pathways were found controling flowering and the floral quartet model were uncoverd specifying flower organ identities.The six major patheways includes the aging pathway,the Gibberellin pathway,the photoperiod pathway,the vernalization pathway,the ambient temperature pathway and the autonomous pathway.The floral quartet model-the identity of the floral organs is specified by quaternary protein complexes composed of A-,B-,C-,D-,E-function proteins.Wintersweet flowers blossom in winter,but the study of the molecular mechanism of floral induction and flower organ specification is still limited.Former transcriptomic analysis of flower development in wintersweet revealed that a large number of candidate genes expressed differentially during flower development.Among them,some C3H-type zinc finger protein genes,such as CpCZF1 was found expressed higher in flower bud stage and lower in senescing stage.So we isolated CpCZF1 from flower of wintersweet,analylized its expression patten in wintersweet and characterized its fuction in transgenic Arabidopsis.Results of Yeast two hybrid screen showed that another CCCH-type zinc finger protein CpCZF2 interact with CpCZF1,and the interaction was further confirmed in tobacco cells by employing a BIFC system.Functional analysis reveal that both genes are involved in regulation of floral induction and stamen identity specifications in transgenic Arabidopsis.The main results in this research are as follows:?1?Isolation and sequence analysis of CpCZF1.Based on the sequence obtained from the transcriptome database of flower development,a 5'RACE was performed to get the 5'sequence of CpCZF1.Sequence analysis showed that the 1447 bp full-length cDNA of CpCZF1?Gene bank accession number:KY435926?contains a 909 bp open read frame?ORF?,a 166 bp 5'-untranslated region?UTR?,and a 372 bp 3'-UTR.The gDNA of CpCZF1 contained three exons that were 88 bp,61 bp,and 760 bp in length,as well as two large introns that had standard gt/ag splicing site were 2909 bp and 5322bp in length.?2?Characterization of the Deduced CpCZF1 protein.The deduced CpCZF1protein was analized by the online software ProtParam 10.0,the resuts showed that the peptide had a molecular forum of C1790H2826N488O529S16,a calculated molecular mass of32.07 kD and a theoretical isoelectric point of 9.35;among the Amino acids,the alanine?Ala?residues compose the most of the Amino acids which had a proportion of12.6%?38/302?,followed by glycine?Gly?,which had a proportion of8.9%?27/302?.The protein with a instability index of 39.68,a Grand average of hydropathicity?GRAVY?of-0.380,with no signal peptide and no transmembrane domain which indicate that CpCZF1,a stable Hydrophilic protein,is Neither transmembrane protein nor secreted protein.Sequence analysis showed that CpCZF1contained four putative conserved domains including two C-X8-C-X5-C-X3-H motifs,one C-X7-C-X5-C-X3-H motif and one RNA or single-stranded DNA binding KH-1domain.?3?Expression analysis of CpCZF1 in wintersweet.The qRT-PCR was performed to detect the expression pattern of CpCZF1 in tissues and in the flowers at different developmental stages.The results showed that the transcripts of CpCZF1 is more abundant in flowers than in vegetative tissues?root,stem,cotyledon,young leaves and mature leaves?and among the flower organs the expression of CpCZF1 is higher in petals and stamens than in pistils;in the developmental stages,the transcripts of CpCZF1 is higher in primodium differentiation stages than in the opening stages,especially in the petal promodium differentiation stage.?4?Construction of the flower cDNA based yeast two hybrid labriray.To screen for the interaction proteins of the CpCZF1,a wintersweet flower cDNA based yeast two hybrid labriray was constructed by following the instruction of the‘Make Your Own“Mate&Plate?”Library System'kit.The library has 4.38 x106 independent clones;titer of the library used is 3.9 x107 cfu/mL;the ratio of the insertions between200-2500 bp is 94.9%,and 59%of the insertions longer than 500 bp.16 candidate clones which encode 7 proteins were screened from the library,online BLAST showed that these 7 proteins may involve in RNA metabolism,protein translation,Chlorophyll synthesis,photosynthesis,growth and development;stress responses and senescence.The interaction of CpCZF1 and CpCZF2 was confirmed by the yeast two hybrid and BIFC system which indicate that the interaction was real in both yeast and plant cells.?5?Isolation and sequence analysis of CpCZF2.Based on the sequence obtained from the yeast two hybrid screen and the transcriptome database of flower development,a pair of specific primer was used to amplify and to confirm the cDNA sequence of CpCZF2,and PCR was perfomed to amplify fragment by fragment to get the DNA sequence of CpCZF2.Sequence analysis showed that the 1637 bp full-length cDNA of CpCZF2?Gene bank accession number:KY435926?contains a 1056 bp open read frame?ORF?,a 221 bp 5'-untranslated region?UTR?,and a 360 bp 3'-UTR.The3171 bp gDNA of CpCZF1 contained two exons that were 802 bp and 254 bp in length,as well as one intron that had standard GT/AG splicing site was 2115 bp in length.?6?Characterization of the Deduced CpCZF2 protein.The deduced CpCZF1protein was analized by the online software ProtParam 10.0,the results showed that the peptide had a molecular forum of C1745H2652N494O510S15,a calculated molecular mass of39.19 kD and a theoretical isoelectric point of 8.74;among the Amino acids,the Aspartic?Asn?residues compose the most of the Amino acids which had a proportion of 8.8%?31/351?,followed by alanine?Ala?,which had a proportion of 8.0%?28/351?.The protein with an instability index of 41.07,with no signal peptide and no transmembrane domain,indicate that CpCZF2,a stable Hydrophilic protein,is neither transmembrane protein nor secreted protein.Sequence analysis showed that CpCZF2contained three putative conserved domains including two C-X8-C-X5-C-X3-H motifs and one C-X7-C-X5-C-X3-H motif.?7?Expression analysis of CpCZF2 in wintersweet.The qRT-PCR was performed to detect the expression pattern of CpCZF2 in tissues and in the flower of different developmental stages.The results showed that the transcripts of CpCZF2 is more abundant in flower organs than in vegetative tissues?root,stem,cotyledon,young leaves and mature leaves?and the expression of CpCZF2 is higher in stamens than in pistils and in petals;in the developmental stages,the transcripts of CpCZF2 is higher in primodium differentiation stages than in the opening stages;the transcripts is the highest in the petal primodium differentiation stage and the lowest in the sepal primodium differentiation stage.?8?Subcellular localization and transcriptional activity assays.The coding sequens of CpCZF1 and CpCZF2 was fused with a reporter gene GFP into the vector pCAMBIA1300 to generate the constructs 35S:CpCZF1-GFP and 35S:CpCZF2-GFP respectively.The Agrobacterium tumefaciens with the fused constructs were used to infect the lower epidermis cells of tobacco leaves,and the GFP was observed in the nucleuses while the GFP of the control construct 35S:GFP was observed in the nucleues and the cytoplasm which indicate that CpCZF1 and CpCZF2 were located in the nucleus.The transcriptional activity of CpCZF1 and CpCZF2 was detected in the yeast GAL4 system,the transcriptional activator VP16 was used as the control,?-Galactosidease activity of the yeast strain Y2HGold which transformed with the constructs BD-CpCZF1,BD-CpCZF2,BD,BD-VP16,BD-VP16-CpCZF1 and BD-VP16-CpCZF2 was detected.The?-Galactosidease activities of the yeast cells transformed with BD-Cp CZF1 and BD-CpCZF2 was almost the same as the yeast cells transformed with empty vector BD which indicate that CpCZF1 and CpCZF2 has no transcriptional activation activaty,and the?-Galactosidease activity of BD-VP16-CpCZF1 and BD-VP16-CpCZF2 was lower than that of BD-VP16 which indicate that the?-Galactosidease activity of BD-VP16 was repressed by CpCZF1 and CpCZF2 respectively.All in all,the results showed that CpCZF1and CpCZF2 have transcriptional repression activaties in yeast.?9?Overexpression of CpCZF1 and CpCZF2 affects flower development in transgenic Arabidopsis respectively.The codiong sequence of CpCZF1 and CpCZF2was fused to the modified plant binary vector pCAMBIA2301G to generate the constructspCAMBIA2301G-CpCZF1andpCAMBIA2301G-Cp CZF2.Plant transformation was conducted by the Arabidopsis floral-dip method,the transgenic plants were selected by the kanamycin and then confirmed by the PCR amplification.The qRT-PCR was performed to detect the expression levels of the CpCZF1 and CpCZF2 in the transgenic plants respectively.Two transgenic lines of OE-CpCZF1 and OE-CpCZF2 which expressed differentiationly were selected for phenotype observation.Early flowering,fewer rosette leaves and abnormal stamens were observed in both OE-CpCZF1 and OE-CpCZF2 plants.Expression analysis of the flowering key genes and the stamen identity specification related genes in transgenic plants and wild type plants showerd that the transcripts of the flowering promoting genes-FT,SOC1,AP1,LFY were up-regulated in transgenic plants inflorescence while the expression of the flowering repressor FLC was down-regulated;the expression of stamen identity specification related genes-PI,SEP1-3,AG,AP1,RBE were up-regulated.?10?Sequence cloning,bioimformatic analysis and expression assay of the promoter of CpCZF1 and CpCZF2.hi-TAIL PCR was performed to clone the upstream sequence of CpCZF1 and CpCZF2.A 928 bp of the upstream?ATG?sequence of CpCZF1 was cloned,cis-element prediction was analized by the online software of plantcare,and results showed that the promoter contained a lot of the TATA-box,CAAT-box,light-response cis-elements and few special cis-element-Gibberllin acid-,salicylic acid-,defends and stress response cis-element.Expression analysis showed that the expression of CpCZF1 in young leaves of wintersweet was induced by the treatment of Gibberllin acid and salicylic acid respectively.At the same time,a 1345bp of CpCZF2 promoter was obtained,cis-element prediction showed that besides the TATA-box,CAAT-box,light-response elements,there are Gibberllin acid-,salicylic acid-,abscisic acid-response elements and defense and stress response cis-elements;construct of ProCpCZF2:GUS was transformed to Arabidopsis to detect the expression pattern of CpCZF2 in the tissues of Arabidopsis,and results showed that the GUS activity was detected in all the tissues including endosperm,radicle,cotyledon,roots,stems,leaves,flowers,but much higher in the meristem,sepals and stamens;the expression of CpCZF2 in young leaves of wintersweet was induced by the treatment of Gibberllin acid and Salicylic acid and Abscisic acid respectively.In summary,CpCZF1 and CpCZF2 are two CCCH type zinc finger protein genes expressed higher in flower and the flower bud at the floral organ primodium differentiation stage.Results of yeast two hybrid and BIFC showed that CpCZF1interact with CpCZF2 in yeast and plant cell.Overexpression of CpCZF1 and CpCZF2in transgenic Arabidopsis caused early flowering and abnormal stamen development,further expression analysis showed that the key genes which promote flowering were up-regulated while the repressor FLC was down-regulated,and the stamen identity specification related genes were up-regulated in different degrees in transgenic Arabidopsis.Results of subcellular locoalization and transcriptional activity assays inplay that both CpCZF1 and CpCZF2 were located in the nucleus and had transcriptional repression activaties in yeast.Promoter activity analysis showed that promoters of CpCZF1 and CpCZF2 were inducible;ProCpCZF2 is also a constitutive promoter.All in all,the expression patterns and the phenotypes of the transgenic plant imply that both CpCZF1 and CpCZF2 might play roles in flower development of wintersweet,but the mechanism still to be explored. |
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